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Technical Briefs |
1 University Women’s Hospital/Department of Research, University Hospital Basel, Basel, Switzerland; Departments of2 Obstetrics and Gynecology and3 Genetics, University of Stellenbosch, Stellenbosch, South Africa;
aaddress correspondence to this author at: Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, University Hospital Basel, Spitalstrasse 21, CH 4031 Basel, Switzerland; fax 41-61-265-9399, e-mail shahn{at}uhbs.ch
Preeclampsia, a severe disorder of human pregnancy of unknown etiology, remains a major cause of fetal and maternal mortality (1)(2). Because of the heterogeneous nature of this disorder, it has been suggested that preeclampsia could be subclassified into two distinct forms to better understand the underlying causes (3). These two forms, termed early and late onset, are defined as the development of symptoms before or after 34 weeks of pregnancy, respectively (3). Results of several studies have indicated that the early-onset form is more severe, frequently leading to the delivery of growth-retarded premature babies, whereas the late-onset form is more evanescent and clinically of lesser importance (4).
Previous studies have indicated that cell-free fetal DNA and mRNA concentrations are increased in preeclampsia (5)(6)(7)(8). Because these studies focused on either cell-free DNA or RNA alone, we examined the concentrations of these nucleic acids simultaneously in cases with early- and late-onset preeclampsia.
Approval for this study was granted by the respective Institutional Review Boards (Basel and Stellenbosch), and export of biological material was approved by the South African Department of Health. Written informed consent was obtained in all instances. In our study, maternal blood samples were collected from 37 pregnant women with manifest preeclampsia. Preeclampsia was defined by new-onset blood pressure of at least 140/90 mmHg in 2 determinations 4 h apart or by a diastolic blood pressure >110 mmHg, as well an associated proteinuria of at least 300 mg/24 h after 20 weeks of gestation (6). Early-onset preeclampsia was defined as having symptoms before 34 weeks of pregnancy, whereas late-onset was defined as having symptoms after 34 weeks of pregnancy (3)(4). Thirty-two maternal blood samples were drawn from normotensive pregnant women who all delivered healthy babies at term. These were matched by gestational age to the preeclampsia study group at the time of blood sampling. All pregnancies were singleton.
The plasma samples were separated by centrifugation and stored frozen as described previously (9). All samples were shipped by air freight on dry ice from Cape Town to Basel for analysis. Circulatory fetal DNA was quantified by a real-time PCR assay for the SRY gene located on the Y chromosome (6) (for full details, see Table 1
in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue9/). Total circulatory DNA was quantified by a real-time PCR assay for the ubiquitous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, which is present in all genomes (6) (see Table 1
in the online Data Supplement). Because circulatory maternal DNA makes up the bulk (>95%) of the circulatory DNA in a maternal plasma sample, this assay is indicative of the amount of circulatory maternal DNA (10). The concentrations of circulatory DNA are given in genome-equivalents per milliliter (GE/mL) of maternal plasma. The presence of circulatory placentally derived fetal mRNA was assayed by a real-time reverse transcription-PCR assay for corticotropin-releasing hormone (CRH) mRNA transcripts as described previously (7)(9) (see Table 1
in the online Data Supplement). The concentrations of circulatory CRH mRNA are given in copies/mL of maternal plasma. The quality of total circulatory mRNA was assayed by a real-time reverse transcription-PCR assay for the ribosomal 18S gene according to the manufacturer’s instructions (Applied Biosystems; data not shown). Stringent anticontamination procedures were used throughout. No false-positive results were recorded.
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The data were analyzed by SPSS® for Windows. Because our analysis indicated that the data did not follow a gaussian distribution, they were examined by the Mann–Whitney U-test for nonparametric data with P <0.05 being regarded as statistically significant. The data concerning concentrations of circulatory DNA and mRNA are presented by box plots (Fig. 1
).
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The maternal characteristics and gestational age at time of sampling and delivery are listed in Table 1
, with the cases with preeclampsia stratified into early- (n = 23) and late-onset forms (n = 19). It is noteworthy that many women affected by the early-onset form of preeclampsia delivered prematurely.
Our analysis of circulatory nucleic acid (DNA and mRNA) concentrations indicated that these were significantly higher in both preeclampsia study groups than in the matched control group (Table 1
and Fig. 1
). A feature readily apparent in this analysis is that the extent of the increase of all 3 circulatory nucleic acids examined, fetal and maternal (total) DNA as well as fetal CRH mRNA, was more pronounced in the cases with early-onset preeclampsia than in those with the late-onset form of the disorder (Table 1
and Fig. 1
). Because increased release of these fetal and maternal circulatory nucleic acids in preeclampsia has been proposed to be attributable to some form of cell turnover, death, or damage (6)(11), their increased presence in cases with early-onset preeclampsia compared with those with the late-onset form suggests that the early form of preeclampsia may be associated with more cellular damage than the late form. In this context, it is worth noting that in our analysis of the 2 control groups, the pattern was reversed: the concentrations of circulatory fetal DNA and mRNA were higher in the control samples taken closer to term (>34 weeks of gestation) than in those taken at an earlier gestational age (<34 weeks of gestation). Therefore, if a correction is made for gestational age, then the difference observed between the early- and late-onset preeclampsia study groups may become even more pronounced.
From our data it is very clear that the amounts of circulatory fetal DNA and CRH mRNA in the preeclampsia study groups were significantly increased compared with the respective control groups. This is particularly true for the group with early-onset preeclampsia, where very little overlap was found to occur with the matched control group. In this manner, a cutoff value of 300 GE/mL of maternal plasma for circulatory fetal DNA and 300 copies/mL of maternal plasma for circulatory CRH mRNA could theoretically be used to distinguish between the study and control groups in our current data set. However, because we found no significant correlations between these two fetal cell-free nucleic acids under any of the conditions examined (data not shown), the efficacy of such an approach may be superior when these two markers are examined in parallel vs their single analysis because the latter may lead to an erroneous result.
Although such an approach may be appealing, it should be noted that we examined only one circulatory fetal mRNA species, CRH mRNA. It is therefore unclear what the patterns for other circulatory fetal mRNA molecules would be, particularly those for which alterations in expression during pregnancy have been noted, e.g., the ß-subunit of human chorionic gonadotropin (12). This latter aspect may be of particular importance when examining circulatory fetal mRNA concentrations in those pregnancies for which increased concentrations of circulatory fetal DNA have been noted early in pregnancy, well in advance of the onset of preeclampsia symptoms (13)(14)(15). In such cases, it is possible that the parallel quantitative assessment of circulatory fetal DNA concentrations in combination with several fetal mRNA species may assist in the improved identification of pregnant women at risk of developing preeclampsia. For this reason it will be necessary to increase our knowledge of cell-free placentally derived mRNA molecules in maternal plasma, their expression changes during pregnancy and pregnancy-related pathologies, and their relationship with circulatory fetal DNA under such conditions (16).
Acknowledgments
We thank Drs. B. Huppertz, C. Rusterholz, D. Huang, and B. Zimmermann for critical reading of the manuscript and fruitful discussions. We also thank Erika van Papendorp for help with recruitment and management of the samples. This study was funded by a grant from the Swiss National Science Foundation (3200-066913).
References
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