Diagnostic Performance of RT-PCR for Fetal Sex Determination Using Cell-free Fetal DNA in Maternal Plasma During the Last Third of the First Trimester in Mares

Introduction: Accurately determining fetal sex early in mares is essential for horse breeding. In this study, we investigated the detection of cell-free fetal DNA (cffDNA) in horse plasma using real-time polymerase chain reaction (RT-PCR) during pregnancy. Materials & Methods: Blood samples were obtained from ۳۰ pregnant mares in the last third of the first trimester of gestation at stables located in the provinces of Semnan, Golestan, and Tehran. In addition, blood samples were taken from one male foal and one female foal with no history of mating to serve as positive and negative controls, respectively. The phenotypic sex of the foals was recorded after parturition and compared with RT-PCR results performed on maternal plasma to evaluate the accuracy of fetal sex determination using the sex-determining region Y (SRY), equine Y‑linked microsatellite marker YH۱۲ (Eca.YH۱۲), and X-linked microsatellite locus TKY۲۷۰ markers. Results: A total of ۳۰ pregnant mares were sampled, with ۲۷ carrying to term and three aborting. Fetal sex was confirmed postnatally (۱۱ males, ۱۶ females). The SRY marker had a sensitivity of ۷۲.۷%, specificity of ۱۰۰%, and accuracy of ۸۸.۹%. The Eca.YH۱۲ marker yielded the same results as SRY and did not enhance sensitivity when used in combination. The X-linked marker TKY۲۷۰ was detected in all samples, indicating that the samples were of high quality and could serve as an internal control. These markers supported our results, strengthening the diagnostic process.  Conclusion: RT-PCR for the SRY gene detected cffDNA in the last third of the first trimester and worked well, especially for identifying female fetuses. These findings were further supported by the results obtained from the Eca.YH۱۲ and TKY۲۷۰ genetic markers.

کلیدواژه ها:

cffDNA ، Fetal sex determination ، Mare ، RT-PCR ، SRY gene