Down Syndrome Screening Evolution

TOG Article: Evolution in screening for Down syndrome

Volume 21, Issue 1 January 2019

This article discusses different methods evolved over time for screening of Down Syndrome with some details for latest cell-free DNA testing.

To download original article (free access): Evolution DS Screening

Introduction 

• The most common reason for invasive testing is to diagnose chromosomal aneuploidies
• Only done for high risk pregnancies as there is associated risk of miscarriage
• Down’s syndrome (DS) is the result of an extra chromosome 21
• Critical factors in screening test are detection rate and false positive rates (FPR)

• Detection rate: ability of a test to give a positive result for those who have the disease
• Screen-positive rate: proportion of affected and unaffected persons having a positive result
• FPR: unaffected proportion yielding a positive result

Screening by maternal age

• Screening for DS introduced in 1970s.
• Women aged 40 years or more considered high risk, but it was not possible to offer diagnostic tests to entire population
• Once it was identified that risk of miscarriage with amniocentesis is low, cut-off for screening changed to women aged 35 years or older which was 5% of pregnant women and 30% of affected fetuses
• Currently, >20% pregnant women are at least 35 years and this group includes 50% of total fetuses with DS

Screening by maternal serum biochemistry in the SECOND trimester

• Trisomy 21 is associated with

• Increased beta-HCG and inhibin A
• Decreased AFP and unconjugated estriol

• At FPR of 5%, detection rates are

• 30% if used only maternal age 
• 60-65% using maternal age, beta-HCG & AFP (double test)
• 65-70% using double test plus unconjugated estriol (triple test)
• 70-75% using triple test plus inhibin A (quadruple test)

Screening using ultrasound and biochemistry in the FIRST trimester

• Trisomy 21 is associated with

• Increase nuchal translucency assessed by USG at 11-13 wks 
• Free beta-HCG almost double
• PAPP-A reduced to half

• Maternal age combined with nuchal translucency and serum biochemistry —> detection rate about 90% with 5% FPR

Screening in twin pregnancies

• Detection rate of trisomy 21 in 1st trimester combined test in twin pregnancy is similar to singleton 
• In monohorionic (MC) FPR is twice as high as singleton. Risk is calculated for each fetus and average of two is given for whole pregnancy
• In dichorionic (DC) an individual risk is given for each fetus

Additional ultrasound markers

• In addition to Nuchal translucency other markers can also be used like 

• absence of nasal bone
• increased resistance to flow in ductus venous
• tricuspid regurgitation

• If combined with first trimester screening, they can give detection rates up to >95% with FPR <3% (not yet implemented at national level)

Screening for trisomies 18 and 13

At 11-13 weeks assessment

• Relative prevalence of

trisomy 18 to trisomy 21 : 1 in 3 (every 3 babies with T21 there is one with T18)

trisomy 13 to trisomy 21:  1 in 7 (every 7 babies with T21 there is one with T13)

• Serum beta HCG

• Increased in T21 
• Decrease in T13 & T18

• Detection rate at FPR of 5%

• T21 — 90%
• T13/18 — 95%

Screening using cell-free DNA in maternal blood

• Cell-free DNA (cfDNA) in maternal plasma is a mixture of DNA fragments arising from dying cells in mother and placenta
• Proportion of fetal to total cell-free DNA usually about 10%
• If pregnancy effected with fetal trisomy, the number of fragments derived from extra chromosome will be higher 
• cfDNA technology detects the increase in total amount of cfDNA fragments from one chromosome compared with other chromosomes; but it cannot differentiate whether its maternal or fetal DNA
• For testing to be effective minimum fetal fraction should be 4%
• cell-free DNA screening is by far superior to ALL previous methods
• It is a screening test NOT a diagnostic test, so results are provided in form of risk assessments (low/high risk)

Failed cell-free DNA tests

• 1-5% singleton pregnancies have no result after 1st sample (due to low fetal fractions)

• on repeat sampling result is obtained in 60%

• Low fetal fractions are mainly due to obesity and small placental mass
• In trisomy 13 & 18 placenta is small and poorly functioning, so low fetal fraction and high failure rates in these pregnancies
• If failed cell-free DNA result, pregnancies are at increased risk of trisomy 13 & 18 but not DS
• If test fails must find out the reason why it was performed and whats the repeating cost
• Majority of tests are done for extra reassurance to mothers who are low risk on combined test but have anxiety; these would usually choose repeat testing
• Those who choose not to repeat the test; USG testing for T13/18 is recommended

• if any features found, an invasive test is advised 

Conditions that can be screened with cell-free DNA testing

• Trisomy 21, 13, 18
• Fetal sex chromosome aneuploidies and some microdeletions such a DiGeorge Syndrome 
• Sex chromosome aneuploidy has high rate of fetal mosaicism of up to 50%
• Test may also reveal maternal aneuploidy such as 47XXX; 90% women are unaware of it

Twin Pregnancies

• Prevalence of twin —> 3% of live births
• Dizygotic more common than monozygotic 70:30 (excluding ART pregnancies)
• Cell-free DNA testing is challenging in twins
• USG can tell about chorionicity but not zygosity
• MC pregnancy

• both fetuses release same amount of cell-free DNA in maternal circulation
• does not effect cf-DNA analysis
• cfDNA can be safely offered with expected performance same like singleton

• DC pregnancy

• mostly fetuses are non-identical and release discordant amount of cell-free DNA
• discordance can vary by 2-fold
• fetal fraction to give successful cell-free DNA result should the lower fetal fraction of two fetuses and not the total fetal fraction. It increases the failure rate by 3-fold as compared to singleton pregnancies
• DC twin parents to be warned about inadequate data for accuracy of test

Options for clinical implementation

• Can either be offered to all pregnant women or to a subgroups of women based on first trimester combined test (current practice)
• If offered to all women

• Financial implications
• Maximum information can be gained at 10-11 wks 
• Results would be available at the time of combined test (12wks) which would allow screening for all trisomies and fetal defects as well as pregnancy complications all within first trimester
• If found high, CVS is also a valid option for confirmation
• If found low risk, parents can be reassured

• Offered to subgroups of women (current practice)

• Cell-free DNA testing can be offered as an alternative to invasive testing in high risk women
• UKNSC recommends that risk cut-off from combined test for offering cell-free DNA testing should be 1:100
• However, this group is very small (only 3%) and has only 87% fetuses with Trisomy 21.
• If desired detection rate
• 94% then cut-off to offer cfDNA at least 1:500 (about 8% of population)
• 96% then cut-off to offer cfDNA at least 1:1000 (about 13% of population)

• Preferred alternative in contingent screening is to divide the population into three groups based on results of combined test

• Very high-risk → risk is 1:10; about 1% of population → consider invasive testing
• Intermediate-risk → cfDNA testing (more accurate test for common trisomies)
• Low-risk → nothing else to do

Ref: TOG

Contingent screening method

• Above screening method was implemented in two NHS hospitals
• If all women in high-risk group opted for invasive testing → detection rate 87% at 3.4% FPR
• If all women in high-risk and intermediate-risk group opted for invasive testing → detection rate 98% at 0.25% FPR
• In high-risk group

• 38% opted for invasive test
• 60% opted for cfDNA test

• In intermediate-risk group

• 92% opted for cell-free DNA

• Therefore, prenatal diagnosis of trisomy 21 was made only in 92% affected pregnancies because many parents chose not to do any further tests or had TOP
• Live births were 32% of affected pregnancies

Conclusion

• It is feasible to incorporate the option of cfDNA testing into current established first trimester combined screening test
• It would improve detection rates and reduce invasive testing rate
• Parental choices effect the extent of improvement

Ref: TOG