CfDNA of feto-placental origin is not detectable in plasma of pregnant mares by optimised sexing-qPCR.

Abstract

Non-invasive prenatal testing is a cell-free-DNA (cfDNA)-based genetic screening, well-established in pregnant women, utilising cfDNA of feto-placental origin (cffDNA) isolated from maternal plasma (Christiaens et al, Prenatal Diagnosis. 2021;41:1316-1323). Fetal aneuploidy is described as causative for equine pregnancy loss (Shilton et al, Scientific Reports. 2020;10:13314), suggesting cffDNA-screening in mares is diagnostically warranted. The aims of the current study were to (i) optimise isolation methodology and examine presence of cffDNA in maternal plasma and (ii) develop a non-invasive cffDNA-based test to determine fetal sex in pregnant mares. Peripheral blood (10ml) from pregnant mares, carrying fetuses of confirmed sex (D70-80:n=4; last trimester:n=15), non-pregnant mares (n=2) and geldings (n=4) was collected into Streck®tubes. Samples were double-centrifuged within 24hrs, and supernatant transferred into Eppendorf® DNA-low-bind-tubes. CfDNA was isolated from multiple plasma aliquots per individual (QIAamp, ccfDNA-kit), applying two different temperatures (56°C, 21°C) for the magnetic-bead-binding step of the protocol. CfDNA was quantified by fluorometry (Qubit®fluorometer, 1XdsDNA-HS-assay). Control DNA was isolated from adult peripheral blood mononuclear cells (PBMC) and fetal tissue. CfDNA-isolation resulted in a median yield of 0.99ng/ml plasma (n=80 isolations) and did not differ (P>0.05) between aliquots of one individual or gestational stages but was significantly higher (P=0.0037) with an incubation temperature of 56°C (n=40), compared to 21°C (n=40). Quantification of cfDNA (n=6) by automatedelectrophoresis (cfDNA-Screentape, Agilent® TapeStation) was in alignment with fluorometry. A qPCR was optimized and validated for GAPDH and the Y-sequence TSPY utilizing 0.1ng and 0.2ng of DNA (indicated by cfDNA-concentrations, isolated above) from adult PBMC (n=41) and fetal tissue (n=6) of confirmed sex. A correct female / male classification was achieved for 95.1% (0.2ng) and 87.2% (0.1ng) of adult and 100% of fetal (0.1ng) DNA. Sensitivity (adult:0.2ng:100%; 0.1ng:100%), specificity (adult:0.2ng:100%; 0.1ng:95.2%) and PPV (adult:0.2ng:100%; 0.1ng:92.8%) were high. Next, qPCR accuracy was examined utilizing plasma cfDNA of non-pregnant mares (n=2) and geldings (n=4). Sensitivity, specificity and positive predictive value (PPV) for Y-chromosomal detection were 100%. In women, a minimum of 2-4% cffDNA is deemed diagnostic for prenatal testing. To determine TSPY detection at this ratio, male DNA was diluted in female DNA and TSPY accurately found at 4-10% but not 2%-dilutions. Finally, cfDNA from the plasma of pregnant mares (n=19) was analysed. All samples amplified GAPDH, however all clinically confirmed male pregnancies (6 / 19) were TSPY-negative. In conclusion, cfDNA was isolated from equine plasma and the yield improved by incubation at 56°C during the magnetic-bead-binding. A highly sensitive sexing-qPCR was validated for equine adult, fetal and cfDNA, but cffDNA was not detected in plasma of pregnant mares carrying male fetuses. CffDNA was not clearly observed by electrophoresis, suggesting isolation of cfDNA was of maternal but not feto-placental origin.