Prenatal Diagnosis of Chromosome Abnormalities

Chorionic Villus Sampling

Placental villi are composed of multiple layers, including the chorionic membrane that makes up the outermost layer of fetal tissue and forms the villi for vascularisation of the placenta. In CVS, villus tissue is removed under the guidance of ultrasound by either the transcervical or transabdominal method. Whereas the transcervical method involves inserting a catheter through the vagina and cervix, the transabdominal method uses a thin needle similar to an amniocentesis. The position of the uterus and placenta dictate which method is used. A small sample of chorionic villi is retrieved and sent for cytogenetic analysis. Two preparations types are possible for a CVS sample, direct and cultured. Direct preparation analyses the rapidly dividing trophoblast cells of the placenta and may produce results in 24 to 48 hours. Cultured preparations examine the mesenchymal cells from the villi and typically require 7 to 10 days for results. Cultured cells usually produce better karyotype preparations and are the preferred approach for many laboratories, although some may set up both direct and culture. DNA can be extracted for chromosomal microarray from either direct or cultured cell preparations, and results are generally available in 7 to 10 days for direct and 14 to 21 days for the cultures.

Amniocentesis

The amniotic sac surrounds the fetus and holds the amniotic fluid. Amniotic fluid contains a variety of cell types both from the fetus and the amnion. Amniocentesis involves removing a sample of amniotic fluid through a thin needle inserted through the maternal abdomen and into the gestational sac under ultrasound guidance. Amniocentesis is the most widely used procedure for obtaining fetal specimens. It is most commonly performed between 15 and 17 weeks’ gestation but not before 14 weeks because of the association with talipes equinovarus and the higher risk for miscarriage. Cells are cultured in flasks and on coverslips and may take up to 1 week before there are a sufficient number of cells for cytogenetic analysis. The success rate for culture of amniocentesis samples is approximately 99%, and the reliability of the cytogenetic diagnosis is also around 99%. Early amniocentesis (performed before 14 weeks’ gestation) does carry a 1% to 2% risk for clubfeet ; therefore CVS is the preferred procedure for patients seeking early prenatal diagnosis. As with CVS, DNA can be extracted directly from the cells within the amniocentesis sample without culture and used for chromosomal microarray analysis. Results from a direct preparation are usually available in 7 to 10 days and in cultured cells in 14 to 21 days.

Percutaneous Umbilical Blood Sampling

Percutaneous umbilical blood sampling is an invasive procedure that involves obtaining a fetal blood sample. PUBS may also be referred to as cordocentesis, umbilical vein sampling and fetal blood sampling, and it was originally developed for prenatal diagnosis of blood disorders such as haemoglobinopathies. PUBS specimens are obtained through an ultrasound-guided needle inserted transabdominally into the umbilical cord at the site of insertion into the placenta, the intrahepatic portion of the umbilical vein or rarely a free loop of cord. Several studies to assess the safety of PUBS showed the procedure carried a 1% to 2% risk for fetal death or spontaneous abortion, significantly higher than CVS or amniocentesis. Recent studies have shown that the risk may be higher in fetuses with abnormalities such as nonimmune fetal hydrops or intrauterine growth restriction. For this reason, it is reserved for cases in which diagnostic information cannot be obtained through CVS, amniocentesis and ultrasound or when the results of these previous diagnostic tests were inconclusive. Occasionally, it is used in later gestations when rapid results are important for management. Unlike CVS and amniocentesis, PUBS procedures are performed after 18 weeks’ gestation. Because culture time is shorter for blood samples, results are generally available in 3 to 4 days for classic cytogenetics analysis and 7 to 10 days for chromosomal microarray.

Advanced Maternal Age

The association between increasing maternal age and Down syndrome was recognised as early as 1933 by Penrose. Advanced maternal age is generally accepted to be 35 years or older at the time of delivery. The age of 35 years was designated as the cutoff because the risk for finding a chromosome abnormality and the risk for causing a miscarriage from amniocentesis were roughly the same. Below the age of 35 years, more miscarriages could be expected compared with the number of liveborn children with a chromosome aneuploidy. The increase in fetal chromosome abnormalities in aging women is due to an increase in nondisjunction during meiosis. Fig. 24.1 shows the direct relationship between maternal age and increased risk for chromosome abnormality at the time of CVS and amniocentesis. In twin gestations, considering maternal age subgroups and twin zygosity, a significantly lower-than-expected Down syndrome incidence is seen for both monozygotic and dizygotic pregnancies than previous empiric calculations would suggest.

Advances in cytogenomic technologies such as chromosomal microarrays, noninvasive prenatal screening (NIPS), and discovery of copy number variants (CNVs) with no maternal age-related risk have led to an increasing proportion of younger women (maternal age <35 years) who are considered to be at low risk for aneuploidy to opt for prenatal cytogenetic screening and diagnostic testing.

Abnormal Screening Result

Although advanced maternal age increases the risk for fetal chromosome abnormalities, the majority of pregnancies occur in younger women because they are in their prime reproductive years. As a result, the majority of liveborn children with chromosome abnormalities are found in young mothers. To address this issue, noninvasive screening techniques were developed as a way to identify which pregnancies have an increased risk for fetal aneuploidy without incurring the risk for miscarriage from invasive CVS or amniocentesis procedures. However, because the combined incidence of fetal chromosome abnormalities (i.e. whole-chromosome aneuploidies, submicroscopic CNVs, uniparental disomy (UPD)) is higher than the risk for miscarriage from invasive procedures (0.11%–0.22%), any pregnant woman may choose diagnostic prenatal cytogenetic testing. However, most low-risk women use noninvasive screening to assess their risk before deciding whether or not to proceed with a CVS or amniocentesis. Several screening methods are currently offered to help identify pregnancies at a higher risk for fetal chromosome abnormalities.

Noninvasive Screening Using Biochemical Analytes and Assessment of Nuchal Translucency

Pregnant women have several options for noninvasive screening in both the first and second trimesters. First trimester screening involves a combination of maternal blood testing and ultrasound analysis of the fetus and can be done as early as 11 weeks. Maternal blood is tested for the levels of free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein A (PAPP-A) and combined with sonographic measurement of the nuchal translucency, the space at the back of the fetal neck. Such screening can identify 80% to 90% of fetuses with Down syndrome. Second trimester screening analyses a different set of maternal serum biomarkers. Quad screening using AFP, hCG, unconjugated estriol and inhibin A identifies approximately 67% to 76% of fetuses with Down syndrome. In all cases, the false-positive rate is around 5%. First and second trimester screening can be done as independent tests or in combination. When both first and second trimester screening tests are combined and presented as a single risk assessment report, it is called integrated screening . Sequential screening is when all screening tests are performed, but the patient may decide if the first trimester results are elevated to proceed to diagnostic testing or defer until all results are complete in the second trimester. In general, the threshold for first trimester diagnostic testing is higher (e.g. 1 in 60 to 1 in 100) than that used later (1 in 270).

Contingent screening uses first trimester screening results to guide the next steps of prenatal screening and diagnostic testing. If a pregnancy is identified as high risk using first trimester screening, it is recommended the patient consider invasive testing such as CVS or amniocentesis. If the pregnancy falls within an intermediate-risk category, the patient may proceed to second trimester screening. Finally, if the pregnancy is deemed low risk for a chromosome abnormality during first trimester screening, then no further screening would be recommended. Screening via maternal serum markers is limited to the most common chromosomal aneuploidies and has a false-positive rate of 5%. In general, screening using a combination of first and second trimester markers has a slightly higher detection rate and lower false positive rate than single trimester screening but depending on the approach chosen may delay diagnostic testing in screen positive cases.

Noninvasive Prenatal Screening Using Cell-Free Fetal DNA

Recently, the ability to analyse fetal cell-free DNA (cffDNA), present in the maternal circulation of pregnant women, has paved the way for the development of NIPS for fetal chromosomal aneuploidies (13, 18, 21 and sex chromosome anomalies) and selected microdeletions. cffDNA is placental in origin, and its absolute amount is a small proportion of the total cell-free DNA (cfDNA) component of the maternal plasma, which is a mixture of both maternal cfDNA and cffDNA (<1 μg in 20 mL of whole blood). The current NIPS technologies do not separate cffDNA from the maternal cfDNA. Analysis is performed on the entire cfDNA complement in the maternal blood without extracting or enriching the fetal fraction. NIPS relies on the use of next-generation sequencing (NGS) (whole genome or targeted) that looks for the presence of extra sequences from chromosomes of interest such as 13, 18 and 21. If the mother is phenotypically normal, the additional sequences are inferred to be derived from the fetus. Two major sequencing approaches are utilised; the first is referred to as ‘counting’ because all sequences are counted, and the DNA sequences from the chromosomes of interest are compared with a reference chromosome or chromosome set to determine whether they are in excess or missing. The second approach uses single nucleotide polymorphisms (SNPs) to assess the genotype patterns that are indicative of aneuploidy. Each of these approaches is coupled with advanced statistical algorithms and sophisticated bioinformatics software that assesses for abnormal amounts of chromosome-specific cfDNA in the maternal circulation in pregnancies with fetal aneuploidy. cffDNA is detectable in maternal plasma by 6 weeks’ gestation, increases during the pregnancy at a fetal fraction rate of 0.1%/week between 10 to 21 weeks and 1%/week after 21 weeks, and is eventually cleared from the maternal circulation within a few hours of delivery. The accuracy of any type of NIPS is limited in cases of multiple gestations, and as such, the American College of Obstetrics and Gynecology does not recommend NIPS for women with multiple gestations. Current data suggest that NIPS-based aneuploidy screening in singleton pregnancies has superior performance in screening for trisomy 21 compared with all other existing methods such as combining maternal age, first or second trimester ultrasound findings and first or second trimester serum biochemical analysis. Therefore this screening is an attractive alternative to traditional serum screening for aneuploidy. Second, compared with trisomy 21, detection rates for trisomies 18, 13 and sex chromosome aneuploidies are lower. A recent meta-analysis with the published studies on the performance of cfDNA testing in screening for aneuploidies in singleton pregnancies revealed weighted pooled detection rates and false-positive rates of 99.2% and 0.09%, respectively, for trisomy 21; 96.3% and 0.13% for trisomy 18; 91.0% and 0.13% for trisomy 13; 90.3% and 0.23% for monosomy X and 93.0%; and 0.14% for sex chromosome aneuploidies other than monosomy X. NIPS was initially validated for clinical use in high-risk patients. However, recent literature adds to its utility in the general obstetric population. It is important to note that cffDNA-based NIPS is a screening method, and given the potential for false-positive and false-negative results, it is recommended that screen-positive patients should be followed up by diagnostic testing using CVS or amniocentesis. The current limitations of NIPS include screening for only a few aneuploidies and select microdeletions, the potential for inaccurate results and a higher cost. Pregnant women opting for NIPS should be appropriately counselled about its limitation to screen for only a few aneuploidies (13, 18 and 21), about the difference between a screening test and confirmatory diagnostic testing and about the potential of NIPS to reveal incidental maternal findings such as a maternal chromosome abnormality or even uncover maternal cancer.

Abnormal Fetal Ultrasound Findings

Ultrasound is important for noninvasive monitoring of all pregnancies, from early confirmation of pregnancy to assessing fetal status through delivery. A recent meta-analysis showed an overall detection rate between 46% and 51% for fetal anomalies before 14 weeks. The detection rate in low risk or unselected populations was 32% and in high-risk groups around 60%. Neck anomalies, such as increased nuchal translucency or cystic hygroma, have the highest detection rate (92%). Abdominal defects such as omphalocele and gastroschisis are also detected at a high rate on early ultrasound. Brain, spine and heart defects had around 50% detection rate, and limb, genitourinary tract and facial defects each had only about a 34% detection rate. It is also important to note that some anomalies (e.g. agenesis of the corpus callosum, cerebellar hypoplasia, renal agenesis, duplex kidneys) will not become apparent until the second trimester and are not reliably detected on early ultrasound. Anatomy scans between 18 and 20 weeks’ gestation are the standard of care for assessing the health of fetuses, but with advances in early screening, many women choose to have an ultrasound in the first trimester.

Chromosome aneuploidies are often associated with multiple congenital abnormalities, which are often apparent on fetal ultrasonography ( Tables 24.1 and 24.2 ). In the 1990s, practitioners began using sonogram measurements of the nuchal translucency as a marker for Down syndrome. Other notable markers for chromosomal aneuploidies include cystic hygroma, nasal bone hypoplasia, echogenic intracardiac focus, exomphalos and fetal growth restriction. As with serum biomarker screening, a positive finding increases the risk but does not mean the fetus is affected with a chromosome abnormality. Generally, the more markers identified by ultrasound, the higher the risk for an affected fetus.