BACKGROUND: Hemolytic disease of the fetus and newborn (HDN) is caused primarily 
by feto-maternal RhD incompatibility. Although all RhD negative pregnant women 
undergo routine antenatal RhD prophylaxis at 28 weeks of gestation, and 
following delivery if the newborn is RhD positive, HDN has not been eradicated. 
Here, we investigated fetal Rhesus D (RHD) genotype in maternal plasma during 
the first trimester of pregnancy in our area.
METHODS: Plasma samples were obtained from 111 RhD negative pregnant women, 
between 9 and 13 weeks of gestation. DNA from maternal plasma containing 
cell-free fetal DNA (cffDNA) was analyzed by quantitative PCR (qPCR) to detect 
RHD exons 5 and 7. A beta-globin (HBB) sequence was quantified to estimate total 
DNA concentration. qPCR results were compared with newborn RhD determined in 
cord blood serum. The influence of several gestational parameters on DNA 
concentration was also analyzed.
RESULTS: The specificity and sensitivity of the assay was 93% and 100%, 
respectively, with 97% diagnostic accuracy. Cell-free DNA concentrations during 
the first trimester of pregnancy were not affected by the gestational parameters 
studied (free-beta fraction of human chorionic gonadotropin and 
pregnancy-associated plasma protein A concentrations, fetal sex, materno-fetal 
ABO blood group incompatibility, maternal weight and gestational age).
CONCLUSIONS: Non-invasive fetal RHD genotyping during the first trimester of 
pregnancy can be determined with a high specificity, thus representing a 
valuable tool for improving the management of RhD negative pregnant women. As a 
high percentage of pregnant women participate in the routine first trimester 
combined screening program for aneuploidies, the fetal RHD study could be of 
immediate implementation, since the same blood collection could be used.