ECAUSE OF THE INHERENT DIF-ficulties with research on human fetuses, the analysis of fetal gene expression has in large part been limited to the examination of tissue from human abortuses and the assessment of animal models for genes and developmental pathways that are conserved across species. Fetal monitoring in vivo is limited to noninvasive methods such as the measurement of uterine size or anatomic evaluation by fetal sonography. In addition, genetic analysis can be performed on amniotic fluid components, including amniocytes, which typically require time-consuming expansion in vitro before use, and cellfree proteins in the amniotic fluid, such as ␣-fetoprotein, which can serve as biomarkers for genetic anomalies. The cellfree component of the amniotic fluid is discarded after these analyses and is therefore available for research and future clinical applications.Cell-free fetal DNA in the serum and plasma of pregnant women was first described by Lo et al 1 in 1997 after others demonstrated the presence of circulating tumor-specific DNA sequences in cancer patients. [2][3][4] After it was shown that cell-free fetal DNA is also present in the urine of pregnant women, 5 we hypothesized that amniotic fluid, as a reservoir of fetal urine, would contain fetal DNA. In a preliminary study, we demonstrated that much larger quantities of fetal DNA are present in amniotic fluid than in maternal serum (100-to 200-fold difference). 6

Metaphase karyotype analysis of fetal cells obtained by amniocentesis or chorionic villus sampling is the current standard for prenatal cytogenetic diagnosis, particularly for the detection of trisomy 21. We previously demonstrated that large quantities of cell-free fetal DNA (cffDNA) are easily extracted from amniotic fluid (AF). In this study, we explored potential clinical applications of AF cffDNA by testing its ability to hybridize to DNA microarrays for comparative genomic hybridization (CGH) analysis. cffDNA isolated from 11 male fetuses showed significantly increased hybridization signals on SRY and decreased signals on X-chromosome markers, compared with female reference DNA. cffDNA isolated from six female fetuses showed the reverse when compared with male reference DNA. cffDNA from three fetuses with trisomy 21 had increased hybridization signals on the majority of the chromosome 21 markers, and cffDNA from a fetus with monosomy X (Turner syndrome) had decreased hybridization signals on most X-chromosome markers, compared with euploid female reference DNA. These results indicate that cffDNA extracted from AF can be analyzed using CGH microarrays to correctly identify fetal sex and aneuploidy. This technology facilitates rapid screening of samples for whole-chromosome changes and may augment standard karyotyping techniques by providing additional molecular information.

Our laboratory continues to be actively involved in the development of new biomarkers for prenatal diagnosis using maternal blood and amniotic fluid. We have also developed a mouse model that demonstrates that cell-free fetal (cff) DNA is detectable in the pregnant maternal mouse. In human maternal plasma and serum we have analyzed factors that are important in the clinical interpretation of cff DNA levels. Maternal race, parity, and type of conception (natural or assisted) do not affect cff DNA levels, but maternal weight does. We have also analyzed the relationship between placental volume, using a three-dimensionsal ultrasound examination, and cff DNA levels. Surprisingly, there is no association between these values. Finally, we are using specific disease models (such as congenital diaphragmatic hernia and twin-to-twin transfusion) to understand the effects of gestational age and specific pathology on fetal gene expression by analyzing cell-free mRNA levels in maternal plasma. In the amniotic fluid we have focused on improvements in recovery of cff DNA and mRNA. By optimizing recovery we have made some interesting observations about differences in fetal DNA between blood and amniotic fluid. In addition, we have successfully hybridized cff DNA in amniotic fluid to DNA microarrays, permitting assessment of fetal molecular karyotype. We also have preliminary data on fetal gene expression in amniotic fluid. Finally, we remain actively involved in promoting noninvasive prenatal testing in the United States, such as encouraging the use of fetal DNA for fetal rhesus D assessment. On the other hand, we are cautious and concerned about the accuracy of "at-home" kits for fetal gender detection.