PRENATAL DIAGNOSIS Prenat Diagn 2006; 26: 1219–1223. Published online 7 November 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/pd.1592 Accuracy of fetal gender determination in maternal plasma at 5 and 6 weeks of pregnancy Ciro Dresch Martinhago 1,2,3 , Ricardo Manoel de Oliveira 3 , Maria do Carmo Tomit˜ ao Canas 1 , Laura Diniz Vagnini 1 , Jo˜ ao Batista Alcantara Oliveira 4 , Claudia Guilhermino Petersen 4 and Jos´ e Gon¸ calves Franco Junior 4,5 * 1 CPDP—Paulista Center of Diagnosis and Research—Ribeir˜ ao Preto, SP Brazil 2 Doctoral Postgraduate Program of Gynecology, Obstetrics and Mastology, Faculty of Medicine of Botucatu/UNESP—Botucatu, SP Brazil 3 RDO Medical Diagnosis—S˜ ao Paulo, SP Brazil 4 CRH—Center for Human Reproduction Professor Franco Junior—Ribeir˜ ao Preto, SP Brazil 5 Department of Gynecology and Obstetrics, Faculty of Medicine of Botucatu/UNESP—Botucatu, SP Brazil Objective To assess the viability of the early diagnosis of fetal gender in maternal plasma before 7 weeks of pregnancy by real-time polymerase chain reaction (real-time PCR), starting at 5 weeks of pregnancy. Method Peripheral blood was collected from pregnant women, starting at 5 weeks of gestation. After centrifugation, plasma was separated for fetal DNA extraction. DNA was analyzed by quantitative real-time PCR for two genomic regions, one on the Y chromosome (DYS-14) and the other shared by both sexes (ß-globin), by the TaqMan Minor Groove Binder (MGB) probe assay. The results of the examinations were compared to fetal gender determined after delivery. Results A total of 79 examinations of fetal DNA in maternal plasma were performed for 52 pregnant women. Accuracy according to gestational age was 92.6% (25 of 27 cases) at 5 weeks, and 95.6% (22 of 23 cases) at 6 weeks. These results also demonstrate that fetal DNA is present at low concentrations in maternal plasma at 5 weeks (8.5 genome equivalents (GE)/mL) and 6 weeks (34.1 GE/mL) of pregnancy. Conclusion Quantitative real-time PCR and TaqMan MGB probes specific for the detection of fetal gender in maternal plasma starting at 5 weeks of gestation have good sensitivity and excellent specificity. Copyright  2006 John Wiley & Sons, Ltd. KEY WORDS: real-time PCR; TaqMan Minor Groove Binder; fetal DNA; maternal plasma; fetal sexing INTRODUCTION The passage of fetal cells into maternal blood is a well-known phenomenon that was first determined at least 35 years ago. This source of fetal material is thought to be a possible tool for the detection of fetal abnormalities (Walknowska et al., 1969). Almost 10 years ago, researchers described the presence of fetal DNA in maternal plasma and serum for the first time (Lo et al., 1997). In addition, several investigators emphasized the importance of this finding as a source of fetal material in maternal blood, which might provide the opportunity for future noninvasive examination for the possible diagnosis of fetal disorders such as chromosome diseases (fetal aneuploidies), paternally inherited genetic disorders, or genetic diseases of autosomal recessive inheritance. The detection of fetal DNA was confirmed by the presence of a region of Y chromosome. Pregnant women with a male fetus had a positive examination and those with a female fetus had a negative examination. Approximately one year after researchers first described *Correspondence to: Jos´ e Gon¸ calves Franco Junior, Avenue Pro- fessor Jo˜ ao Fiusa n ◦ 689—Alto da Boa Vista, Ribeir˜ ao Preto —S˜ ao Paulo CEP 14025-310 Brazil. E-mail: franco@crh.com.br the presence of fetal DNA in maternal plasma and serum, the same investigators succeeded in quantifying fetal DNA in relation to maternal DNA by means of a new molecular biology technique denoted as real-time quantitative polymerase chain reaction (PCR) (Lo et al., 1998). Fetal DNA was extracted from the plasma of pregnant women and this DNA was later analyzed by real-time PCR for the detection of two genomic regions, one on Y chromosome and the other in a region common to both sexes called ß-globin. Amplification of the ß-globin region serves as a positive control of the reaction, and the region of Y chromosome is detected when the pregnant woman carries a male fetus. On this basis, a male fetus is diagnosed when the signal of amplification occurs in this region of Y chromosome, whereas a female fetus is diagnosed by exclusion of its occurrence. The methodology applied to quantify the fetal DNA was based on a ‘standard curve’ in which a quantified DNA known to be male is used. This DNA is diluted and, in the case of the present study, the dilutions ranged from 1000 to a single copy of Y chromosome. Thus, the standard curves and the DNA extracted from the plasma of pregnant women, which presents its curve among the ‘standard’ ones, were run simultaneously and the DNA was then quantified. Using this methodology, the authors were able to demonstrate that during the first and second trimester of pregnancy, Copyright  2006 John Wiley & Sons, Ltd. Received: 7 April 2006 Revised: 25 August 2006 Accepted: 26 September 2006 Published online: 7 November 2006