Detection of fetal DNA and RNA in placenta-derived syncytiotrophoblast microparticles generated in vitro.
Pregnancy is associated with the release of microparticles by the syncytiotrophoblast membrane into the maternal circulation (7). These particles, frequently termed STBM, are released by turnover of the syncytiotrophoblast monolayer covering the entire vinous tree (8-11). This process of normal physiologic syncytiotrophoblast turnover involves the release of apoptotic material into the maternal circulation by the extrusion of syncytial knots and the associated release of STBM (8-11). The amount of material that is released by apoptotic shedding of syncytial knots (and STBM) is several grams per day (9), and the circulating concentrations are increased significantly in preeclampsia (11).
STBM particles have been suggested to evoke the mild maternal inflammatory response accompanying normal pregnancies (12), and increased release has been proposed to play a role in the etiology of preeclampsia by triggering maternal endothelial cell damage (13, 14).
As these particles are difficult to detect and prepare from maternal blood samples, use is frequently made of in vitro-prepared particles to study their physiologic activity (13). 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. For this reason, we examined whether fetal nucleic acids are physically associated with STBM.
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. In brief, vinous explants were cultured in a 1:1 mixture of DMEM and Ham's F-12 medium (Gibco Invitrogen Life Technologies) supplemented with 10 g/L antimycotics and antibiotics (Gibco Invitrogen Life Technologies), 100 mL/L fetal calf serum, 25 kIU/L heparin (Roche Diagnostics), 50 kIU/L aprotinin (Fluka Chemicals), and 2 mmol/L Mg504 for 72 h at 37[degrees]C in 5% C[O.sub.2], after which the culture supernatant was collected and stored at -70[degrees]C. Mechanically dissected STBM were prepared by washing vinous tissue three times in phosphate-buffered saline (PBS) containing 100 mmol/L Ca[Cl.sub.2], after which the tissue was manually dissected and rinsed overnight at 4[degrees]C in 100 mL of 0.15 mol/L NaCl supplemented with 10 g/L antimycotics and antibiotics. After rinsing, the tissues were discarded, and the supernatant was collected and stored at -70[degrees]C. For the collection of STBM from placental perfusion, the intervillous space (maternal compartment) of a single cotyledon was perfused with an in vitro system, using a medium composed of NCTC-135 tissue culture medium diluted with Earle's buffer (1:1) with added glucose (1.33 g/L), dextran 40 (10 g/L), 40 g/L bovine serum albumin, heparin (2.5 kIU/L), and clamoxyl (250 mg/L). The perfusates from the intervillous space were collected and stored at -70[degrees]C.
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. The final pellet, containing the STBM, was washed once with PBS, resuspended in 1 mL of sterile PBS containing 50 g/L sucrose, and stored at -70[degrees]C until use.
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). DNA was extracted from STBM by use of the High Pure PCR Template Preparation Kit (Roche Diagnostics). Total RNA was isolated using High Pure RNA Isolation Kit (Roche Diagnostics) and eluted in 50 [micro]L of elution buffer. cDNA was reverse-transcribed from 500 ng of total RNA by use of a commercial reverse transcription system (Promega).
Real-time quantitative PCR and real-time quantitative RT-PCR were used for all DNA and mRNA quantifications as described previously (17,19, 20). The real-time PCR and real-time RT-PCR reactions were set up according to the manufacturer's instructions (Applied Biosystems) in a reaction volume of 25 [micro]L. Each sample was analyzed in duplicate, and the corresponding calibration curve was run in parallel with each analysis. 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). In this regard, the highest concentration of fetal DNA was detected in STBM prepared by in vitro vinous explant cultures (Fig. 1A), whereas the highest concentration of fetal CRH RNA was present in STBM obtained by perfusion of a placental cotyledon (Fig. 1B).
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. The amounts of fetal DNA in the supernatants cleared by ultracentrifugation were approximately fourfold higher than those in the matching STBM preparations. Provided that these results can be extrapolated to the release of fetal DNA into maternal plasma, then it is possible that the major proportion of circulatory fetal DNA may exist in a completely particle-free form. On the other hand, very little CRH mRNA was detected in the STBM-free vinous explant supernatants (~10% of that present in the STBM preparation). Again, provided that the observations we have made with in vitro-generated STBM correspond to the in vivo situation, then it is possible that fetal mRNA species may be largely associated with membrane particles, as has been reported previously (6). It is also likely, that these few mRNA species present in the cleared culture supernatants are associated with very small microparticles that are not effectively harvested by high-speed ultracentrifugation.
[FIGURE 1 OMITTED]
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 presence of fetal DNA and mRNA species in all three STBM preparations, particularly in those obtained by perfusion of the maternal compartment of the placenta under near-physiologic conditions, implies that cell-free fetal nucleic acids may similarly be associated with STBM in vivo. This facet, however, needs to be confirmed by the analysis of STBM isolated from maternal blood samples, currently a technically demanding undertaking.
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). In normal pregnancy, the shedding of placental particles occurs continuously as part of the self-renewal of the syncytiotrophoblast monolayer, a process that involves apoptosis of the aged nuclei and fusion of cytotrophoblast cells (9). In preeclampsia, this process is altered in that syncytiotrophoblast apoptosis rates are dramatically increased, which has been suggested to contribute to the increased release of STBM, possibly by apo-necrotic pathways (21).
Therefore, provided that circulatory fetal nucleic acids are indeed associated with STBM in vivo, then it is possible that the analysis of the fetal DNA and RNA content of STBM in the maternal circulation in normal and pathologic pregnancies may yield new insights into the underlying mechanisms leading to their release by the syncytiotrophoblast. Furthermore, if this proviso concerning the presence of fetal nucleic acids with STBM in vivo is true, then it may also provide a new strategy for the enrichment of these fetal analytes from maternal blood samples.
We thank Drs. Corinne Rusterholz and Bernhard Zimmermann for helpful discussions. We would like to thank the staff of Women's Hospitals in Aachen, Basel, and Bern for invaluable help in collecting placentas.
Previously published online at DOI: 10.1373/clinchem.2004.040196
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Anurag Kumar Gupta,  Wolfgang Holzgreve,  Berthold Huppertz,  Antoine Malek,  Henning Schneider,  and Sinuhe Hahn  * ( Laboratory for Prenatal Medicine, University Women s Hospital/Department of Research, University of Basel, Basel, Switzerland;  Department of Anatomy, University Hospital, RWTH, Aachen, Germany;  University Women s Hospital, Inselspital, Bern, Switzerland; * address correspondence to this author at: Laboratory for Prenatal Medicine, University Women s Hospital/Department of Research, Spitalstrasse 21, CH4031 Basel, Switzerland; fax 41-61-265-9399, e-mail firstname.lastname@example.org)
Table 1. Concentrations of fetal DNA and CRH mRNA in STBM prepared by villous explant culture, mechanical dissection, and placental perfusion. (a) Copies/mg of STBM STBM preparation method DNA (SRY) mRNA (CRH) Villous explant culture Median 94 530.5 4986 Range 9444-158 135 1232-10 500 Mechanical dissection Median 2593 121 144.5 Range 307-7745 17 868-331 607 Placental perfusion Median 620 222 352.5 Range 268-2975 98 190-275 981 (a) Six placentas were used for each STBM preparation. Fetal DNA (SRY locus) and mRNA (CRH) concentrations were determined by real-time PCR and real-time RT-PCR, respectively.
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|Title Annotation:||Technical Briefs|
|Author:||Gupta, Anurag Kumar; Holzgreve, Wolfgang; Huppertz, Berthold; Malek, Antoine; Schneider, Henning; Ha|
|Date:||Nov 1, 2004|
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