Establishment of A Non-invasive Prenatal Diagnosis Method for Chromosomal Abnormalities

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We verified that fetal hematopoietic progenitor cells circulate with a significant frequency in peripheral blood of women in pregnancy as well as puerperium period, as follows.1) The objects were 15 women who were delivered of male infants at term. Three to 9 ml of their peripheral blood was drawn within 1 hour, at 4 days, and at 1 month post-delivery. Mononuclear cells separated from these blood samples by Ficoll-hypaque centrifugation were cultured in methylcellulose medium containing PHA-LCM and erythropoietin. After 2 to 3 weeks of culture, colonies formed in medium were counted and individually collected into hypo-osmotic solution in PCR tubes. DNAs were extracted by boiling, and DNA fragments in the SRY gene on Y chromosome and the ZP3 gene on chromosome 7 were simultaneously amplified by nested PCR method. Amplified products were electrophoresed on agarose gels and visualized by ethydiumbromide staining. We could distinguish the colonies derived from fetal hematopoietic progenit … More ors by the presence of both the SRY and ZP3 bands.Mononucleated cells separated from 1 ml of peripheral blood taken on delivery day (n=13) formed 42.7*19.6 (mean *1 SD) colonies in all. Further PCR amplification revealed that the number of fetus-derived colonies exhibiting the male band pattern was 4.8*2.4 per 1 ml of maternal blood. 7.5*2.9 and 1.1*1.1 of fetal colonies were also detected in the women at 4 days (n=2) and I month (n=5) after their deliveries, respectively.2) The same colony analysis was applied to 17 pregnant women having male infants. Their peripheral blood was drawn from 14 to 35 weeks of gestation. The frequency of fetus-drived colonies was 3.7*2.3%, which was converted to be 4.1*3.1 (range 0-10.0) per 1 ml of maternal blood. There was a significant correlation between fetal colony numbers per 1 ml of maternal blood and gestational age (R=0.58, p=0.015). The analysis of cases in the first trimester is under way. The above results indicated the possibility that we can collect fetal cells exhibiting 100 % of purity and ability of prolifiration from maternal blood, It is effortless to perform subsequent cytogenetic analysis using these fetal cells. Therefore, it was suggested that we could establish noninvasive mass-screening method for chromosome abnormalities by generalizing and facilitating our analysis system. Less