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 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.
In our study, STBM prepared by placental perfusion may be regarded as being the closest representatives of those generated under normal physiologic conditions in that here STBM are collected directly from the intervillous space, the site where they would typically enter the maternal circulation.
The difference we observed in fetal DNA and mRNA content in the three STBM preparations may be attributable to the manner in which these particles are generated, in that those obtained by perfusion or in vitro culture are generated predominantly by apoptotic cell turnover, in contrast to STBM isolated by mechanical disruption, in which release of STBM may involve necrotic pathways (15).
In this context it is worth noting that the release of STBM differs in normal pregnancy compared with preeclampsia (9, 21).