18.05.01 Prenatal sampling and considerations

Question 1

What are some properties of cell foetal DNA?

Answer 1

1) cffDNA is foetal DNA circulating freely in the maternal blood stream
2) Can be obtained through non-invasive sampling
3) Appears in the maternal blood from ~5/40
4) Present in 200bp fragments (this is smaller than mat. DNA which allows for differentiation)
5) Derived from apoptotic trophoblasts which make up the placenta.
6) 2-6% of maternal plasma. Some studies suggest 10-20%

Question 2

What is the goal of prenatal testing?

Answer 2

To allow expectant couples to make an informed choice regarding their pregnancy

Question 3

What factors will influence the decision of what/if prenatal sample type to take?

Answer 3

1) Referral reason
2) Gestation
3) Amount of material
4) Risk to foetal and maternal health
5) Rapidity and accuracy of genetic results

Question 4

What is CVS material? When in pregnancy it is usually obtained and how?

Answer 4

Placental tissue made of trophoblasts and mesenchyme cells.

11+0 - 13+6 / 40

Typically 10-25mg obtained either transabdominally or transcervically.

Question 5

What are the risks associated with CVS sampling?

Answer 5

1-2% risk of miscarriage
~1.5-3% will show CPM
Maternal cell contamination possible

Question 6

What is amniotic fluid? When in pregnancy it is usually obtained and how?

Answer 6

Foetus is surrounded by the amnion - fluid filled sac - which acts as a protective barrier, maintains temperature and promotes symmetrical growth and lung development.

Usually taken from 15 weeks in pregnancy

UP to 20ml sample taken transabdominally

Question 7

What are the risks associated with AF sampling?

Answer 7

0.5-1% miscarriage
Can lead to infection and placental/foetal damage.
Samples taken at earlier gestation have higher culture fail rate, higher miscarriage risk and greater number of extra-embryonic cell growth (rather than foetal)

Question 8

What is the composition of amniotic fluid?

Answer 8

Fluid is heterogenous mix of shed foetal cells e.g. from lung, respiratory tract and skin
65& extra-embryonic membranes and amniocytes from the amnion
25% epitherlial
15% fibroblasts

Gives a more accurate representation of the foetal than CVS

Question 9

When can foetal blood samples be taken? Why might this be the preferential sample type?

Answer 9

18-20/40
Performed when veins are developed by insertion of a needle into the umbilical cord (aka - percutanenous umbilical cord blood sampling, cordocentesis)

Used when other techniques are inconclusive (e.g. mosaic) and a result is required rapidly.

Commonly used for suspected blood disorders e.g. foetal anaemia, immunology or deminse

Question 10

What are the risk of FBS?

Answer 10

Risk of foetal loss is ~2%

High risk of complication so only used in pregnancies with a very high risk of genetic defects

Question 11

What other sample types might be used to determine the foetal karyotype?

Answer 11

1) POC
2) Skin
3) Pleural effusion

Question 12

What factors will influence the level of MCC present in a sample?

Answer 12

1) Sampling technique
2) Sample quality
3) Operator
4) Method of sample processing

Question 13

Why is the rate of MCC significantly lower in cultured AF samples than in direct AF samples?

Answer 13

Culture conditions favour the growth of amniocytes and reduce/eliminate blood cells.

However culture AF more likely to represent a homogenous cell type reflecting pure foetal origin.

Question 14

Culture AF more likely to represent a homogenous cell type reflecting pure foetal origin that direct AF samples, why?

Answer 14

Maternal blood cells senesce during culture, however prolonged culture may allow overgrowth of maternal fibroblasts and epithelial cells (potentially leading to MCC).

Discarding the first draw can reduce this possibility.

Question 15

How is the possibility of MCC reduced in CVS and POC culture?

Answer 15

By careful separation of the maternal decidua from chorionic villis before culture initiation.

Question 16

What is the desired coverslip confluency prior to testing?

Answer 16

75%

Should always have back up cultures for repeats of confirmations

Question 17

What may be indicated from the presence of MCC in the absence of bloodstaining?

Answer 17

Presence of solid maternal tissues

Question 18

When should MCC be suspected?

Answer 18

1) Mix of XX and XY genotypes (or mixed genotypes on QF-PCR)
2) An abnormal cell line is present with a normal cell line (karyotype)
3) Female karyotype is discordant with a previous prenatal diagnosis of foetal sex
4) Uncertainty of the origin of tissue used in culture
5) Slow cell growth, small number of colonies

Question 19

When should MCC testing be performed?

Answer 19

For the validity of all ‘molecular’ PND. Low level MCC may not necessarily be an issue

MCC checks on all prenatal testing

Analysis of maternal sample is required to state the significant MCC has been excluded. In some instances a paternal sample may be required but this could raise issues of non-paternity

Question 20

What is sufficient evidence to exclude MCC?

Answer 20

Evidence from a minimum of two microsatellite markers

MCC assay should be capable of detecting MCC to 10%

Question 21

Which prenatal assays are most sensitive to MCC?

Answer 21

TP-PCR

MLPA

Question 22

What is the definition of CPM?

Answer 22

The presence of abnormal cells restricted to the extra-embryonic tissues.

80% of mosaicism cases of autosomal trisomy are CPM. Can be numerical or structural.

Abnormal cell lines confined to:
Trophoblast (50%)
Villus mesenchyme (30%)
Trophoblast and mesenchyme (20%)

Detected in 1-2% of ongoing pregnancies at 10-12 weeks

Question 23

What are the two ways in which CPM can arise?

Answer 23

1) Mitotic CPM: Mitotic non-disjucntion in a trophoblast cell or a non-foetal cell from the ICM creating a trisomic cell line in the tissue which is destined to become the placental mesoderm.
2) Meiotic CPM: trisomy rescue. If a trisomic conception undergoes trisomy rescue in some cells, including those destined to be the foetus, remaining trisomies may be confined to the placenta.

Question 24

Which autosomal trisomies are assoicated with mitotic CPM?

Answer 24

2, 3, 7, 8.

Question 25

Which autosomal trisomies are associated with meiotic CPM?

Answer 25

16 and 22

Question 26

What factors influence the pattern or normal and abnormal cells in the developing embryo?

Answer 26

1) Reduced or improved replication rates of the trisomic cells could affect the number of abnormal cells compared to normal cells.
2) The abnormal cells may fail to differentiate or function properly.
3) There may be no selection against the abnormal cells, but their presence could compromise the pregnancy on a whole (e.g. abnormal placentation, IUGR, IUD etc).
4) When CVS is performed, usually samples of both cells lines (cytotrophoblasts and mesenchymal cells) are obtained. Chromosomal analysis of these cell lines can be performed by means of direct preparations (e.g. FISH), short-term cultures (cytotrophoblasts), or long-term cultures (mesenchymal cells) of the chorionic villi.

Question 27

Three types of CPM have been described based on which placental cells are affected. Summarise these.

Answer 27

Type I (40%): abnormal cell line confined to the cytotrophoblast. Usually associated with a normal pregnancy outcome

Type II (40%): Affects only mesenchymal cells of stromal villus core.

Type III (7%): confined to the placental (CPM) or to the foetus (TFM).

Question 28

Which types of CPM are related to mitotic nondisjunction events?

Answer 28

Types I and II

Question 29

What complications are associated with Type III CPM?

Answer 29

Result of a meiotic error and trisomy rescue.

Risk of UPD

Question 30

What is the significance of CPM to prenatal array-CGH investigations?

Answer 30

Lead to false positive results for submicroscopic changes
e.g. Report of STS deletion prenatally, not confirmed neonatally.

Prenatal reports should carry a rider to state that CPM cannot be excluded. Confirmation on cultured CVS of AF should be performed.

Question 31

What is pseudomosaicism? What are the different levels?

Answer 31

Abnormal cell lines that occur after cell culture and which are seen in the progeny of one colony of cells.

Level 1: a single abnormal cell observed in a single culture = likely cultural artefact, not reported

Level 2: two or more cells with the same abnormality within a single culture. Usually not reported but depends on which chromosome is involved and the US finding.

Level 3: 2+ cells with same abnormality in 2+ cultures likely to represent true mosaicism. Reported.

Question 32

What is the clinical relevance of CPM?

Answer 32

1) Observation in aneuploidy in CVS and normal foetal karyotype for CVS is a risk for UPD
2) Most pregnancies with CPM continue to term with no complications
3) Some pregnancies with CPM show prenatal or perinatal complications linked to sub-optimal placental function resulting in decreased intrauterine growth rates.
4) The pregnancy loss rate in pregnancies with CPM diagnosed by CVS is higher than pregnancies without placental mosaicism.

Question 33

What factors should be considered when predicting the likely effects of CPM (if any)?

Answer 33

1) Origin of error – somatic errors are associated with less severe consequences.
2) Level of mosaicism – there is a correlation between a high number of aneuploid cells and poor pregnancy progress.
3) Specific chromosomes – the influence of CPM on fetal growth is chromosome specific. Certain chromosomes carry imprinted genes involved in growth or placental function, which may contribute to impaired pregnancy progress when CPM is detected. CPM involving sex chromosomes usually has no adverse effect on foetal development.
4) Type of chromosome abnormality – marker chromosomes are more often confirmed in the fetus than trisomies.

Question 34

How can CPM be minimised in prenatal testing?

Answer 34

1) Mesenchymal core culture results are more likely to reflect a true fetal mosaicism than direct preparation.
2) More than one frond from the placental biopsy should be selected to ensure that the majority of the sample is not comprised of villus tips.
3) Fronds from different areas of the biopsy should be selected to increase the likelihood of identifying whether different areas of the biopsy have a different chromosome complement.
4) Including material from the whole biopsy after either fine chopping or enzymatic digestion allows mesenchymal cells to be enriched in the material for testing.
5) If mosaicism is found on either culture or direct preparation, follow-up amniocentesis should be offered in addition to detailed ultrasound scans.

Under no circumstances should a decision to terminate a pregnancy be based entirely on a CVS mosaic result.