Patients who carry chromosome abnormalities are one of the most difficult groups of patients to treat, due to the high levels of abnormal embryos they produce. Many couples around the world opt for PGD as an alternative to prenatal diagnosis, and several thousand babies have been born. However, below we will try to describe the main methods of prenatal diagnosis of fetal used today in most medical institutions of Ukraine.
During the second trimester of pregnancy, a number of markers have been found that help to identify pregnancies with chromosome abnormalities. Down’s syndrome pregnancies show lower maternal serum alpha-fetoprotein (AFP) and un-conjugated estriol, whereas human chorionic gonadotropin (hCG) levels are two times higher than normal. Taking into account the patient’s age, a triple screening test to measure AFP, free beta-hCG and un-conjugated estriol can increase the detection rate to 70%, thereby reducing the number of women who require invasive prenatal diagnosis.
Pregnancy-associated plasma protein A (PAPP A) is reduced in Down’s syndrome pregnancies. Non-invasive prenatal testing (NIPT) that screen cell-free DNA in maternal serum is now also available.
During the first trimester of pregnancy, ultrasound detection of nuchal translucency measuring greater than 3 mm is associated with a chromosome abnormality. This is caused by fluid accumulation at the back of the fetal neck. In conjunction with maternal age, studies have shown this to give a detection rate of 86% with a false positive rate of 4.5%. When used in combination with first and second trimester serum screening, a detection rate of over 90% has been reported.
Approximately, half of all major structural abnormalities can now be detected by usage of ultrasound in the first trimester, including acrania/anencephaly, abdominal wall defects, holo-prosencephaly, and cystic hygromata. Other anomalies become evident later in the second trimester, as organ systems develop: cardiac malformations, duodenal atresia, hydrops, choroid plexus cysts, nuchal edema, renal pyelectasis, omphalocele, hypoplastic midphalanx of the fifth finger, and short femur and humerus can be used to screen for aneuploidy.
Digital imaging combined with computer analysis of two-dimensional ultrasound pictures can be used to create three—and four-dimensional images, which allow more in-depth assessment of cardiac and neural abnormalities, and show a clearer picture of what specific abnormalities look like.
Non-invasive Prenatal Testing (NIPT)
Antenatal screening for the autosomal trisomies, and potentially also for many other genetic conditions, is undergoing major developments as non-invasive prenatal testing (NIPT) has been introduced in many areas. In Ukraine, non-invasive prenatal testing (NIPT) has been approved for use as a second-line screening test for those identified by conventional screening (based on ultrasound and maternal serum markers) as having an increased chance of fetal Down syndrome (or trisomy 13 or 18). The major benefit is that only those with an increased chance of an affected fetus on both tests (conventional serum screening and non-invasive prenatal testing [NIPT]) will have amniocentesis, so there will be many fewer procedures—and many fewer procedure-related pregnancy losses.
A different approach is being used in some private clinics in Ukraine and elsewhere. Non-invasive prenatal testing (NIPT) is being used there as a first-line screening test. However, the performance of non-invasive prenatal testing (NIPT) is not as satisfactory as a first-line test, because the prior risk of a trisomy is lower, so that the positive predictive value (PPV) of a “positive” screening test is around 80% as a first-line test, whereas it is at least 90% as a second-line test. Furthermore, if additional testing for a wider range of CNVs is requested as well, then the performance becomes much less satisfactory so that the number of “unnecessary” invasive tests, to confirm or exclude a potential diagnosis of a microdeletion syndrome, based on the non-invasive prenatal testing (NIPT) result, would increase again. However, non-invasive prenatal testing (NIPT) on its own is certainly more effective as a screening test than earlier methods.
What must be clearly understood by all concerned is that non-invasive prenatal testing (NIPT) is not a diagnostic test in this screening context. Whether screening by non-invasive prenatal testing (NIPT) is offered as a first-line or second-line screening test for autosomal trisomy, it is not diagnostic. Therefore, a pregnancy should not be terminated in the mistaken belief that a positive screening test based on non-invasive prenatal testing (NIPT) has no need for confirmation. Any positive non-invasive prenatal testing (NIPT) screening test requires confirmation by additional (usually invasive) testing.
If the purpose of antenatal screening for Down syndrome is taken to be the opportunity to avoid having an affected child, the evidence currently suggests that conventional screening approaches will allow this in a considerably greater proportion (60%-70%) of pregnancies than age-related screening alone. Screening that employs non-invasive prenatal testing (NIPT) will have a higher sensitivity and higher PPV and could be used to avoid more than 95% of cases. How this can be weighed against any adverse effects on women being screened, on our patients with Down syndrome and other genetic conditions, and on society as a whole, are open questions. More evidence, social as well as scientific, is urgently needed as such screening becomes more comprehensive.
Good practice necessitates the full provision of up-to-date information to allow informed choices by women and their partners. It is therefore most important that professionals understand the use of several key terms in the evaluation of population screening tests. These include the sensitivity, specificity, and the positive and negative predictive values of the test.
Focusing on the performance of a screening test may sound rather dull and dry but it is crucial, especially when different providers promote their services in the marketplace. This is because providers will often seek to present their test’s performance in terms of its “accuracy.” This, however, is most unhelpful: if that were applied to Down syndrome screening in the general population, then simply giving every pregnancy a low-risk result would score an accuracy of substantially greater than 99%. It is therefore essential that professionals who provide such services understand how these tests operate as screening tests, how they should be explained to patients and how their performance should be evaluated.
Diagnosis of Inherited Disorders
Couples who have already had an affected pregnancy or child, or have a family member affected with the disease, are aware that they are at risk of transmitting an inherited disease. These couples, as well as those who have a positive serum or ultrasound screen, can be offered prenatal diagnosis. Chorionic villus sampling and amniocentesis have been the traditional methods of choice for prenatal diagnosis, and more recently, new molecular biology techniques have facilitated the development of non-invasive prenatal diagnosis tests that can screen fetal DNA in the maternal circulation.
Chorionic Villus Sampling (CVS)
CVS can be performed by the trans-cervical route between 10 and 12 weeks of gestation, or from 12 weeks onwards by the trans-abdominal route. Cell sample is removed from the placenta and used to diagnose chromosomal, metabolic and DNA analysis. The disadvantages of the procedure are that it cannot be used for neural tube and other congenital abnormalities, and some studies have suggested a risk of limb reduction deformities if it is performed too early or by inexperienced operators. There is also a 1–2% risk of miscarriage, which is fairly higher than the risk after amniocentesis. In 1.5% of cases, the karyotype of the placenta is found to be different from that of the embryo (confined placental mosaicism).
Amniocentesis for prenatal diagnosis is usually performed in the second trimester, from 15 weeks onwards. Under ultrasound guidance, 15–20 mL of amniotic fluid is aspirated. This can be used for the diagnosis of chromosome abnormalities, measurement of specific substances, detection of inborn errors of metabolism such as Tay-Sachs disease, measurement of enzyme activity and diagnosis of neural tube defects. Its disadvantages include the potential of causing fetal loss (1%) and rarely there may be continued leakage of the amniotic fluid. The main limitation of this technique is that results are available only very late in the pregnancy (17–20 weeks), so that a termination must be induced during the second trimester.
Cell-Free Fetal DNA Screening (cfDNA)
Cell-free fetal DNA can be detected in maternal plasma as early as seven weeks’ gestation, and this has been successfully used to provide non-invasive prenatal testing (NIPT) for common aneuploidy; with low false positive rates. Cell-free DNA is extracted from a maternal blood sample, and real-time polymerase chain reaction (PCR) with fluorescently labeled specific probes for target genes is used to identify and quantify fetal-specific sequences present in low copy numbers.
Accuracy depends on the condition under investigation: the test can accurately diagnose Rhesus D status, and fetal sex determination can identify X-linked disorders. Sensitivity and specificity are less for trisomy 21, 18 and 13 due to the influence of biological factors such as confined placental mosaicism (CPM). cfDNA screening is therefore currently considered as a screening test for aneuploidy, although accuracy is improving with developments in technology.
Fetal Blood Sampling, Cordocentesis or PUBS
These techniques are used less frequently than CVS or amniocentesis. Samples can be taken from 18 weeks’ gestation to term. Fetal blood is taken from the cord or intra-hepatic umbilical vein and used for fetal karyotyping (quick result), evaluation of fetal status (if an infection is thought to be present) and hematological abnormalities (Rh or immune hemolytic disease).
The most common indication is karyotyping for single or multiple congenital abnormalities and mosaicism.
Fetal Tissue Sampling
Using ultrasound guidance, it is possible to biopsy skin, liver, muscle and fluid collections from the urinary tract, abdomen, thorax and cystic hygroma.
Diagnostic Testing for Prenatal Diagnosis
Tests used to establish a fetal diagnosis following CVS or amniocentesis include PCR and karyotyping. PCR analysis can detect single-gene defects and triplet repeat disorders and determine fetal gender. A karyotype is performed for any diagnosis which involves chromosome identification, i.e., in those patients carrying chromosome abnormalities or who are at risk of age-related aneuploidy. In some situations, fluorescent in-situ hybridization (FISH) or DNA micro-array-based technology is used to complement the karyotype result.
Polymerase Chain Reaction (PCR)
PCR is a molecular method developed in the 1980s, which is used for amplification of the DNA of thousands of times. For prenatal diagnosis, PCR can be used to detect the normal or mutated gene by amplifying the region around the mutation. This is achieved using primers that have a sequence which is complementary to a region of the gene. Primers that will bind to either side of the mutation are selected, and copies of the DNA sequence in between (target gene) are generated via repeated cycles of heating and cooling:
Heating to 96ºC to denature the DNA and create separate strands,
Cooling to 55º C to allow primers to bind to the DNA target (primer annealing),
Heating to 72ºC to allow DNA synthesis (primer extension).
This PCR cycle is repeated a number of times (25–35 times) to produce billions of copies of the DNA sequence for analysis. The reaction is made possible by using a thermo-stable polymerase enzyme that can withstand the high temperatures needed for denaturation: the first such enzyme to be used was Taq polymerase, isolated from a heat-tolerant bacterium that lives in hot springs and hydrothermal vents. A similar enzyme, PfuDNA polymerase, has superior thermostability and higher fidelity when copying DNA.
Once the DNA sequence of interest has been amplified, the PCR products are analyzed using a number of different techniques. The simplest method, which can be used to detect an insertion or deletion, is to separate the PCR products by polyacrylamide gel electrophoresis. Other techniques include single-stranded conformational polymorphism (SSCP), amplification refractory mutation system (ARMS) and hetero-duplex analysis, which can detect even just a single base change within a gene. The technology has further evolved over the past decade, with a range of more sophisticated DNA micro-array molecular biology tools available.
Karyotype analysis examines the chromosomes of a cell. For prenatal diagnosis, the sample obtained by CVS or amniocentesis is cultured to increase the number of cells, and mitotic inhibitors are used to arrest some of the cells in metaphase. Agents which elongate the metaphase chromosomes are also used.
Slide preparations of the nuclei are treated with Giemsa stain, which results in a specific banding pattern for each chromosome. Using this method, missing or extra chromosomes, translocations, inversions, etc. can be identified. Occasionally the results of a karyotype may be inconclusive, and more advanced techniques are now used to help elucidate the diagnosis.
Karyotyping has also been used to check the number of chromosomes in diagnosis of age-related aneuploidy.