The present study was undertaken to evaluate a nested polymerase chain reaction (PCR) for detection of Y chromosome-specific fetal DNA in maternal plasma and urine of pregnant women during different gestational stages. DNA isolated from plasma and urine samples of 80 pregnant women (between 7 and 40 weeks' gestation) underwent amplification for Y chromosome-specific 198 bp DNA by nested PCR. The postpartum analysis of fetal gender showed that 55 women carried male and 25 female fetuses. Among the 55 women bearing male fetuses, Y chromosome-specific signals were detected in 53 (96%) plasma and 21 (38%) urine samples. Moreover, out of 25 women bearing female fetuses, 3 (12%) and 1 (4%) women had Y chromosome-specific signal in plasma and urine, respectively. Analysis of results with respect to gestational age revealed that there was no significant difference in the detection of Y chromosome-specific DNA between different trimesters in maternal plasma of women bearing male fetuses. These results showed that fetus-specific DNA was detected with high sensitivity (96%) and specificity (88%) in the maternal plasma by nested PCR, and therefore the method could be useful as a non-invasive procedure for fetal sex determination and prenatal diagnosis.

Objective: To establish and evaluate nested polymerase chain reaction (PCR) for detection of Y-chromosome-specific fetal DNA in maternal blood circulation during pregnancy and its clearance several days after delivery. Materials and Methods: Genomic DNA was isolated from a male donor to amplify Y-chromosome-specific DNA sequences by using four pairs of oligonucleotide primers targeting single or multiple copy genes in standard and nested PCR. The nested PCR was evaluated for detection of Y-chromosome-specific sequences in the maternal blood circulation of pregnant women bearing a male fetus during pregnancy and at 2 and 24 h and 7 days after delivery. Results: Although both standard and nested PCR assays were established by using genomic DNA from a male donor to detect Y-chromosome-specific DNA sequences, the nested PCR was 10 times more sensitive than standard PCR. Further experiments showed that nested PCR was able to detect Y-chromosome-specific DNA in the peripheral blood cells as well as in the plasma and serum of pregnant women carrying a male fetus. When tested with plasma samples from women carrying male fetuses during pregnancy, nested PCR was positive with 53/55 (96%), 22/22 (100%), 16/22 (73%) and 0/30 (0%) specimens obtained before and at 2 and 24 h and 7 days after delivery, respectively. These results showed that fetus-specific Y-chromosome DNA was cleared from maternal circulation within 7 days of delivery. Conclusion: Our results suggest that nested PCR is useful in fetal sex determination and that prenatal diagnosis can be done without interference from previous pregnancy.