In this context, we have recently extensively examined three different modes of STBM preparation: mechanical dissection of fresh placental vinous tissues; in vitro cultures of vinous explants; and perfusion of single placental cotyledons (15).
All three preparations lead to the production of STBM as confirmed by the presence of the syncytiotrophoblast-specific protein placental alkaline phosphatase, physiologic activity on human endothelial cell cultures, and their morphology, as seen by scanning electron microscopy (15).
Intrigued by the seemingly parallel increased release of STBM and circulatory fetal nucleic acids in preeclampsia (2, 7, 14, 16, 17) and its potential relationship to the placental distress associated with the disorder, we examined whether these two events may be more intimately associated.
In our study, after the receipt of informed consent and Institutional Review Board approval, we prepared, by the three previously described methods (15)), STBM from placentas from normal full-term pregnancies in which healthy males were delivered.
STBM from these three preparations were harvested by a three-step centrifugation procedure at 4[degrees]C: 10008 for 10 min, 10 000g for 10 min, and 70 000g for 90 min.
We examined the presence of fetal DNA and RNA in these STBM. The amount of fetal DNA was measured by a TagMan[R] real-time PCR assay for a Y-chromosome-specific sequence (SRY) (17), whereas the presence of fetal mRNA was quantified by a similar quantitative reverse transcription-PCR (RT-PCR) assay for the corticotropin-releasing hormone (CRH) gene, which is known to be expressed in the placenta (18).
The protein content in each STBM preparation was quantified with the advanced protein assay reagent (Cytoskeleton).
Absolute concentrations of CRH mRNA and SRY DNA were expressed as copies/mg of STBM.
Our analysis showed that all STBM preparations contained both fetal DNA and mRNA, although the concentrations of each of these fetal analytes differed in the three preparations (Table 1).
Although we took great care to harvest as many of the STBM as possible by the use of high-speed ultracentrifugation, we were still able to detect considerable amounts of fetal DNA in the STBM-free supernatant of vinous explant preparations.