This chapter explains normal fetal development during pregnancy and clinical conditions and disorders associated with pregnancy, including pregnancy detection, neural tube defects (anencephaly and spina bifida), threatened abortion, ectopic pregnancy, Down syndrome (trisomy 21), trisomy 18, trisomy 13, Turner’s syndrome, premature labor, preterm delivery, placental insufficiency, fetal growth retardation, fetal death, stillbirth, miscarriage, fetal distress, preeclampsia, eclampsia, placental abruption, maternal diabetes, gestational diabetes, Rhesus disease, thalassemia, Smith-Lemli-Opitz syndrome, steroid sulfatase deficiency and Cornelia de Lange syndrome. The Barker hypothesis of fetal origins of adult disease is explained and illustrated. For each immunoassay analyte of interest in this field the biological function is explained, with the clinical applications of the test and its limitations. Typical assay technology is described. The type of sample and frequency of use are included, with an example reference interval (for background information only). The analytes included are alphafetoprotein, human chorionic gonadotropin, estriol, inhibin, pregnancy-associated plasma protein A, placental growth factor and progesterone. Profiles of analyte concentrations during pregnancy are provided where relevant, with an explanation of the use of the Multiple of the Median. The metabolic pathways involved in estrogen synthesis are provided. The final section explains screening for aneuploidy, particularly Down syndrome (trisomy 21) and the importance of nuchal translucency using ultrasound.

Since the middle of 2012 a significant development in the application of molecular tests has led to the introduction of a new method of screening for aneuploidy which is likely to introduce a new paradigm shift in the way that screening services are provided across many countries. The application of sensitive molecular counting methods such as massive parallel sequencing of DNA has been applied to testing for aneuploidy. A number of companies in the US have introduced commercial testing for aneuploidies (initially Trisomy 21 and more recently Trisomy 13, 18 and in some cases Turner’s syndrome and other sex aneuploidies). These tests are performed in CLIA-approved commercial testing centers in the USA by companies such as Natera, Sequenom, Ariosa, Verinata. In China another company, BGI, is also offering this test in the Asian/Pacific region and in Europe.

Essentially, the test involves extracting cell-free DNA from a blood sample collected from the mother at any time from about 8 weeks gestation onwards. The blood sample is usually collected into a special tube that prevents cell breakdown and thus preserves the cell-free DNA. The cell-free DNA contains DNA both from the mother and a small amount (usually of the order of 10%) that comes from the fetus. The extracted cell-free DNA is then amplified, the fragments sequenced and the sequences of the base pairs assigned to their respective chromosomes using a highly sophisticated bioinformatics software tool that decides which chromosome a specific sequence comes from. The relative representation of the various numbers of sequences coming from each chromosome is in this way “counted”. In a situation where the fetus has Trisomy 21 the fetal DNA will contain an overrepresentation of sequences from Chromosome 21 and thus the number of total sequences from chromosome 21 in the mothers blood sample will be marginally increased compared to normal.

Initial studies with this type of technology showed it was feasible in high-risk singleton populations to have a high level of detection of Trisomy 21 (around 99%) with a false-positive rate of around 0.2%. In some instances it was not possible to produce a definitive result – usually because the proportion of fetal DNA in the maternal blood was less than 4%. Also in some instances – particularly in the case of placental mosaicism – the result reflected the karyotype of the placenta and not the fetus, i.e., gave a false-positive result. When the technology was applied to Trisomy 18, again results in high-risk singleton populations suggested detection rates of around 95% for a similar false-positive rate of 0.3%, but application to Trisomy 13 has been less successful with detection rates of around 80%. More recently, application to general low-risk populations has shown similar levels of performance. The test therefore appears to be a very good screening test – however it is clearly not diagnostic and women who are shown to be “positive” using this test still need to go on and have this confirmed by conventional amniocentesis or chorionic villus sampling and karyotyping of the fetal tissue.

The test currently is quite expensive and these companies at the moment charge anything from $300 to £1,000 per test. This is largely because the cost of sequencing is still quite high – although the cost is expected to fall – and secondly there is a commercial monopoly of this testing. Currently a couple of studies in the US and some in Europe are looking at how this new screening test may be used in a National Health Care setting. Whilst the economic studies have argued that unless the cost of the test can be reduced to around $50-100 it is unlikely that government funded health programs could afford to offer this test to all women as a front-line screen – and furthermore this test would not replace an ultrasound examination – it is more likely that the test would be introduced as a second-line screen in those women identified by combined biochemical and ultrasound screening as having an intermediate risk (say 1:151 to 1:1000). Therefore women with a high risk (1:2-1:150) would either have the DNA test or go straight to amniocentesis/chorionic villus sampling, women in the intermediate group would be offered the DNA test and then those with a positive result from DNA would have further invasive testing. Using this Contingent Model it would be expected that around 10% of women would need to consider the DNA test and with this strategy invasive testing could be reduced to around 0.3% of the pregnant population and achieve a detection rate of around 98% at a cost that the service could afford. By reducing the invasive testing rate this saving alone could probably fund the additional DNA tests if the cost per test fell to around $100.  This is an improvement over existing Combined Biochemistry & Ultrasound testing, which achieved a detection rate of around 88% for a 2.5% false-positive rate.

Testing logistics in a national health care setting are still to be worked out – as is whether the test is appropriate in women with twins. Some limited studies have shown the feasibility in twin pregnancies but more data is required before this could be shown to be convincing. Other problems such as screening in the presence of a vanished twin (which may result in a DNA contribution from the dead twin) are also still to be resolved.

In Health Care systems that have less well developed ultrasound and combined biochemical screening in the first trimester (for example the USA) it may be that this test will become the screening method of choice.

It is clear however that this new technology will introduce a further layer of complexity to screening in pregnancy over the next few years.

1. Benn, P., Cuckle, H., Pergament, E. Noninvasive prenatal testing for aneuploidy: current status and future prospects. Ultrasound Obstet. Gynecol. 42, 15-33 (2103).
2. Morris, S., Karlsen, S., Chung, N. Hill, M., Chitty, L.S. Model based analysis of costs and outcomes of Non Invasive Prenatal Testing for Down’s Syndrome using Cell Free Fetal DNA in the UK National Health Service (2014). PLoS ONE 9(4); e93559.
3. Hill, M., Wight, D., Daley, R., Lewis, C., McKay, F., Mason, S., Lench, N., Howarth, A., Boustred, C., Lo, K., Plagnol, V., Spencer, K., Fisher, J., Kroese, M., Morris, S., Chitty, L.S. Evaluation of non-invasive prenatal testing (NIPT) for aneuploidy in an NHS setting: a reliable accurate prenatal non-invasive diagnosis (RAPID) protocol. BMC Pregnancy & Childbirth 14, 229 (2014).

Professor Kevin Spencer BSc MSc DSc CSci CBiol CChem EurClinChem FSB FRSC FRCPath is Head of Department, Consultant Biochemist and Clinical Lead in Biochemistry at Barking Havering and Redbridge University Hospitals NHS Trust. He also is a Visiting Professor in Reproductive Biochemistry at King's College, London and Director of Biochemical Screening at the Fetal Medicine Foundation (London). He is Honorary Consultant Scientist in the Fetal Medicine Unit St Thomas's Hospital, London and Honorary Consultant Biochemist to Heatherwood & Wexham Park Hospitals NHS Foundation Trust.

This chapter also contains some material originally contributed by the late Professor Tim Chard to the first edition of The Immunoassay Handbook. He wrote one of the first books on immunoassay: Introduction to Radioimmunoassay and Related Techniques (Laboratory Techniques in Biochemistry and Molecular Biology), 4^th edition, Elsevier, 1990.

Pregnancy, fetal development, amniocentesis, neural tube defect, ultrasound, threatened abortion, ectopic pregnancy, Down syndrome, trisomy 21, trisomy 18, trisomy 13, sex chromosomal aneuploidy, Turner’s syndrome, anencephaly, spina bifida, premature labor, preterm delivery, placental insufficiency, fetal growth retardation, fetal death, stillbirth, miscarriage, fetal distress, preeclampsia, eclampsia, placental abruption, maternal diabetes, gestational diabetes, Rhesus disease, thalassemia, Smith-Lemli-Opitz syndrome, steroid sulfatase deficiency, Cornelia de Lange syndrome, Barker hypothesis, alphafetoprotein, human chorionic gonadotropin, estriol, inhibin, pregnancy-associated plasma protein A, placental growth factor, progesterone, nuchal translucency.