Systematic micro-array based identification of placental mRNA in maternal plasma: towards non-invasive prenatal gene expression profiling.

T he discovery of fetal DNA in the plasma of pregnant women has led to the development of promising approaches for non-invasive prenatal diagnosis. However, as fetal and maternal DNA species co-exist in maternal plasma, these DNA based diagnostic applications depend largely on the use of genetic markers that would allow the discrimination between fetal and maternal DNA (for example, the Y chromosome of a male fetus), and thus, a particular genetic marker could generally only be used in a proportion of pregnancies. This situation has prompted a quest by many laboratories to develop fetal nucleic acid markers that are independent of sex or polymorphism. The detection of fetal RNA in maternal plasma offers new possibilities for non-invasive prenatal investigation. This field has recently taken on new momentum as robust methods for plasma RNA extraction have been developed and circulating RNA has been shown to be surprisingly stable, possibly through an association with particulate matter. Furthermore, recent studies have identified the placenta as a significant source of such circulating fetal RNA. Hence, placental expressed mRNA transcripts, such as those coding for human placental lactogen (hPL), human chorionic gonadotropin b subunit (bhCG), and corticotropin releasing hormone (CRH), have been shown to be detectable in maternal plasma. Quantitative assays have been developed for the measurement of these circulating mRNA transcripts. The pregnancy specificity of these mRNA species has been demonstrated by their rapid clearance from maternal plasma after delivery. 11 Thus, the detection in maternal plasma of mRNA transcripts derived from the plasma offers new avenues for the development of fetal specific nucleic acid markers that are independent of sex and polymorphism for the noninvasive prenatal assessment of all pregnancies. The clinical value of such an approach has been demonstrated by the observation of elevated CRH mRNA concentrations in the plasma of pre-eclamptic pregnant women, when compared with normal pregnancies matched for gestational age. Despite the promise shown by circulating placental mRNA in maternal plasma, several important questions remain to be explored. First, it is important to demonstrate empirically that circulating placental mRNA in maternal plasma would allow non-invasive placental gene expression profiling. Second, the previous choice of hPL, bhCG, and CRH was essentially the result of the ‘‘candidate transcript’’ selection, based on the biological characteristics of a particular transcript. To streamline the development of further plasma RNA markers, a high throughput, generic approach is required. In this study, we systematically addressed each of these two important questions. We first investigated the feasibility of an approach based on an oligonucleotide microarray (Affymetrix) for the efficient development of new placental specific mRNA markers that could be detected in maternal plasma. We further sought to provide direct empirical evidence that maternal plasma RNA analysis did indeed allow the performance of non-invasive prenatal gene expression profiling.