This study examined how maternal and fetal characteristics affect the percentage of cell-free fetal DNA (fetal fraction) found in maternal plasma at 11-13 weeks of gestation. They found the fetal fraction was lower in women with higher weight, in Afro-Caribbean women, smokers, and fetuses with a smaller crown-rump length. The risk of a fetal fraction below 4%, resulting in a failed non-invasive prenatal test, increased substantially with maternal weight above 100kg. The results suggest non-invasive prenatal testing should be performed alongside standard screening to avoid missed testing opportunities if the initial non-invasive test fails due to low fetal fraction.

1. UOG Journal Club: January 2013
Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks’
gestation: relation to maternal and fetal characteristics
G Ashoor, A Syngelaki, LCY Poon, JC Rezende and KH Nicolaides
Volume 41, Issue 1, Date: January 2013, pages 26–32
Journal Club slides prepared by Dr Asma Khalil
(UOG Editor for Trainees)

2. Chiu RW et al., BMJ 2011; Chen EZ et al., PloS One 2011; Ehrich M et al., AJOG 2011; Palomaki GE et al., Genet Med 2011; Palomaki GE et al., Genet Med 2012;
Ashoor G et al., AJOG 2012; Bianchi D et al., Obstet Gynecol 2012; Norton ME et al., AJOG 2012; Sparks AB et al., AJOG 2012
Trisomy 21 Trisomy 18 Trisomy 13
DR FPR DR FPR DR FPR
Chiu et al. 2011 100% 2.1%
Chen et al. 2011 91.9% 2% 100% 1.1%
Ehrich et al. 2011 100% 0.2%
Palomaki et al. 2011 98.6% 0.2%
Palomaki et al. 2012 100% 0.3% 91.7% 1%
Ashoor et al. 2012 100% 0% 98% 0% 80% 0.05%
Bianchi et al. 2012 98.9% 0% 92.1% 0% 68.8% 0%
Norton et al. 2012 100% 0.03% 97.4% 0.07%
Sparks et al. 2012 100% 0% 100% 0%
DR, detection rate; FPR, false positive rate
Screening by cell-free (cf) DNA testing of maternal blood in high-risk populations:
Background

3. Nicolaides KH et al., AJOG 2012
Ashoor G et al., UOG 2013
Detection rate False positive
rate
Trisomy 21 99.5% 0.1%
Trisomy 18 92–100% 0–0.3%
Trisomy 13 68–100% 0–1.1%
Screening by cfDNA testing of maternal blood in low-risk populations:
Background

4. Strengths
• Highly accurate in the detection of
trisomies 21 and 18 and to a lesser
degree trisomy 13
• The screening performance for
trisomies 21 and 18 is far superior to
that of all currently available
screening methods (DR>99% and
FPR <1%)
Limitations
•The main limiting factor currently is
the high cost
• Fails to provide results if the fetal
fraction < 4%
Background
Non-invasive prenatal testing (NIPT)

5. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
Objectives
• Examine the possible effects of maternal and fetal characteristics on
the fetal fraction in maternal plasma cfDNA at 11–13 weeks
• Estimate the proportion of pregnancies at high risk of NIPT failure
because the fetal fraction is < 4%

6. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
• Prospective first-trimester screening for aneuploidies and
adverse pregnancy outcomes in singleton pregnancies
(n=1,949)
• Stored maternal plasma at −80 °C
• Collected in ethylenediamine tetraacetic acid BD vacutainer TM tubes
• Centrifuged at 2000g for 10min and subsequently at 16 000g for
10min, within 15min of collection
Methodology

7. 0
50
100
150
200
2.3 4.0 6.3 9.0 12.3 16.0 20.3 25.0 30.3
Fetal fraction (%)
Frequency
• The median fetal
fraction was 10.0%
•  with weight
•  in Afro-Caribbean
•  with fetal CRL
•  7.5% in smokers
•  25.0% in trisomy 21
Results

8. 0
5
10
15
20
25
Fetalfraction(%)
40 50 60 70 80 90 100 110 120 130
Maternal weight (kg)
140 150 160 170 180
99th
75th
50th
25th
97th
95th
90th
10th
5th
3rd
1st
4
Weight
(kg)
Failure
(%)
40 0.4
60 1.5
80 4.7
100 12.1
120 25.1
140 43.1
160 62.8
180 79.5
Results

9. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
Results
No significant contribution to fetal fraction from:
• Maternal age
• Fetal gender
• NT thickness
• Method of conception
• Linear association with free β-hCG and PAPP-A

10. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
Discussion
The inverse association between fetal fraction and maternal
weight could be attributed to:
• Dilutional effect
• Accelerated turnover of adipocytes (releases cfDNA of maternal
origin into the circulation thereby lowering the proportion of fetal
cfDNA)

11. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
• NIPT is far superior to currently available screening methods. The
greatest risk factor for low fetal fraction and failure is obesity. The
opportunity for testing and counseling in the same visit is missed when
NIPT fails.
• Alternatively, NIPT can be performed along with the combined screening
blood test at 10-11 weeks. Hence results are available at the scan
assessment, where CVS can be performed if high risk. Where NIPT fails,
the parents would still have the first trimester combined test result.
• This policy might exaggerate the problem of low fetal fraction because it is
done earlier when the CRL and fetal fraction are smaller
Implications for practice

12. Fetal fraction in maternal plasma cf-DNA is affected by
 Maternal characteristics (weight, ethnicity, smoking)
 Fetal characteristics (CRL, aneuploidy)
Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks: relation to
maternal and fetal characteristics
Ashoor et al., UOG 2013
Conclusions

13. Discussion points
• What maternal characteristics would influence the fetal fraction in maternal plasma cell-
free fetal DNA test after 14 weeks’ gestation?
• Apart from trisomy 21, what other fetal chromosomal abnormalities would influence the
fetal fraction in maternal plasma cell-free DNA in multiple pregnancies?
• Can we offer the non-invasive prenatal testing (NIPT) using cell-free fetal DNA to twin
pregnancies or pregnancies conceived using IVF with egg donation?
• What sort of NIPT result would be expected with placental mosaicism?
• Could invasive prenatal diagnosis be avoided with a low-risk NIPT result where the fetus
has an isolated increased nuchal translucency?
• What impact would the introduction of NIPT as a screening tool have on the numbers of
prenatal invasive tests performed?
• Would there be a role for first-trimester combined nuchal plus biochemistry testing if NIPT
were used universally as a routine screening tool.
• What would be the management options for a woman that had fetal DNA in maternal
plasma test when first seen at 11 weeks’ gestation, and who received a ‘failed’ result at 13
weeks?