en ژنتیک/ بیوتکنولوژی Genetics/ Biotechnology پژوهشي Research <div style="text-align: justify;"><strong>Background and Aims:</strong> New advances in the use of cell-free fetal DNA (cffDNA) in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. One of the applications of prenatal diagnosis is fetal gender determination. Early prenatal determination of fetal sex is required for pregnant women at risk of X-linked and some endocrine diseases. The present study was carried out to perform an efficient polymerase chain reaction&nbsp;(PCR) method in order to improve sensitivity, specificity and accuracy of non-invasive fetal gender detection using fetal DNA in maternal plasma during 8th -12th weeks of pregnancy.<br> <strong>Materials and Methods:</strong> Thirty-five pregnant women with 8 to 12 weeks of pregnancy were selected for prenatal fetal sex determination. Maternal peripheral blood was collected and cffDNA was extracted from 3-ml of maternal plasma. Two multi copy Y-chromosome-specific region (DYS and DAZ) and a single copy gene (SRY) were amplified by real-time quantitative PCR. Amplification was labeled as positive, negative, or inconclusive according to a stringent algorithm.<br> <strong>Results:</strong> Using this method, the sensitivity and specificity of the real-time PCR assay was 100% and 93.8% for prenatal fetal sex detection, respectively.<br> <strong>Conclusions:</strong> It is concluded that fetal sex can be determined with a high level of accuracy by our algorithm, after 8 weeks of gestation with cffDNA analysis.</div> Cell-free DNA, Real-Time PCR, Sex-determination 85 92 http://ijml.ssu.ac.ir/browse.php?a_code=A-10-330-1&slc_lang=en&sid=1 Najmeh Davoodian نجمه داوودیان 0000-0002-4714-6619 Yes Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Ali Kadivar علی کدیور 0000-0002-0993-397X No Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran Department of Clinical Science, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran Heidar Heidari Khoie حیدر حیدری خویی 0000-0002-3059-1492 No Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Sima Hematian Khayat سیما حکمتیان خیاط No Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Mahboobeh Heidari Nasirabadi محبوبه حیدری نصیرابادی No Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran.