Fetal Fraction Estimate in Twin Pregnancies Using Directed Cell-free DNA Analysis

Objective: To estimate fetal fraction (FF) in monozygotic and dizygotic twin pregnancies.

Methods: Maternal plasma samples were obtained from 35 monochorionic twin pregnancies with male fetuses (monozygotic) and 35 dichorionic pregnancies discordant for fetal sex (dizygotic) at 11-13 weeks' gestation. Cell-free DNA was extracted and chromosome-selective sequencing with digital analysis of selected regions (DANSR™) was carried out. The fetal-fraction optimized risk of trisomy evaluation (FORTE™) algorithm was used to estimate FF using polymorphic alleles. In dizygotic twins the FORTE algorithm was modified to estimate the smallest FF contribution of the 2 fetuses. In both types of twins, FF was also determined by analysis of Y-chromosome sequences.

Results: In monozygotic twins, the median total FF was 14.0% (range 8.2-27.0%) and in dizygotic twins the median smallest FF was 7.9% (4.9-14.0%). There were significant associations in FF between the methods using polymorphic alleles and Y-chromosome sequences for both monozygotic (r=0.951, p<0.0001) and dizygotic (r=0.743, p<0.0001) twins.

Conclusions: The study demonstrates the feasibility of an approach for cfDNA testing in twin pregnancies. This involves estimation of total FF in monozygotic twins and estimation of the lower FF of the 2 fetuses in dizygotic twins.