A Novel Facet of Noninvasive Fetal Genotyping

Today, fetal RHD genotyping by cell free fetal deoxyribonucleic acid (cfDNA) amplification in the serum of RhD-negative (RH:−1) mothers is used to tailor the administration of anti-D prophylaxis.[1]

Fetal RHD genotyping performed at 27 + 1 weeks' gestation in a 27 year old, 3 gravidity, 0 parity patient with A RH:-1 blood type demonstrated a higher than expected amplification signal of all three tested exons (5, 7, and 10) ([Fig. 1A] and [1B]). A false positive result due to a maternal RHD variant was ruled out by molecular genotyping with single strand (SS)-polymerase chain reaction showing classical RHD*01N.01 and RHCE*01 alleles, and no preanalytical factor altering the analysis could be identified. One week later, the patient underwent an emergency cesarean section for severe preeclampsia with fetal growth restriction. Placental histology showed signs of maternal and fetal vascular hypoperfusion.

Fig. 1 Representative amplification curves from real-time polymerase chain reaction (RT-PCR). (A) For RHD exon 7 for the patient's sample (blue, cycle threshold [CT] 28.7) to positive (red, CT 34.6) and negative (green, CT noncomputable) controls. The positive control mimics a fetal signal by diluting RH1+ plasma into RH-1 plasma at a 1/8 ratio. RHD exons 5 and 10 gave similar results (data not shown). (B) Typical RHD positive fetal (red, CT 33.7) and maternal RHD variant signals (blue, CT 29.7) illustrating the initial suspicion of a maternal RHD variant due to similar amplification patterns. (C) Beta-globin amplification curves for the patient's male fetus (light blue, CT 24.7), healthy male (dark blue, CT 27.9) and female fetuses (red, CT 28.3), all at the same gestational age. (D) SRY amplification curves from the same samples (CT 30.7, 34.9 and noncomputable). The two latter results show a higher than expected cell free fetal deoxyribonucleic acid (cfDNA) signal for gestational age and confirm its fetal origin.

We postulated that the strong RHD positive signal observed was due to unusually large quantities of cfDNA released in the maternal circulation, caused by placental hypoperfusion and necrosis, especially since the result of the sex-determining region of the Y chromosome (SRY) sequence showed comparable levels of amplification ([Fig. 1C] and [1D]).

At the time of clinically overt preeclampsia, the total amount of cfDNA is elevated compared to healthy pregnancies and correlates with disease severity.[2] This case, together with previous publications,[3] [4] demonstrates a novel potential source of aberrant positive RHD signal in maternal blood. It also highlights the value of quantitative cfDNA as a widely available, early indicator for placental hypoperfusion and preeclampsia, in addition to other biological markers.[5]

Conflict of Interest

None declared.

• References

• 1 Schimanski B, Kräuchi R, Stettler J. et al. Fetal RHD screening in RH1 negative pregnant women: experience in Switzerland. Biomedicines 2023; 11 (10) 2646
• 2 Kolarova TR, Gammill HS, Nelson JL, Lockwood CM, Shree R. At preeclampsia diagnosis, total cell-free DNA concentration is elevated and correlates with disease severity. J Am Heart Assoc 2021; 10 (15) e021477
• 3 Lo YM, Leung TN, Tein MS. et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999; 45 (02) 184-188
• 4 Vlková B, Turňa J, Celec P. Fetal DNA in maternal plasma in preeclamptic pregnancies. Hypertens Pregnancy 2015; 34 (01) 36-49
• 5 Verlohren S, Brennecke SP, Galindo A. et al. Clinical interpretation and implementation of the sFlt-1/PlGF ratio in the prediction, diagnosis and management of preeclampsia. Pregnancy Hypertens 2022; 27: 42-50

Publication History

Article published online:
31 March 2025