Shiho Okazaki

Prediction of preeclampsia by analysis of cell-free messenger RNA in maternal plasma

OBJECTIVE The purpose of this study was to predict the occurrence of preeclampsia in a series of patients at gestational week 15-20 weeks, with the use of a panel of messenger RNA markers. STUDY DESIGN Data from 62 patients with preeclampsia who were asymptomatic at the time of blood testing and 310 control subjects were analyzed. Multivariable analysis was performed with discriminant analysis. RESULTS Univariable analysis identified vascular endothelial growth factor receptor 1 as the marker with the highest detection rate; placenta-specific 1 with the lowest. Mean estimated score for preeclampsia was 9.4 for control subjects and 72.5 for subjects who experienced preeclampsia. A receiver operating characteristic curve that was obtained with the estimated score for preeclampsia as a test variable yielded a detection rate of 84% (95% CI, 71.8-91.5) at a 5% false-positive rate with an area under the curve of 0.927 (P < .001). Again, detection rate and score for each patient for classification as preeclamptic correlated with severity. CONCLUSION A panel of messenger RNA is able to detect subjects who will experience preeclampsia.

Measurement of mRNA of trophoblast-specific genes in cellular and plasma components of maternal blood

Background: Placental mRNA in maternal plasma is suitable for quantitative analysis regardless of fetal gender and genetic polymorphism status. Methods: We obtained 155 blood samples from pregnant women to compare human placental lactogen (hPL) and β-subunit of human chorionic gonadotropin (βhCG) mRNA and protein levels between the cellular and plasma components of maternal blood. To assess clearance of hPL mRNA expression, we obtained blood samples from nine women immediately before and after delivery by caesarean section. mRNA was extracted from the cellular and plasma components of all samples, and hPL and βhCG mRNA expression was analysed by reverse transcription-PCR assay. Results: The concentration of βhCG mRNA in the cellular component positively correlated with the plasma concentration of βhCG protein and βhCG mRNA (p = 0.001 for both). The concentration of hPL protein in the plasma correlated with the hPL mRNA concentration of the cellular component (p<0.05). For both hPL and βhCG, the mRNA concentration of the cellular component was greater than that of the plasma component (22.9-fold higher for hPL and 4.3-fold higher for βhCG). The half life of hPL mRNA clearance was significantly longer for the cellular fraction (mean half life = 203.8 min, range 150–3465 min) than for the plasma fraction (mean half life = 32.2 min, range 15–385 min) (p = 0.008). Conclusion: The present findings indicate that the concentration of hPL and βhCG mRNA is significantly higher in the cellular component of maternal blood samples than in the plasma component. Cellular mRNA in maternal blood is useful for non-invasive evaluation of placental function.

Cellular mRNA expressions of anti-oxidant factors in the blood of preeclamptic women

To assess the alterations of mRNA expressions associated with oxidative stress in the cellular component of blood from pregnant women with pre‐eclampsia.

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood.

Prenatal diagnosis of aneuploidy and single‐gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non‐invasive prenatal diagnostic procedures. One potential non‐invasive approach involves analysis of cell‐free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD‐negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non‐invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin‐based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non‐invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non‐invasive prenatal diagnosis.

Placenta-derived, cellular messenger RNA expression in the maternal blood of preeclamptic women.

OBJECTIVE: To perform gene expression profiling and real-time quantitative reverse-transcription polymerase chain reaction (PCR) analysis to identify biomarkers of preeclampsia in cellular messenger RNA (mRNA) from maternal blood. METHODS: We performed a microarray analysis with five maternal blood samples from women with preeclampsia and five matched control subjects. Up-regulated gene expression was further analyzed through reverse-transcription PCR analysis with 28 consecutive blood samples from women affected with preeclampsia and 29 controls. RESULTS: Both pregnancy-specific &bgr;1 glycoprotein and trophoblast glycoprotein were selected based on microarray analysis. Reverse-transcription PCR analysis detected significantly increased mRNA concentrations among women in the preeclampsia group. When stratified according to mild or severe preeclampsia, 19.2-fold and 41.8-fold increases in pregnancy-specific &bgr;1 glycoprotein and 8.3-fold and 10.6-fold increases in trophoblast glycoprotein were observed, respectively. Among women with hemolysis, elevated liver enzymes, and low platelet count syndrome, 51.6-fold and 13.1-fold increases in pregnancy-specific &bgr;1 glycoprotein and trophoblast glycoprotein were observed, respectively. In the preeclampsia group, pregnancy-specific &bgr;1 glycoprotein correlated with severity of proteinuria (P<.001) and systolic blood pressure (P=.01). CONCLUSION: The mRNA expression of pregnancy-specific &bgr;1 glycoprotein and trophoblast glycoprotein is up-regulated in cells circulating within blood from women with preeclampsia, and pregnancy-specific &bgr;1 glycoprotein expression is positively correlated with the clinical severity of preeclampsia. LEVEL OF EVIDENCE: II

Evaluation of physiological alterations of the placenta through analysis of cell-free messenger ribonucleic acid concentrations of angiogenic factors

OBJECTIVE Placental messenger ribonucleic acid (mRNA) has been shown to circulate in maternal plasma. We investigated concentrations of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), and endoglin in subjects with preeclampsia, compared with normal pregnancies. STUDY DESIGN Peripheral blood samples were obtained from preeclampsia (n = 43) and control subjects (n = 41). Plasma ribonucleic acid was subjected to analysis by reverse transcription-polymerase chain reaction assay to examine the mRNA distribution among women with preeclampsia and control subjects during weeks 35-41 of gestation. RESULTS Concentrations of VEGF, VEGFR-1, and endoglin mRNA of women with preeclampsia were significantly increased. The mRNA values were observed to correlate directly with the severity of hypertension and proteinuria. VEGFR-1 mRNA was markedly elevated in women with preeclampsia and hemolysis, elevated liver enzymes, and low platelet syndrome. CONCLUSION The mRNA concentrations of VEGF, VEGFR-1, and endoglin were observed to correlate directly with the severity of preeclampsia.

Clinical Potential for Noninvasive Prenatal Diagnosis Through Detection of Fetal Cells in Maternal Blood

Fetal cells circulate in maternal blood and are considered a suitable means by which to detect fetal genetic and chromosomal abnormalities. This approach has the advantage of being noninvasive. Since the early 1990s, nucleated erythrocytes (NRBCs) have been considered good target cells for a number of techniques, including fluorescence-activated cell sorting and magnetic cell sorting, using antibodies such as anti-transferrin receptor and anti-gamma-hemoglobin antibodies, followed by analysis with fluorescence in situ hybridization or polymerase chain reaction. In the late 1990s, the National Institute of Child Health and Human Development Fetal Cell Isolation Study assessed the reliability of noninvasive prenatal diagnosis of fetal aneuploidy using NRBCs isolated from maternal circulation. This study revealed the limitations of NRBC separation using antibodies specific for NRBC antigens. A more recent study has demonstrated the efficiency and success of recovery of NRBCs using a galactose-specific lectin, based on the observation that erythroid precursor cells have a large quantity of galactose molecules on their cell surface. Thus, recent advances in this field enhance the feasibility of this diagnostic method. This review article focuses on various methods of detection of fetal cells within the maternal circulation, as well as the status of previous and current studies and the prospective view for noninvasive prenatal diagnosis using fetal cells from the maternal circulation.