Genetic testing identifies changes in genes or chromosomes. These changes may result in structural abnormalities or have functional implications for the developing fetus or the child after birth. An abnormal result may allow parents to prepare for a child who may require additional medical or social needs and to allow maternity caregivers to optimise the delivery. Prenatal diagnosis may be regarded as an important bridge between obstetrics and paediatrics. In other situations, an abnormal result may allow parents the opportunity to make reproductive choices, increasing their autonomy, and consider termination of pregnancy. Over the past two decades, genetic testing has rapidly shifted from the second to the first trimester, owing to the advancement of high-resolution ultrasound and genetic analysis technology.

Screening versus diagnostic testing

Offering a genetic test should be nondirective and it should be clear that testing is voluntary. Informed consent via pre-test counselling should be obtained to enable parents to balance the risk, limitations and benefits of a test. It is important to clarify to parents the differences between the screening and diagnostic nature of the tests offered. The purpose of a screening test is to detect potential indicators of a condition and a positive result indicates a suspicion of a condition. A prenatal screening test is non-invasive and, therefore, procedure-related complications can be avoided whereas a diagnostic test, which involves an invasive procedure, is to establish the presence or absence of the condition.

In accordance with the RANZCOG statement, it is recommended that all pregnant women should be provided with the opportunity to discuss¹:

• the range of chromosomal abnormalities together with the characteristics of the available prenatal screening and diagnostic tests; and
• prenatal diagnostic testing for other genetic conditions.

Screening tests

The following screening test options are available and meet the performance standard for the common chromosomal abnormality (trisomies 13, 18, 21 and sex chromosomes).^{2 3 4 5}

Combined first trimester test (11+0 to 13+6 weeks)

A combination of maternal age, sonographic measurement of nuchal translucency (NT) and biochemical markers – pregnancy-associated plasma protein-A (PAPP-A) and chorionic gonadotropin (hCG) – is used to calculate a risk (low or increased). Most developed countries have adopted the combined first trimester test as the standard of care.

Advantages include the following:

• early detection;
• high sensitivity of around 85 per cent, given a false positive rate of five per cent for trisomy 21;
• at time of early NT scan, the incorporation of extra sonographic markers (such as the assessment of nasal bone, ductus venosus waveform and tricuspid regurgitation) allows better detection to 96 per cent and a reduction of false positive rate to 2.5 per cent for trisomy 21;
• abnormal maternal serum markers, for example, a decreased levelof PAPP-A (<0.4 MoM) and hCG(<0.5 MoM) may be associatedwith increased frequency of adverse obstetric outcome. The optimal method to manage them is unclear; however, educating women about the signs and symptoms of certain complications such as preterm labour, vaginal bleeding, pre-eclampsia and decreased fetal movements may be of value.⁶

Disadvantages include the following:

• NT – variability in quality of measurement;
• and barriers to access – cost and availability.

Second trimester quadruple test (15-20 weeks)

A combination of maternal age and the measurement of maternal serum alpha-fetoprotein (AFP), unconjugated oestriol, hCG and inhibin-A level.

Advantages include the following:

• available for women who present late in the second trimester;
• an unexplained elevation of maternal serum AFP (>2.5 MoM), hCG (>4 MoM), inhibin-A (>2 MoM) or decreased level of unconjugated oestriol (<0.5 MoM) may be associated with an increased frequency of adverse obstetric outcomes. Again, no clear management strategy is available, with some authors suggesting closer fetal/maternal surveillance, and its benefit is still subject to debate.⁶

Disadvantages include the following:

• later detection;
• lower detection rate of 80–83 per cent, given a false positive rate of five per cent for trisomy 21.

Fully integrated test

This is an integration of first and second trimester maternal serum screen, NT and maternal age: a single result is provided after all tests are completed. The fully integrated test offers the benefit of a higher detection rate of 96 per cent, given a false positive rate of five per cent for trisomy 21, but has the disadvantage of later detection.

Step-wise sequential test

This is a modified version of the fully integrated test. The result is available after the first combined screening test, so an early diagnostic test can be offered for those with a high-risk result (for example, a less than one-in-50 risk). Those who are not at the highest risk go on to the second trimester testing to complete the integration. The advantage of this is that women at the highest risk benefit from early detection while women at a lower risk benefit from the high detection rate.

Integrated and sequential screening strategies are not funded and not routinely used in Australia and New Zealand.

Cell-free fetal DNA testing

In cell free fetal DNA (cffDNA) testing, fragments of cffDNA released from the placenta into the maternal blood are used for analysis. Quantitative difference in the DNA sequences that map to individual chromosomes can be used to distinguish fetuses with trisomy and those without. It can be used as either a primary screening test or as a secondary test after a high-risk first trimester screening result.

Advantages include the following:

• early detection – from ten weeks onward;
• a superior detection rate, with a sensitivity of 99.5 per cent and specificity of 99.8 per cent for trisomy21. Numbers of procedure-related miscarriage can thus be reduced.

Disadvantages include the following:

• up to five per cent inconclusive result, largely owing to inadequate fetal fractions in maternal blood sample(for example, sample taken at early gestation or maternal obesity);
• cost for patients;
• an NT scan is not included in the test, so there is a lack of information on anatomical abnormality in fetus;
• a false positive/negative result could be owing to confined placental mosaicism.

According to the RANZCOG statement, first trimester combined screening remains the recommended modality for twin pregnancy screening. Its sensitivity range is 72–80 per cent for trisomy 21 (without incorporation of nasal bone assessment). Second trimester maternal serum screening can also be offered, if women have missed the opportunity in the first trimester. Owing to the smaller number included in the studies, cffDNA testing in twin pregnancies has not been evaluated as extensively. Sensitivity for trisomy 21 is only 90 per cent and the failure rate can be more than five per cent. Therefore, it is important women take these limitations into account when choosing a screening test. For higher order multiple pregnancies, serum markers and cffDNA testing cannot be used, therefore only ultrasound markers in the first trimester can be offered.

Diagnostic tests

Moving on to diagnostic testing, some women who are at a higher risk for aneuploidy or other genetic conditions may choose to go straight to this option for a definitive result. Diagnostic testing can also be offered after a high-risk screening result or in a fetus with major structural anomalies.

Chorionic villi sampling (11–14 weeks)

Ultrasound-guided placental biopsy via a needle aspiration for chromosome or DNA analysis, chorionic villi sampling (CVS) is usually performed via a transabdominal approach, although some practitioners may still use a transcervical approach.

Advantages include the following:

• early diagnosis;
• an abnormal result allows women the options of surgical or medical termination of pregnancy.

Disadvantages include the following:

• the weighted polled procedure-related rate of miscarriage is 0.22 per cent(much lower than previously quoted)⁷;
• there is a one to two per cent risk of confined placental mosaicism, which requires further testing, such as amniocentesis, to exclude fetal mosaicism; and
• a posterior placenta may not be accessible, making CVS not possible.

Amniocentesis (15 weeks gestation onward)

Amniotic fluid is withdrawn via needle under ultrasound guidance. Amniotic fluid contains fetal urine, secretions, exfoliated cells and transudate that can be used for chromosome and DNA analysis. Advantages include a lower procedure-related miscarriage rate (0.11 per cent), lower than previously quoted or that of CVS⁷, and it is not restricted by the location of placenta. However, there is the disadvantage of later diagnosis.

Laboratory testing techniques ^{8 9}

Quantitative fluorescent polymerase chain reaction

Quantitative fluorescent polymerase chain reaction (QF-PCR) technique consists of amplifying polymorphic markers located on the chromosomes of interest to determine the number of copies present per cell. Usually only chromosomes 13, 18, 21 and sex chromosomes are tested. Only a small amount of DNA is required for analysis and the result is available within 24 hours. Other merits include its ability to detect maternal cell contamination, triploidy and mosaicism. (This test is not available in our unit.)

Fluorescence in situ hybridisation

This is another technique for rapid aneuploidy detection (chromosome 13, 18, 21 and sex chromosomes); the result can be available within 48 hours. Fluorescence in situ hybridisation (FISH) uses fluorescently labelled probes targeted to a unique sequence of DNA on the chromosomes of interest. These probes selectively bind and the cells are examined under a microscope to determine gain or loss of that specific chromosomal region of interest. Like QF-PCR, it can also detect mosaicism, triploidy and other common chromosomal microdeletions, such as 22q11 deletion. The main drawback, in comparison to QF-PCR, is the cost and it is a more labour-intensive process.

G-band karyotyping

This involves culturing cells in vitro and harvesting chromosomes for analysis. This is a low-resolution, whole genome study that can detect abnormality at a resolution of approximately 10MB, that is major chromosomal aneuploidies. The advantage of this test, compared to the rapid aneuploidy tests, is that all of the chromosomes are examined so that whole or partial chromosome aneuploidy will be detected. Balanced translocations will also be detected. However, it is limited by its low resolution and is a labour-intensive test. Results are usually available within ten to 14 days.

Chromosomal microarray

Many units nowadays across Australasia have shifted from conventional karyotyping to chromosomal microarray analysis, particularly in the setting of fetal structural anomalies identified on ultrasound. This is a high-resolution whole genome study, with a resolution of approximately 25KB. It can identify aneuploidy as well as the location and type of specific genetic changes that are too small to be detected by conventional karyotype. Therefore, it can yield more genetic information.

The result is usually available within two weeks. Two types of technologies are used: comparative genomic hybridisation (CGH) and single-nucleotide polymorphism (SNP). CGH detects copy number variants, such as trisomy, but it cannot detect triploidy. SNP detects homozygosity or heterozygosity (identical or different segment of DNA) and therefore can detect triploidy and uniparental disomy. Unlike conventional karyotyping, it cannot detect balanced translocation. One drawback is that it may detect too much genetic information, including CNV of unknown or uncertain clinical significance, which may lead to substantial patient anxiety. Therefore, pre-test counselling and informed consent is essential before women undergo this test.

Testing for single gene disorders

If there is a high clinical suspicion of a single gene disorder based on ultrasound findings or when a specific gene mutation associated with a single gene disorder has been identified in the family and the fetus is known to be at risk, fetal DNA can be analysed. In the case of a suspected single gene disorder a specific gene or genes of interest are analysed in an attempt to identify a pathogenic mutation. In the case of a known familial mutation, targeted analysis of part of the gene is performed to determine whether the mutation is present or absent. This type of testing is usually done in consultation with the clinical genetic service.

The role of the clinician

Parents can choose to gather more information about their unborn child in early pregnancy by means of genetic testing. All of these tests, either screening or diagnostic, involve limitations, risks and benefits. It is our duty to provide women with accurate information so that they can make informed choices for a test that will best suit their needs.

1. Prenatal screening and diagnosis of chromosomal and genetic abnormalities in the fetus in pregnancy. RANZCOG statement C-Obs 59. March 2015.
2. Malone FD, Canick JA et al. A comparison of first trimester screening, second trimester screening, and the combination of both for evaluation of risk of Down syndrome. N Engl J Med. 2005; 353:2001-2011.
3. Wald NJ, Rodeck C et al. First and second trimester antenatal screening for Down’s syndrome: the results of the Serum, Urine and Ultrasound Screening Study (SURUSS). J Med Screen. 2003; 10:56-104.
4. Nicolaides KH. Screening for fetal aneuploidies at 11 to 13 weeks. Prenat Diagn. 2011; 31(1):7-15
5. Hui L & Hyett J. Noninvasive prenatal testing for trisomy 21: Challenges for implementation in Australia. Aust NZ J Obstet Gynaecol. 2013; 53(5):416-24.
6. Lakhi N, Govind A et al. Maternal serum analytes as markers of adverse obstetric outcome. The Obstetrician & Gynaecologist. 2012; 14:267-73.
7. Akolekar R, Beta J et al. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2015; 45(1):16-26.
8. New Molecular Techniques for the Prenatal Detection of Chromosomal Aneuploidy. SOGC technical update. No. 210, July 2008.
9. The use of chromosomal microarray analysis in prenatal diagnosis. Committee Opinion No. 581. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2013; 22:1374-7.