Noninvasive Prenatal Diagnosis using Cell-Free Fetal DNA in Maternal Plasma: Clinical Applications

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

Effective Method for Extraction of Cell-Free DNA from Maternal Plasma for Non-Invasive First-Trimester Fetal Gender Determination: A Preliminary Study

PURPOSE: To find the most effective method for extraction of cell-free DNA (cf-DNA) from maternal plasma, we compared a blood DNA extraction system (blood kit) and a viral DNA extraction system (viral kit) for non-invasive first-trimester fetal gender determination. MATERIALS AND METHODS: A prospective cohort study was conducted with maternal plasma collected from 44 women in the first-trimester of pregnancy. The cf-DNA was extracted from maternal plasma using a blood kit and a viral kit. Quantitative fluorescent-polymerase chain reaction (QF-PCR) was used to detect the SRY gene and AMEL gene. The diagnostic accuracy of the QF-PCR results was determined based on comparison with the final delivery records. RESULTS: A total of 44 women were tested, but the final delivery record was only obtained in 36 cases which included 16 male-bearing and 20 female-bearing pregnancies. For the blood kit and viral kit, the diagnostic accuracies for fetal gender determination were 63.9% (23/36) and 97.2% (35/36), respectively. CONCLUSION: In non-invasive first-trimester fetal gender determination by QF-PCR, using a viral kit for extraction of cf-DNA may result in a higher diagnostic accuracy.

Determinación del sexo fetal en el primer trimestre de la gestación: estudio prospectivo / Fetal gender determination in the first trimester of gestation: a prospective cross-sectional study

Objetivos: Valorar la precisión de la determinación ecográfíca del sexo fetal entre las 11 y las 14 semanas en una cohorte no seleccionada de gestantes. Métodos: Se realizó un estudio prospectivo transversal en una serie consecutiva de 636 gestaciones entre las 11 y 14 semanas, en gestantes que acudieron para el estudio ecográfco rutinario de primer trimestre. Se examinó la región genital del feto en un plano mediosagi-tal, visualizando el signo sagital y la dirección en la que apunta el tubérculo genital (craneal en los varones, caudal en las mujeres) ambos indicadores del sexo fetal. La confrmación clínica del sexo fetal se obtuvo después del parto por confrmación telefónica, o por el cariotipo en casos con amniocentesis por indicaciones convencionales. Resultados: Se consiguió asignar el sexo fetal correctamente en el 86,3 por ciento de los fetos. La precisión de la asignación ecográfca del sexo fetal fue mayor en varones que en mujeres (el 90,6 por ciento de los varones fueron asignados correctamente vs el 83,1 por ciento de las mujeres, p<0,05), y se incrementó con la edad gestacional desde un 72 por ciento entre las 11 y 11+6 semanas, un 92 por ciento entre las 12 y las 12+6 semanas, hasta un 95 por ciento entre las 13 y las 13+6 semanas. Conclusión: La determinación ecográfíca del sexo fetal tiene una elevada tasa de precisión a partir de las 13 semanas, lo que sugiere que las pruebas invasivas para la exclusión de enfermedades ligadas al X podrían obviarse cuando se identifca un feto varón a esta edad gestacional. Sin embargo, en fetos identifcados como mujeres esta decisión deberá posponerse hasta una edad gestacional más avanzada.

Objective: To assess the accuracy of fetal gender determination by ultrasound at 11-14 weeks of gestation in a large cohort of unselected population. Methods: A prospective cross-sectional study was performed in 636 consecutive pregnancies at 11-14 weeks of gestation, attending for frst trimester ultrasound screening. The genital region was examined in the mid-sagittal plane to detect the sagittal sign and the direction in which the genital tubercle pointed (cranial for males, caudal for females) both as markers of fetal gender. The clinical confrmation of fetal gender was obtained from telephonic interviews after delivery or from karyotype in cases scheduled to amniocentesis for conventional indications. Results: Fetal gender was correctly determined by ultrasound in 86.3 percent of the fetuses. The accuracy of sex assignment was higher in male fetuses than in female fetuses (90.6 percent of the males fetuses vs 83.1 percent of the females were correctly assigned, p<0.05) and increased with gestational age from 72 percent at 11-11+6 week's to 92 percent at 12-12+6 week's, and 95 percent at 13-13+6 week's gestation (p<0.01). Conclusion: Prenatal gender assignment by ultrasound has a high accuracy rate at 13 to 13+6 weeks. These results suggest that invasive testing can probably be performed in fetuses identifed as males at this gestational age. However in fetuses identifed as females, the decision regarding invasive testing should be postponed until a higher gestational age is achieved.

The analysis of fetal gender and BclI polymorphism with fetal cells in maternal blood / 대한산부인과학회잡지

OBJECTIVE: We used nucleated erythrocytes in maternal blood for prenatal determination of the fetal gender as the preliminary experiment for the screening of fetal genetic status and the BclI DNA polymorphism in an attempt to clarify the origin of erythrocytes in maternal blood. METHODS: In seventeen pregnant women, venous blood was withdrawn and the nucleated erythrocytes were recovered by magnetic activated cell sorting (MACS) and immunostaining. After isolation of nucleated erythrocytes by micromanipulation, we performed nested PCR for amelogenin gene to identify the fetal gender and performed BclI DNA polymorphism to clarify the origin of erythrocytes. RESULTS: We could amplify the minute DNA in a single cell by primer extension preamplification and nested PCR of amelogenin gene in 94 (48.7%) cells and could identify the fetal gender by 58.8%. BclI DNA polymorphism revealed that the several cells, which did not reveal the specific band of Y chromosome in spite of the pregnancy of male fetuses, must be the cells from mother. CONCLUSION: Through this study, we could conclude that several nucleated erythrocytes in maternal blood circulation can originate from mother, therefore we must develop the new method to identify the nucleated erythrocyte of fetal origin. Considering that we must apply for the larger number of pregnant women to screen, the procedure was multi-step and complex. Therefore, we must design the new scheme to utilize the nucleated erythrocytes in maternal blood.