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Prenatal diagnosis

Prenatal diagnosis for populations at risk of genetic disorders has been established within

antenatal care, with the aim of confirming a normal pregnancy. Populations at risk for

having an abnormal baby include women aged 35 or more, carriers for X- linked inherited

disorders such as Duchenne muscle dystrophy and Fragile-X syndrome, parents who are

carriers for chromosomal abnormalities, and many other family histories of genetic

disorders. Prenatal diagnosis is helpful for:

Managing the remaining weeks of the pregnancy

Determining the outcome of the pregnancy

Planning for possible complications with the birth process

Planning for problems that may occur in the newborn infant

Deciding whether to continue the pregnancy

Finding conditions that may affect future pregnancies

Variable screening tests have been used to investigate the high-risk pregnancy, where

some tests are invasive while others are non-invasive.

Non-invasive tests Biochemical markers

Isolation of Foetal cells from maternal circulation

Ultrasound

Invasive tests Amniocentesis

Chorionic villus sampling

Foetal blood sampling

Foetal tissue sampling / foetoscopy

1- Non-invasive methods:

These tests together with maternal age are used as screening rather than diagnostic tests.

These will limit the invasive tests to those considered high risk pregnancies and includes

the following:

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1.1- Maternal serum or blood tests

A- The triple test, is routinely used in the second trimester to investigate women at risk

of having a Down syndrome (DS) foetus, and includes maternal serum alpha feto-protein

(MSAFP), human chorionic gonadotrophin (hCG) and unconjugated estriol (uE3). AFP

can be measured at 15-18 weeks of gestation. MSAFP testing is based on the fact that the

DS foetus tends to be small, have a smaller placenta, and thus releases less AFP into the

serum of the pregnant woman. This is not a definitive test. If the MSAFP test is low it

suggests the risk of a DS foetus especially in woman with high risk (age 35 or more). If

MSAFP alone is tested, 20 % of DS foetuses will be identified. If MSAFP and human

chorionic gonadotrophin (HCG) are determined, 50 - 60 % of DS foetuses will be

identified. If MSAFP, HCG and estradiol (E2) are tested, 60 -70 % of DS foetuses will be

identified. A positive screening test suggests only that the risk of DS is increased, and the

definitive testing of karyotyping via amniocentesis is indicated.

On the other hand, quadruple test [triple test with the fourth indicator inhibin-A (INHA)]

is highly effective in identifying several foetal anomalies other than Down syndrome such

as trisomies 18 and 16, Turner syndrome, triploidy and foetal death.

The first trimester screening of free beta-human-chorionic gonadotropin and pregnancy

associated plasma protein-A (PAPPA) is a highly indicative test for a risk of DS

especially when associated with the first trimester ultrasound finding of foetal nuchal

translucency (NT).

1.2- Isolation of foetal nucleated cells from maternal circulation is a promising non-

invasive prenatal test enabling investigation of foetal chromosomes in early pregnancy.

Several studies have successfully isolated nucleated foetal cells (NFCs) from maternal

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circulation. Yet, NCFs are not used routinely not only due to the scarcity of these cells in

maternal circulation but also due to the need to confirm their foetal origin. Therefore by

improving the techniques of isolation and identification of NFCs. We have used NFCs to

investigate chromosomal aneuploidy with a relative low false positive rate. And used

FISH on non-dividing interphase nuclei to investigate the numerical but not structural

chromosome anomalies. In addition, foetal DNA has been identified, especially that for

male foetus, in maternal serum and plasma during pregnancy. Several studies have used

foetal DNA to screen paternally inherited single-gene disorders or mutated genes that can

be distinguished from maternal components. Studies have successfully investigated

several disorders such as Duchenne muscle dystrophy, foetal RhD blood type, sickle cell

anaemia and beta-thalassemia in heterozygous carriers.

1.3- Genetic Sonogram: Ultrasonographers can determine several congenital anomalies

suggestive of chromosomal and non-chromosomal defects. First trimester sonographic

assessment of foetal anomalies is an important test for prenatal diagnosis. It requires

understanding of embryonic development as some normal second trimester findings would

be considered abnormal first trimester findings. The optimal time for scanning is around

12-week of gestation as most of organs can be visualized. Increased nuchal fold thickness

detected in the first trimester is a good indicator not only for chromosomal anomalies

(aneuoploidy) but also for skeletal and cardiovascular disorders. The second trimester

ultrasound findings (18- 20 weeks) assess the foetal measurements and can assess foetal

age to support risk estimation based on maternal serum biochemical markers. Foetal

abnormalities including changes in the head circumstances; heart, nuchal thickness, foetal

and limbs size are indicators of several disorders. DS foetuses for example, have a

relatively thickened nuchal fold, short femur, short humerus, and echogenic intracardiac

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focus. Trisomy 18 (Edward syndrome) foetuses usually show marked growth retardation

and several congenital anomalies that can detected in ~90% of cases. Also trisomy 13

(Patau syndrome) foetuses have distinctive features and anomalies that can be diagnosed

in the second trimester in 90-100% of cases. On the other hand, triploidy is usually lost

early in pregnancy, but can occasionally be seen in the second trimester and frequently has

specific ultrasonographic features. Generally sonographic markers may not be able to

distinguish normal variation from specific disorders, such as growth retardation due to

placental insufficiency; and other confirmative tests should be performed.

2- Invasive techniques: Invasive techniques, procedures used to obtain foetal cells, fluids

or tissue samples for accurate diagnosis. The diagnostic value of these tests has to be

balanced against the risk of causing foetal loss.

2.1- Chorionic villus sampling (CVS) can be done at about 11 weeks, either

transvaginally or transabdominally. Earlier testing (before 11 weeks) is thought to be

associated with a small risk of foetal hazards (such as limb reduction). The CVS test is

done earlier, and is usually faster than amniocentesis. There is a small chance of maternal

cell contamination and a 1-4% risk of miscarriage, infection, placental trauma, or foeto-

maternal haemorrhage after the procedure dependent on obstetric experience.

2.2- Amniocentesis, a sampling of the amniotic fluid (AF) surrounding the foetus, is

routinely done transabdominally at 16- 18 weeks gestation, but can be performed from 11

weeks. A 10- 20 ml sample is withdrawn that contains fluid (for biochemical tests) and

cells, mainly amniotic fluid cells (65%), fibroblast cells (10%) and epithelial cells (25%).

Other foetal and maternal cell types, mainly blood cells, are frequently seen in the sample

but do not usually divide in culture. Miscarriage, intrauterine infection, persistent amniotic

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fluid leak, foeto-maternal haemorrhage, foetal injuries are less frequent complications

after the procedure. Amniocentesis testing for chromosome disorders is 99.8 % reliable for

detecting numerical chromosome abnormality.

2.3- Foetal blood sampling (FBS) is usually done for rapid assessment of foetal

chromosomal anomalies, haemoglobinopathies, coagulopathies, infection and

immunodeficiency later in pregnancy. Blood is taken from the umbilical vein at the

placental insertion under the ultrasound guidance. Foetal loss rate after the procedure is

variable from 0-24%, with 12% losses for growth retarded foetuses.

2.4- Foetal tissue sampling (FTS):

Diagnosis of certain foetal anomalies depends on histological and ultrastructure studies,

based on foetal biopsy. For example foetal skin biopsy in the second trimester is used for

prenatal diagnosis of epidermolysis bullosa lethalis. Foetal loss after these invasive

procedures under ultrasound guidance is as high as 1%.

3- Cytogenetics / molecular genetics and prenatal diagnosis

Development and accuracy of obstetric procedures for collecting samples (CVS, AF, FBS)

and the reliability of chromosomal and/or DNA analysis have increased the demand for

prenatal diagnosis. Despite advances in DNA technology, chromosomal karyotyping is

still the core service of prenatal diagnosis. AF cells require about 1-2 weeks to give

sufficient dividing cells for karyortyping. On the other hand, CVS contains cytotrophoblast

cells that are actively dividing and provide sufficient metaphases in short-term cultures (24

hours) for rapid low resolution karyotyping. CVS mesenchymal core cells are also cultured

and karyotyped after 1-2 weeks to confirm and support the result of the short-term

cultures. Foetal blood contains lymphocytes that grow well in culture. Complications of

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culture failure and maternal contamination are infrequent and represent ~1%. CVS

sometimes shows chromosomal mosaicism, in particular with cytotrophoblast short-term

cultures and represents confined placental mosaicism, but this is less frequent in the

mesenchymal core cultures.

Foetal chromosomal anomalies can be classified into numerical, mosaic and structural,

where viable chromosomal aneuploidy of autosomal and/or sex chromosomes mainly

chromosomes 13, 18, 21, X and Y chromosome is a particular focus of prenatal testing.

Rapid interphase FISH screening of amniocytes has been used successfully in most

prenatal diagnostic units for chromosomes 13, 18, 21, X and Y, to give rapid diagnosis of

numerical chromosome anomalies with a high degree of concordance with karyotyping.

Chromosome specific FISH probes or/ and subtelomeric FISH probes have been used

prenatally to identify, some structural chromosome anomalies.

DNA based techniques using polymerase chain reaction (PCR) tests are employed in

prenatal diagnostic tests, such as multiplex fluorescence PCR (QF-PCR) tests for

identifying chromosomal aneuploidy using DNA samples extracted from different foetal

tissues (CVS, AF, FB, FTS). Also mutated genes that cause inherited genetic disorders

such as Fra-X syndrome, and Duchenne muscle dystrophy have been detected prenatally

by using PCR based tests.

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Some of the more common chromosomal abnormalities that can occur. The descriptions

are for the completely abnormal condition in which all fetal cells contain the abnormal

karyotype.

Maternal Age Trisomy 21 Trisomy 18 Trisomy 13

15 - 19 1:1600 1:17000 1:33000

20 - 24 1:1400 1:14000 1:25000

25 - 29 1:1100 1:11000 1:20000

30 - 34 1:700 1:7100 1:14000

35 - 39 1:240 1:2400 1:4800

40 - 44 1:70 1:700 1:1600

45 - 49 1:20 1:650 1:1500