Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins

Abstract

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Vanished Twins and Misdiagnosed Sex: A Case Report with Implications in Prenatal Counseling Using Noninvasive Cell-Free DNA Screening

“…6 A prospective study of the accuracy of cell-free DNA testing noted 2 cases of discordant cell-free DNA test results for trisomy 21, which were associated with vanished twins. 11 The authors concluded that "(t)he gender of the living and apparently healthy fetus was confirmed to be female and thus, the cffDNA that caused the increased z-score for trisomy 21 can clearly be assigned to a deceased male fetus. "…”

Contingency Screening using Noninvasive Prenatal Test for the Detection of Trisomy 21

“…It is not advisable for low-risk population and multiple gestations at present, due to a small but definite risk of false positive. 5 In some cases, result may be uninterpretable (due to early gestation, maternal obesity leading to low fetal fraction or placental mosaicism). In such cases, diagnostic tests and ultrasound evaluation should be offered, although repeat sampling has also been considered.…”

Modifying Risk of Aneuploidy with a Positive Cell-Free Fetal DNA Result

“…13,14 Several studies have shown that this phenomenon is a source of false positive cfDNA results, [13][14][15] whereby the contribution of additional cfDNA from the demised twin can be detected 8 weeks after the demise, creating an additional complement of fetal DNA. 13,14 The recent introduction of single-nucleotide polymorphism-based screening NIPT may provide a solution to this issue. 13 By scanning for the presence of polymorphic loci, this test can detect the presence of different fetal haplotypes, which can help confirm a previously undetected dizygotic vanishing twin when combined with morphology scan.…”

“…13 Whenever a confirmed singleton pregnancy with additional haplotypes is noted on singlenucleotide polymorphism NIPT results, the patient should be counseled on proceeding with invasive diagnostic testing before definitive management. 14 …”

“…13,14 The demise of the embryo has been reported to be frequently associated with chromosomal anomalies. 13,14 Several studies have shown that this phenomenon is a source of false positive cfDNA results, [13][14][15] whereby the contribution of additional cfDNA from the demised twin can be detected 8 weeks after the demise, creating an additional complement of fetal DNA. 13,14 The recent introduction of single-nucleotide polymorphism-based screening NIPT may provide a solution to this issue.…”