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Technical Briefs |
1 Department of Histology, Medical Embryology, Obstetrics and Gynecology, University of Bologna, Bologna, Italy;
2 Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China;
aaddress correspondence to this author at: Department of Histology, Medical Embryology, Obstetrics and Gynecology, University of Bologna, Policlinico S. Orsola Malpighi, Bologna, Italy; fax 39-051-2094110, e-mail antonio.farina{at}unibo.it
Higher concentrations of circulating nucleic acids (DNA and RNA) in maternal plasma/serum have been reported in pregnancies complicated by preeclampsia (PE) compared with controls matched for gestational age (1)(2)(3)(4)(5). Fetal and total DNA concentrations were also demonstrated to be directly correlated to both gestational age and to the severity of PE (6). Again, both hypertension and proteinuria (the two main symptoms for PE classification) are independently associated with an increase in circulating fetal DNA (6). Most studies, however, have had to rely on Y-chromosome loci, which can be used as fetus-specific markers for male fetuses. For this reason, the identification in maternal plasma of fetal-gender-independent markers, such as mRNA transcripts from genes expressed in fetal placenta, represented an important development in the field (4). The concentrations of mRNA for corticotropin-releasing hormone (CRH) in maternal plasma are significantly higher in PE than in controls (5), but no demonstration of relationships with gestational age and/or severity of PE have been reported.
The aim of this study was to evaluate and quantify the relationships of the concentration of circulating CRH mRNA in maternal plasma with both gestational age and the severity of PE.
We conducted a retrospective study in which the control group was 17 women with uncomplicated pregnancies (35% males) during the third trimester (median, 259 days since last menstrual period; minimum–maximum, 189–281 days). Controls were matched with 17 pregnancies (53% males) affected by PE at the time of blood collection (median, 256 days since last menstrual period; minimum–maximum, 181–279 days). PE was defined as gestational hypertension (systolic pressure >140 mmHg or diastolic blood pressure >90 mmHg on at least two occasions after 20 weeks of gestation) with proteinuria (>0.3 g/day). Severe PE was defined as severe gestational hypertension (systolic pressure >160 mmHg or diastolic pressure 
110 mmHg on at least two occasions after 20 weeks of gestation) with severe proteinuria (>5 g/day). Seven cases had mild PE, and six cases had severe PE. The PE status of one case was not documented. All women presented either at the University of Bologna (Bologna, Italy) or The Chinese University of Hong Kong, Prince of Wales Hospital (Hong Kong, China). All study participants provided written informed consent for the use of their biological specimens for research purposes. The ethics committees of both participating centers approved this protocol.
We mixed 1.6 mL of plasma with 2 mL of Trizol (Invitrogen) and 0.4 mL of chloroform (7). The mixture was centrifuged at 12 000g for 15 min at 4 °C, and the aqueous layer was transferred to new tubes. One volume of 700 mL/L ethanol was added to one volume of the aqueous layer. The mixture was then applied to an RNeasy minicolumn (Qiagen) and processed according to the manufacturer’s recommendations. Potential contaminating DNA was digested by use of a RNase-free DNase preparation (Qiagen) as described by the manufacturer. Total RNA was eluted in 30 µL of RNase-free water and stored at –80 °C.
One-step real-time quantitative reverse transcription-PCR was performed with the EZ rTth RNA PCR reagent set (Applied Biosystems) as described previously (5). In brief, 5 µL of extracted plasma was added to 20 µL of reaction mixture, and real-time reverse transcription quantitative PCR for the CRH gene was performed in a PE Applied Biosystems 7700 Sequence detector. Each sample was analyzed in duplicate, and the corresponding calibration curve was run in parallel. Several negative water controls were also included in the analysis. The thermal profile used for the CRH gene was as follows: the reaction was initiated at 50 °C for 2 min, followed by 60 °C for 30 min. After denaturation at 95 °C for 5 min, 40 cycles of PCR were carried out at 94 °C for 20 s and 58 °C for 1 min.
The absolute concentrations of CRH mRNA were expressed as copies/mL of plasma based on the following calculation:
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Sample analysis was performed without knowledge of the gestational age and disease status (affected or not affected). Median CRH mRNA concentrations as a function of increasing gestational age (expressed in days) were calculated by weighted log-linear regression. In addition, PE (PE absent/present) was added to the regression as a "dummy" variable. Thus, this variable could assume a value of either 0 (absent) or 1 (present). All mRNA values in both cases and controls were expressed as multiples of the median (MoM). The MoM values were illustrated in a box-and-whisker plot, and the comparisons across generated groups was performed with nonparametric statistics.
The unadjusted median mRNA concentrations for CRH in controls and in the PE cases were 100 (0–568) copies/mL and 962 (284–5896) copies/mL, respectively. The CRH mRNA concentration directly correlated with gestational age (P = 0.045; t = 2.096) and to PE (P <0.001; t = 6.482; Table 1
and Fig. 1A
). The mean (SD) MoM values were 1.00 (1.48) and 9.35 (11.18) for controls and PE cases, respectively (P <0.001). For cases affected by mild PE, the mean MoM was 8.30 (3.68); for cases affected by severe PE the mean MoM was 10.38 (14.24). The difference was not significant (Fig. 1B
). The median (minimum–maximum) gestational ages were 256 (209–274) and 256 (181–279) days for those pregnancies affected by mild and severe PE, respectively. Because of the small sample size, no definitive conclusions can be reached about the estimated MoM values in mild and severe PE, but it is worth mentioning that the net difference for severe PE when compared with mild PE was + 2 MoM.
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A correlation between placental hormones and their respective maternal plasma mRNAs has been described previously (4). Such a result is important because the abnormal hormonal profiles detected in numerous pathologic conditions would allow a preliminary evaluation of plasma mRNA products having a potential discriminant ability (especially for monitoring and screening) for a specific disease. Placental CRH plays a major role controlling those mechanisms for the maintenance of pregnancy. Plasma CRH is significantly higher in women with preterm deliveries and significantly lower in women who delivered post term, when compared with those who delivered at term (8). Such a mechanism, which begins at the first trimester, is a placental clock that triggers the onset of labor. CRH participates in the control of vascular tone in the human placenta, and it has been found in higher than expected concentrations in those conditions associated with vascular damage as well as PE (9), a disease that affects 
3–5% of pregnancies (10).
The pathogenesis of PE, although poorly understood, has been associated with apoptotic change of the villous trophoblasts (11), possibly secondary to inadequate oxygenation of blood within the intervillous spaces (10). The increases in circulating fetal DNA and RNA observed in PE (2) are likely to be epiphenomena of the apoptosis of such placental tissues, although 5-fold (6) and 10-fold (5) increases, respectively, were shown for fetal DNA and CRH mRNA even after adjustment for possible confounding factors (6). Such increases in CRH mRNA concentrations, if also demonstrated for women who will eventually develop PE but are asymptomatic at the time of blood collection, would make CRH mRNA a promising tool for possible use in a screening program. It must be emphasized that in women who will eventually develop PE, a 2.5-fold increase in circulating fetal DNA has been observed (12). Furthermore, for those women who screen positive for anomalies of the uterine arteries based on Doppler waveform (a current method for PE and fetal growth restriction screening), circulating fetal nucleic acid quantification could be used as an adjunct for better differentiation of those women who will develop the complications (13). CRH mRNA, as described here, is an attractive candidate because of its gender- and polymorphism-independent nature.
Acknowledgments
This work was supported by an Earmarked Research Grant (CUHK4474/03M) from the Research Grants Council of the Hong Kong Special Administrative Region, China, and by Fondazione CARISBO Progetto Triennale-Molecular Genetics of Fetal DNA, and by Fondi ex-60 A.F., Italy.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Purwosunu, A. Sekizawa, K. Koide, A. Farina, N. Wibowo, G. H. Wiknjosastro, S. Okazaki, H. Chiba, and T. Okai Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia Clin. Chem., March 1, 2007; 53(3): 399 - 404. [Abstract] [Full Text] [PDF]
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 S Okazaki, A Sekizawa, Y Purwosunu, M Iwasaki, A Farina, and T Okai Measurement of mRNA of trophoblast-specific genes in cellular and plasma components of maternal blood. J. Med. Genet., September 1, 2006; 43(9): e47 - e47. [Abstract] [Full Text] [PDF]
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R. W.K. Chiu, W.-b. Lui, M.-c. Cheung, N. Kumta, A. Farina, I. Banzola, S. Grotti, N. Rizzo, C. J. Haines, and Y.M. D. Lo Time Profile of Appearance and Disappearance of Circulating Placenta-Derived mRNA in Maternal Plasma Clin. Chem., February 1, 2006; 52(2): 313 - 316. [Abstract] [Full Text] [PDF]
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X. Y. Zhong, S. Gebhardt, R. Hillermann, K. C. Tofa, W. Holzgreve, and S. Hahn Parallel Assessment of Circulatory Fetal DNA and Corticotropin-Releasing Hormone mRNA in Early- and Late-Onset Preeclampsia Clin. Chem., September 1, 2005; 51(9): 1730 - 1733. [Full Text] [PDF]
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H. Masuzaki, K. Miura, K. Yamasaki, S. Miura, K.-i. Yoshiura, S. Yoshimura, D. Nakayama, C. K. Mapendano, N. Niikawa, and T. Ishimaru Clinical Applications of Plasma Circulating mRNA Analysis in Cases of Gestational Trophoblastic Disease Clin. Chem., July 1, 2005; 51(7): 1261 - 1263. [Full Text] [PDF]
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H. Masuzaki, K. Miura, K.-i. Yoshiura, K. Yamasaki, S. Miura, S. Yoshimura, D. Nakayama, C. K. Mapendano, N. Niikawa, and T. Ishimaru Placental mRNA in Maternal Plasma and Its Clinical Application to the Evaluation of Placental Status in a Pregnant Woman with Placenta Previa-Percreta Clin. Chem., May 1, 2005; 51(5): 923 - 925. [Full Text] [PDF]
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) and PE cases (X; A), and CRH mRNA MoM values in controls and women with mild or severe PE (B).