Low Fetal Fraction of Cell-free DNA Identified by Non-invasive Prenatal DNA Testing: Possible Causes, Clinical Significance, and Tactics

Study Objective: To compare the rates of fetal chromosomal abnormalities (CA) detected during initial non-invasive prenatal DNA testing (NIPT) with the rates of CA found through repeat NIPT in patients with low fetal fraction or low quality of cell-free embryonic DNA. Study Design: This was a retrospective cohort study. Materials and Methods: Twenty-one thousand forty-two women who underwent NIPT in Russia between 2013 and 2018 were included in the study. The main group comprised 1,025 of the 1,044 patients with uninformative results (low fetal fraction result, making it impossible to assess the risk of CA), who consented to repeat NIPT. The control group was made up of 19,998 women who had informative results of initial NIPT. The exclusion group comprised women with low fetal fraction who declined repeat screening. The study method was targeted NIPT. Blood samples were taken from a vein and centrifuged to obtain plasma. Fetal cell-free DNA was analyzed by next-generation sequencing (NGS), a method patented by Natera for sequencing single nucleotide polymorphisms. Study Results: Initial NIPT was uninformative in 1,044 (5%) of the patients and repeat procedure yielded informative results in 821 (80.1%) out of 1,025 patients. Among the patients with informative results from the initial study, the rate of chromosomal aneuploidies was 2.4%. In the group of women with informative results from the repeat procedure, fetal CA were detected in 27 (3.3%) cases. In the subgroup of women with informative results only after a third NIPT, the prevalence of CA was 9.3% (seven out of 75 cases). The study showed that in women carrying fetuses with trisomy 18 or 13 or monosomy X, mean fetal fraction in the first trimester was significantly lower than normal. In the second trimester, significantly lower than normal fetal fraction was observed in women carrying fetuses with trisomy 18 or monosomy X. There was a statistically significant difference in fetal fraction levels between patients with body weight <50 kg and those with body weight 80-89 kg or above (р<0.05). Conclusion: The probability of detecting CA by repeat NIPT is significantly higher than in an initial procedure. If initial testing is not informative, it should be repeated. If the second procedure also fails to yield informative results, invasive prenatal diagnosis should be considered. Fetal fraction levels are lower in heavier women. Thus, other methods of prenatal diagnosis should be recommended for overweight and obese women. Keywords: non-invasive prenatal DNA testing, fetal fraction, prenatal diagnosis, Down syndrome.

Download Full-text

Current controversies in prenatal diagnosis 3: is there still a value in a nuchal translucency screening ultrasound in conjunction with maternal plasma non-invasive cell-free DNA testing?

Prenatal Diagnosis ◽

10.1002/pd.4719 ◽

2015 ◽

Vol 36 (1) ◽

pp. 20-24 ◽

Cited By ~ 3

Author(s):

R. Douglas Wilson ◽

Liona C. Poon ◽

Alessandro Ghidini

Keyword(s):

Prenatal Diagnosis ◽

Nuchal Translucency ◽

Maternal Plasma ◽

Dna Testing ◽

Cell Free Dna ◽

Non Invasive ◽

A Value ◽

Free Dna ◽

Screening Ultrasound ◽

Invasive Cell

Download Full-text

Low fetal fraction in cell‐free DNA testing is associated with adverse pregnancy outcome: analysis of a subcohort of the TRIDENT‐2 study

Prenatal Diagnosis ◽

10.1002/pd.6034 ◽

2021 ◽

Author(s):

E.C. Becking ◽

S.A.M. Wirjosoekarto ◽

P.G. Scheffer ◽

J.V.M. Huiskes ◽

M.J. Remmelink ◽

...

Keyword(s):

Pregnancy Outcome ◽

Adverse Pregnancy Outcome ◽

Outcome Analysis ◽

Dna Testing ◽

Cell Free Dna ◽

Free Dna ◽

Adverse Pregnancy ◽

Fetal Fraction

Download Full-text

Non-Invasive Prenatal Diagnosis (NIPD) of Sickle-Cell Disease By Massively Parallel Sequencing of Cell-Free Fetal DNA in Maternal Serum

Blood ◽

10.1182/blood-2019-127945 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2085-2085

Author(s):

Yvonne Daniel ◽

Julia Van Campen ◽

Lee Silcock ◽

Michael Yau ◽

Joo Wook Ahn ◽

...

Keyword(s):

Prenatal Diagnosis ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Massively Parallel Sequencing ◽

Plasma Samples ◽

Cell Disease ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Cell Free Fetal Dna

Sickle cell disease (SCD) is the most common genetic haematological disorder worldwide. Around 300.000 affected infants are born every year, including at least 1000 in the United States. Prenatal diagnosis is currently carried out using amniotic fluid or chorionic villus sampling. These invasive procedures are perceived to have a small risk of miscarriage. The availability of non-invasive prenatal diagnosis (NIPD) is predicted to increase uptake of prenatal diagnosis for SCD, as it has no perceived miscarriage risk. NIPD may also be more readily implemented than invasive prenatal diagnosis in the low-resource countries in which SCD is the most prevalent. However, accurate NIPD of autosomal recessive disorders such as sickle cell disease has proven challenging as this requires detection of fetal inheritance of a maternal allele from a mixed maternal-fetal pool of cell-free DNA. We report the development of a targeted massively parallel sequencing assay for the NIPD of fetal SCD using cell-free fetal DNA from maternal plasma. No paternal or previous offspring samples were required. 44 clinical samples were analysed, including 37 plasma samples from pregnant SCD carriers and 7 plasma samples from women with SCD due to Hb SC. We used a relative mutation dosage based approach for the 37 samples from maternal SCD carriers (Hb AS or Hb AC), integrating Unique Molecular Identifiers (UMIs) into the analysis to improve the accuracy of wildtype and mutant allele counts. We used a separate wildtype allele detection approach for the 7 samples from women with compound heterozygous SCD, in whom the detection of wildtype cell-free DNA indicates the presence of a carrier fetus. The success of the assay was evaluated by comparing results with the established fetal sickle status as determined through either invasive prenatal diagnosis or newborn screening. During development, two key factors improved the accuracy of the results: i) Selective analysis of only smaller cell-free DNA fragments enhanced the fetal fraction for all samples, with greater effects observed in samples from earlier gestations. This approach improved diagnostic accuracy: for 3 out of 44 samples, the genotype was inconclusive or incorrect before size selection, but correct after size selection. ii) Modifications to DNA fragment hybridisation capture optimised the diversity of Unique Molecular Identifier-tagged molecules analysed. This led to improvements in the results obtained for 5 samples, with 3 previously inconclusive samples correctly called and 2 previously discrepant results moved into the inconclusive range. In total, 37 results were concordant with the established fetal sickle status; this included 30/37 samples from carrier women and 7/7 samples from women with sickle cell disease due to Hb SC. The remaining 7 carrier samples gave an inconclusive result, which for 3 samples was attributed to a low fetal fraction. Samples from as early as 8 weeks gestation were successfully genotyped. There were no false positive or false negative results. This study is the largest to use NGS-based NIPD on clinical plasma samples from pregnancies at risk of SCD. Efforts to validate the assay on a larger sample cohort and to reduce the inconclusive rate are warranted. This study shows that NIPD for SCD is approaching clinical utility and has the potential to provide increased choice to women with pregnancies at risk of sickle cell disease. Disclosures Silcock: Nonacus Ltd.: Employment.

Download Full-text