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

Articles in Press

Document Type : Original Articles

Authors

1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran.

2 Department of Pathobiology, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran.

10.22092/ari.2025.370005.3734

Abstract

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 serum using RT-PCR during pregnancy. Blood samples were obtained from 30 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 YH12 (Eca.YH12), and microsatellite locus TKY270 on the equine X chromosome (TKY270) gene markers. A total of 30 pregnant mares were sampled, with 27 carrying to term and three aborting. Fetal sex was confirmed postnatally (11 males, 16 females). The SRY marker had a sensitivity of 72.7%, specificity of 100%, and accuracy of 88.9%. Eca.YH12 marker yielded the same results as SRY and did not enhance sensitivity when used in combination. The X-linked marker TKY270 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. 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.YH12 and TKY270 genetic markers.

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Archives of Razi Institute

Articles in Press, Accepted Manuscript
Available Online from 05 October 2025

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