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The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia.

The discovery of fetal DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis (1). This technology has been used for the noninvasive determination of many disorders, including fetal rhesus D status (2), myotonic dystrophy (3), achondroplasia Achondroplasia Definition

Achondroplasia is the most common cause of dwarfism, or significantly abnormal short stature.
Description
 (4), and [beta]-thalassemia (5). In addition, quantitative abnormalities of fetal DNA in maternal plasma/ serum have also been reported in preeclampsia preeclampsia /pre·eclamp·sia/ (pre?e-klamp´se-ah) a toxemia of late pregnancy, characterized by hypertension, proteinuria, and edema.

pre·e·clamp·si·a
n.
 (6-8), fetal trisomy trisomy /tri·so·my/ (tri´so-me) the presence of an additional (third) chromosome of one type in an otherwise diploid cell (2n + 1). See also entries under syndrome. triso´mic

tri·so·my
n.
 21 (9,10), and hyperemesis gravidarum (11). For the latter investigations, most groups have used Y-chromosomal markers, which are present only in male fetuses, as a fetal-specific marker. This approach has limited the application of this technology to the 50% of pregnant women who are carrying male fetuses.

The demonstration of the presence of fetal RNA RNA: see nucleic acid.
RNA
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 in maternal plasma provides an approach for detecting fetal nucleic acids in maternal plasma that is independent of the gender and genetic polymorphisms present in a fetus (12,13). Recently, methods to enhance our ability to use plasma RNA as a potential molecular diagnostic tool have been developed. Our group has developed a protocol for the quantitative analysis of plasma RNA (14), demonstrated the unexpected stability of circulating RNA (15), and shown that the placenta is an important source of fetal RNA in maternal plasma (16).

In this study, we tested the hypothesis that the ability to measure plasma RNA would provide us with a gender-and polymorphism-independent marker for monitoring pregnancy-associated disorders. Because of its importance, we chose preeclampsia as our model system (17). For the mRNA target, we chose the mRNA of the corticotropin-releasing hormone (CRH CRH corticotropin-releasing hormone.

CRH
abbr.
corticotropin-releasing hormone



CRH

corticotropin releasing hormone.
) [3] locus, which is known to be expressed in the placenta (18) and is released into the maternal circulation (19, 20). Its exact role in human pregnancy is not yet fully understood. A significantly higher CRH peptide content has been reported in placentas of preeclamptic pregnancies (21, 22). Abnormally increased maternal plasma CRH has also been reported by various groups in pregnancies complicated by preeclampsia (23-25). In this study, we investigated whether maternal plasma CRH mRNA might also be increased in preeclamptic pregnancies; if this hypothesis is confirmed, then plasma CRH mRNA might represent a new noninvasive marker for preeclampsia.

Patients and Methods

PATIENTS

Peripheral blood samples were collected with informed consent and Research Ethics Committee approval from pregnant women who attended the Department of Obstetrics and Gynaecology at the Prince of Wales Hospital
This article is about a hospital in Hong Kong. For the hospital in Sydney, Australia, see Prince of Wales Hospital, Sydney. There also exists another Prince of Wales Hospital in the United Kingdom.
, Hong Kong. Women were recruited between April and October 2002.

In the first part of this study, blood samples were obtained from 10 healthy pregnant women during the third trimester of gestation. In the second part of the project, pregnant women with uncomplicated pregnancies were recruited just before elective cesarean section. Peripheral blood samples were taken from these women just before delivery and at 2 h postdelivery. In the third part of the study, two patient groups were studied: (a) 12 preeclamptic women, and (b) 10 control pregnancies. The median gestational ages of the preeclamptic and control groups were 37 weeks (interquartile range, 36.6-38.9 weeks) and 38 weeks (interquartile range, 37.3-38.3 weeks), respectively. Preeclampsia was defined on the basis of a sustained increase in diastolic blood pressure Diastolic blood pressure
Blood pressure when the heart is resting between beats.

Mentioned in: Hypertension
 >110 mmHg on one occasion or >90 mmHg on two or more occasions at least 4 h apart, with the presence of significant proteinuria proteinuria /pro·tein·uria/ (-ur´e-ah) an excess of serum proteins in the urine, as in renal disease or after strenuous exercise.proteinu´ric

pro·tein·u·ri·a
n.
1.
 in women with no history of hypertension. Significant proteinuria was defined as proteinuria >0.3 g/day or [greater than or equal to] 2+ on dipstick dipstick /dip·stick/ (dip´stik) a strip of cellulose chemically impregnated to render it sensitive to protein, glucose, or other substances in the urine.  testing in two clean-catch midstream urine specimens collected at least 4 h apart. The control group included pregnant women with no preexisting medical diseases or antenatal complications.

PROCESSING OF BLOOD SAMPLES

Plasma harvesting was performed immediately on arrival at the laboratory (within 1 h of venesection). Blood samples were processed based on a previously reported protocol (14). In brief, 10-mL blood samples were collected in EDTA-containing tubes and centrifuged at 1600g for 10 min at 4[degrees]C. Plasma was then carefully transferred into plain polypropylene tubes. The plasma samples were recentrifuged at 16 000g for 10 min at 4[degrees]C, and the supernatants were collected in fresh polypropylene tubes.

RNA EXTRACTION

We mixed 1.6 mL of plasma harvested after the centrifugation steps described above with 2 mL of Trizol LS reagent (Invitrogen) and 0.4 mL of chloroform (14). The mixture was centrifuged at 11 900g for 15 min at 4[degrees]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 mini column (Qiagen) and processed according to the manufacturer's recommendations. Total RNA was eluted with 30 [micro]L of RNase-free water and stored at -80[degrees]C. DNase treatment (RNase-Free DNase Set; Qiagen) was carried out to remove any contaminating DNA.

REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 

One-step real-time quantitative reverse transcription-PCR (RT-PCR) was used for all mRNA quantifications (14). The CRH primer sequences were 5'-000TCCCATCTCCCTGGAT-3' (forward) and 5'-TGTGAGCTTGCTGTGCTAACTG-3' (reverse), and the dual-labeled fluorescent probe was 5'-(FAM FAM 5-FU, adriamycin/doxorubicin, mitomycin C Oncology A chemotherapeutic regimen used with varying degrees of failure for advanced gastric CA. See Stomach cancer. )TCCT TCCT Tactical Communications Control Terminal 000GGAAGTCTTGGAAATGGC(TAMRA TAMRA Technical And Miscellaneous Revenue Act of 1988
TAMRA Tetramethyl-6-Carboxyrhodamine (dye) 
)-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. Calibration curves for CRH mRNA quantification were prepared by assaying serial dilutions of HPLC-purified single-stranded synthetic DNA oligonucleotides (Genset Oligos) specifying a 89-bp CRH amplicon (GenBank Accession No. NM 000756), with concentrations ranging from 1 x [10.sup.7] copies to 1 x [10.sup.1] copies. Absolute concentrations of CRH mRNA were expressed as copies/mL of plasma. The sequence of the synthetic DNA oligonucleotides for CRH calibrations was 5'-GAGCCTCCCATCTCCCTGGATCTCACCTTCCACCTCCTCCGGGAAGTCTTGGAAATGGCCAGGGCCGAGCAG TTAGCACAGCAAGCTCACAGCA-3' A calibration curve for glyceraldehyde-3-phosphate dehydrogenase (GAPDH GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase (also seen as G3PDH) ) quantification was prepared as described previously, with results expressed in pg/mL of plasma (14).

The RT-PCR reactions were set up according to the manufacturer's instructions (EZ rTth RNA PCR reagent set; Applied Biosystems) in a reaction volume of 25 [micro]L. The fluorescent probes (Genset Oligos) were used at concentrations of 100 nM. The PCR primers (Genset Oligos) were used at a concentration of 200 nM for both the CRH and GAPDH systems. We used 5 [micro]L of extracted plasma RNA for amplification. Each sample was analyzed in duplicate, and the corresponding calibration curve was run in parallel with each analysis. Samples were also tested to ensure that they were negative for DNA by substituting the rTth polymerase with the AmpliTaq Gold enzyme (Applied Biosystems). No amplification was observed for this control analysis, indicating the specificity of the assays for the respective mRNAs. Multiple negative water blanks were also included in every analysis. All analyses were performed by two of the authors (E.K.O.N. and N.B.Y.T.).

The thermal profile used for the CRH and GAPDH analyses was as follows: the reaction was initiated at 50[degrees]C for 2 min for the included uracil uracil (yr`əsĭl), organic base of the pyrimidine family. It was isolated from herring sperm and also produced in a laboratory in 1900–1901.  N-glycosylase to act, followed by reverse transcription at 60[degrees]C for 30 min. After a 5-min denaturation denaturation, term used to describe the loss of native, higher-order structure of protein molecules in solution. Most globular proteins exhibit complicated three-dimensional folding described as secondary, tertiary, and quarternary structures.  at 95[degrees]C, 40 cycles of PCR were carried out with denaturation at 94[degrees]C for 20 s and 1 min of annealing/ extension at 58 and 62[degrees]C for CRH and GAPDH, respectively.

STATISTICAL ANALYSIS

Statistical analysis was performed with the Sigma Stat 2.03 software (SPSS A statistical package from SPSS, Inc., Chicago (www.spss.com) that runs on PCs, most mainframes and minis and is used extensively in marketing research. It provides over 50 statistical processes, including regression analysis, correlation and analysis of variance. ). The Mann-Whitney test was used for the comparison of maternal plasma CRH mRNA concentrations between preeclamptic and control groups. The Wilcoxon test was used for the comparison of maternal plasma GAPDH mRNA concentrations before and at 2 h postdelivery.

Results

ESTABLISHMENT OF REAL-TIME QUANTITATIVE RT-PCR

To determine the quantitative performance of the CRH RT-PCR assay, we used this system to amplify serially diluted calibrators that were synthetic DNA oligonucleotides based on the CRH sequence. Previous data have shown that such single-stranded oligonucleotides reliably mimic the products of the reverse transcription step and produce calibration curves that are identical to those obtained using T7-transcribed RNA (26). The calibration curve for the CRH amplification systems demonstrated a dynamic range from 2.5 x [10.sup.1] to 1 x [10.sup.6] copies and had a correlation coefficient of 0.983. The sensitivities of the amplification steps of these assays were sufficient to detect 25 copies of the CRH target. To determine the precision of the whole analytical procedure, including the RNA extraction, reverse transcription, and amplification steps, we performed 10 replicate RNA extractions from a plasma sample obtained from a healthy pregnant woman (gestational age, 38 weeks) and subjected these extracted RNA samples to RT-PCR analysis. The CV for the threshold cycle values of these replicate analyses for CRH mRNA was 2.8%. The development and performance of the real-time quantitative GAPDH RT-PCR assay was described previously (14).

DETECTABILITY OF CRH mRNA IN MATERNAL PLASMA

To test whether CRH mRNA transcripts were detectable in maternal plasma, we analyzed plasma samples from 10 pregnant women in the third trimester of pregnancy (gestational age, 37-41 weeks) by the CRH RT-PCR assay. CRH mRNA was detected in all tested samples. The median concentration of plasma CRH mRNA was 73 copies/mL (interquartile range, 51-177 copies/mL). As a positive control, GAPDH mRNA was also detectable in all of these plasma samples.

CLEARANCE OF CRH mRNA FROM MATERNAL PLASMA AFTER DELIVERY

To demonstrate that the maternal plasma CRH mRNA was derived from the fetus, we analyzed maternal plasma for CRH mRNA both before and at 2 h postdelivery. Four women who delivered by cesarean section (gestational age, 38-40 weeks) were studied. CRH mRNA was detected in 100% of predelivery maternal plasma samples, whereas CRH mRNA was not detected in any of the postdelivery samples. GAPDH mRNA was detectable in all plasma samples, thus demonstrating the quality of the samples. No systematic alternation in maternal plasma GAPDH mRNA concentration was observed (Wilcoxon test, P = 0.25). The results are shown in Fig. 1.

[FIGURE 1 OMITTED]

QUANTITATIVE ANALYSIS OF CRH mRNA IN THE PLASMA OF PREECLAMPTIC PREGNANT WOMEN

To compare the concentration of CRH mRNA in maternal plasma of preeclamptic and control pregnant women, we obtained plasma samples from 12 preeclamptic women and 10 control pregnant women with matched gestational age. The median CRH mRNA concentration in the plasma of preeclamptic women and control pregnancies were 1070 copies/mL (interquartile range, 535-1468 copies/ mL) and 102 copies/mL (interquartile range, 51-158 copies/mL), respectively (Fig. 2). The median plasma CRH mRNA concentrations were 10.5 times higher in preeclamptic than control pregnancies (Mann-Whitney test, P <0.001).

Discussion

In this report, we have demonstrated that CRH mRNA is easily detectable in the plasma of pregnant women in the third trimester of pregnancy. The fact that such mRNA is cleared within 2 h postpartum provides evidence that such mRNA is derived from the fetus. These results support our previous data demonstrating that the placenta is an important source for releasing fetal mRNA species into maternal plasma (16).

We also considered the possibility that CRH mRNA produced by the mother, rather than the placenta, might also be detectable in the plasma, but the postpartum data suggest that this is improbable. Furthermore, because CRH is produced by the hypothalamus hypothalamus (hī'pəthăl`əməs), an important supervisory center in the brain, rich in ganglia, nerve fibers, and synaptic connections. It is composed of several sections called nuclei, each of which controls a specific function. , we think that it is unlikely that large amounts of such mRNA will be released into the blood (possibly even requiring the passage of mRNA through the intact blood-brain barrier). Conversely, the relatively large surface area of the placenta would make it a much more plausible source of CRH mRNA release.

The data presented here have demonstrated that the concentration of maternal plasma CRH mRNA is increased in pregnancies complicated with preeclampsia. The median plasma CRH mRNA concentration was increased 10.5 times in preeclampsia, compared with non-preeclamptic pregnancies matched for gestational age. In comparison, our previously published results showed a fivefold increase in circulating fetal DNA in maternal plasma in preeclamptic pregnancies (6).

[FIGURE 2 OMITTED]

Our results suggest that maternal plasma CRH mRNA might be a new molecular marker for preeclampsia. This approach offers an alternative to current studies that involve the measurement of maternal plasma CRH using immunoassays. For immunoassays, the specificity of the method is critically dependent on the specificity of the antibodies used. On the other hand, at least at the present time, the mRNA approach is probably more expensive on a case-by-case basis than a well-established immunoassay system. Future studies should aim at a direct comparison of these potentially complementary approaches in the same patient cohort.

The mechanism producing the increase in such quantitative aberration in plasma RNA requires further investigation. Several theoretical possibilities exist. The first is that increased concentrations of pro-CRH mRNA have been detected in placental tissues in preeclamptic pregnancies (22), which may lead to increased liberation of such transcripts into the plasma. The second possibility is that, because plasma nucleic acids have been postulated to be related to cell death (27-29), it is possible that the increase in cell death within the placenta in preeclampsia (30) may contribute to the increased release of placenta-expressed mRNA species into maternal plasma. Concerning the third possibility, we have recently demonstrated that impaired clearance of maternal plasma fetal DNA is observed in preeclampsia (31). In theory, a similar phenomenon may also exist for plasma RNA clearance in preeclampsia. This is particularly relevant because the data in the present study demonstrate the rapid clearance of CRH mRNA after delivery (Fig. 1), which is similar to the rapid clearance of fetal DNA from maternal plasma after delivery (32).

Compared with fetal DNA measurements in maternal plasma (6-8), quantitative analysis of circulating fetal RNA, such as placenta-expressed mRNA, has the advantage of being applicable to all pregnant women irrespective of fetal gender and polymorphism status. Furthermore, numerous targets can be selected for plasma RNA analysis, including the numerous genes that are known to be expressed in the placenta. It could therefore be worthwhile to systematically investigate the detectability of such transcripts in maternal plasma. In addition, because CRH is a hormone, our data have also opened up the possibility that a similar approach can be used for the investigation of other hormonal systems, with new diagnostic and research opportunities.

This project was supported by the Innovation and Technology Fund (AF/90/99). The Chinese University of Hong Kong The motto of the university is "博文約禮" in Chinese, meaning "to broaden one's intellectual horizon and keep within the bounds of propriety".  has filed patent applications covering aspects of the technology of circulating nucleic acid analysis for prenatal diagnosis and monitoring.

Received December 11, 2002; accepted February 12, 2003.

References

(1.) Lo YMD YMD Yazoo Mississippi Delta
YMD Young Mental Drylaw (Edinburgh gang) 
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(2.) Lo YMD, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734-8.

(3.) Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2000;46:301-2.

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(5.) Chiu RWK RWK Rework
RWK Race War Kingdoms (online game) 
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KCK Kohl's Cares for Kids
KCK Kilkenny College, Kilkenny (Ireland)
KCK Key Certification Key
KCK Key Component Enciphering
KCK Key Confirmation Key
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n.
A cell or an organism characterized by aneuploidy.


Aneuploid
An abnormal number of chromosomes in a cell.
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(23.) Wolfe CD, Patel SP, Linton EA, Campbell EA, Anderson J, Dornhorst A, et al. Plasma corticotrophin-releasing factor (CRF CRF
abbr.
chronic renal failure


CRF Chronic renal failure
) in abnormal pregnancy. Br J Obstet Gynaecol 1988;95:1003-6.

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(27.) Fournie GJ, Martres F, Pourrat JP, Alary a·la·ry  
adj.
Variant of alar.

Adj. 1. alary - having or resembling wings
aliform, wing-shaped, alar

biological science, biology - the science that studies living organisms
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(29.) Hasselmann DO, Rappl G, Tilgen W, Reinhold U. Extracellular tyrosinase Tyrosinase
An enzyme in a pigment cell which helps change tyrosine to DOPA during the process of making melanin.

Mentioned in: Albinism


tyrosinase

an enzyme important in the production of melanin from tyrosine.
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(30.) Leung DN, Smith SC, To KF, Sahota DS, Baker PN. Increased placental apoptosis in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2001;184:1249-50.

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(32.) Lo YMD, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999;64:218-24.

[3] Nonstandard abbreviations: CRH, corticotropin-releasing hormone; RT-PCR, reverse transcription-PCR; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ENDERS K.O. NG, [1] TSE See Tokyo Stock Exchange.

TSE

1. See Tokyo Stock Exchange (TSE).

2. See Toronto Stock Exchange (TSE).
 N. LEUNG, [2] NANCY B.Y. TSUI, [1] TZE K. LAU, [2] NIRMAL S. PANESAR, [1] ROSSA W.K. CHIU, [1] and Y.M. DENNIS LO [1]

Departments of [1] Chemical Pathology and [2] Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR (Segmentation And Reassembly) The protocol that converts data to cells for transmission over an ATM network. It is the lower part of the ATM Adaption Layer (AAL), which is responsible for the entire operation. See AAL.

SAR - segmentation and reassembly
.

* Author for correspondence. Fax 852-2194-6171; e-mail loym@cuhk.edu.hk.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Ng, Enders K.O.; Leung, Tse N.; Tsui, Nancy B.Y.; Lau, Tze K.; Panesar, Nirmal S.; Chiu, Rossa W.K.;
Publication:Clinical Chemistry
Date:May 1, 2003
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