Yuri Hasegawa

Characterization of placenta-specific microRNAs in fetal growth restriction pregnancy.

The aim of this study was to characterize placenta‐specific microRNAs in fetal growth restriction (FGR) pregnancy.

Copy number variation of the antimicrobial-gene, defensin beta 4, is associated with susceptibility to cervical cancer

The aim of this study was to investigate association between copy number variation of the defensin beta 4 gene (DEFB4) and susceptibility to cervical cancer in a population at high risk of persistent oncogenic human papillomavirus (HPV) infection. The study subjects comprised 204 women with cervical cancer, a population having a high risk of persistent oncogenic HPV infection (cervical cancer group), and 200 healthy women from the general population (control group). Copy number variation of DEFB4 in each test sample was determined by relative quantitation using the comparative CT (ΔΔCT) method. Differences between the two groups were evaluated. The median DEFB4 copy number in the cervical cancer group was four and in the control group was five (P=2.77e–4, t-test). The odds ratio of cervical cancer in individuals with four DEFB4 copies or less was higher (odds ratio 2.02; 95% confidence interval odds ratio 1.36–3.02), compared with that in individuals with five or more copies (odds ratio 0.49; 95% confidence interval odds ratio 0.33–0.74). We found copy number variation of DEFB4 was a host genetic factor conferring susceptibility to cervical cancer. A lower DEFB4 copy number was associated with susceptibility to cervical cancer.

Circulating chromosome 19 miRNA cluster microRNAs in pregnant women with severe pre-eclampsia

To clarify the association between circulating chromosome 19 miRNA cluster (C19MC) microRNAs in maternal plasma and severe pre‐eclampsia.

Circulating levels of maternal plasma cell-free pregnancy-associated placenta-specific microRNAs are associated with placental weight

The aim of this study was to investigate the relationship between plasma concentration of cell-free pregnancy-associated placenta-specific microRNAs and clinical variables (placental weight, maternal body mass index, and neonatal birth weight). Circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miR-515-3p, miR-517a, miR-517c and miR-518b) in maternal plasma were measured by quantitative real-time RT-PCR in sixty-two pregnant women. The levels of cell-free pregnancy-associated placenta-specific microRNAs were significantly associated with placental weight, but not associated with body mass index or birth weight. Therefore, the measurement of cell-free pregnancy-associated placenta-specific miRNAs levels in maternal plasma may reflect the pregnancy status related to placenta volume.

Clinical applications of analysis of plasma circulating complete hydatidiform mole pregnancy-associated miRNAs in gestational trophoblastic neoplasia: A preliminary investigation

The aim of this study was to investigate the clinical application of plasma complete hydatidiform mole pregnancy-associated microRNAs (CHM-miRNAs: hsa-miR-520b, hsa-miR-520f and hsa-miR-520c-3p). We measured plasma CHM-miRNA concentration by real-time quantitative reverse transcriptase polymerase chain reaction in two cases of CHM resulting in gestational trophoblastic neoplasia later. As progress of treatments in both cases, the plasma concentrations of CHM-miRNAs showed a decreasing tendency similar to the pattern for serum hCG concentration, but exhibited a transient increasing tendency after each course of chemotherapy, suggesting that the plasma CHM-miRNAs could be an additional follow-up marker for malignant changes of CHM.

Increased Levels of Cell-Free miR-517a and Decreased Levels of Cell-Free miR-518b in Maternal Plasma Samples From Placenta Previa Pregnancies at 32 Weeks of Gestation.

Objective: The aim of this study was to clarify the association between placenta previa and circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miRNAs) in maternal plasma. Method: Twenty singleton pregnancies with placenta previa (placenta previa group) and 26 uncomplicated pregnancies (control group) were recruited. Blood sampling was performed at 32 weeks of gestation, and cesarean delivery in all cases of placenta previa was performed at a mean gestational age of 37 weeks. The maternal plasma concentrations of cell-free pregnancy-associated placenta-specific miRNAs (miR-517a and miR-518b) were measured by absolute quantitative real-time reverse transcription-polymerase chain reaction. Results: Plasma concentrations of cell-free miR-517a were significantly higher in the placenta previa group than that in the control group (P = .011), while the plasma concentration of cell-free miR-518b was significantly lower in the placenta previa group than that in the control group (P = .004). Plasma concentrations of cell-free miR-517a in placenta previa were significantly higher in placenta previa with alert bleeding later group than those in placenta previa without alert bleeding group or control group (P = .030 or .047, respectively) and correlated with the volume of hemorrhage at delivery (R and P value: .512 and .025). Conclusion: Plasma concentrations of cell-free miR-517a and miR-518b at 32 weeks of gestation were altered in pregnant women with placenta previa, and the circulating level of cell-free miR-517a in placenta previa may be a predictive marker for the risks of alert bleeding later and massive hemorrhage at delivery.

Effect of labor on plasma concentrations and postpartum clearance of cell‐free, pregnancy‐associated, placenta‐specific microRNAs

This study aimed to investigate the effect of labor on plasma concentrations of cell‐free, pregnancy‐associated, placenta‐specific microRNAs (miRNAs) before and after delivery.

Circulating Levels of Pregnancy-Associated, Placenta-Specific microRNAs in Pregnant Women With Placental Abruption

The aim of this study was to clarify the association between circulating pregnancy-associated, placenta-specific microRNAs (miRNAs) in maternal plasma and placental abruption. All samples were obtained after receiving written informed consent, and the study protocol was approved by the institutional review board. Maternal blood samples (7 mL) were obtained at 25 to 40 weeks of gestation from 15 cases of placental abruption (placental abruption group) and from 24 cases of uncomplicated pregnancies (uncomplicated pregnancy group). The plasma concentrations of pregnancy-associated, placenta-specific miRNAs (miR-515-3p, -517a, -517c, and -518b) were measured by quantitative real-time reverse transcription-polymerase chain reaction. There were no significant differences in clinical characteristics between the 2 groups. The median concentration of plasma cell-free miR-517c in the placental abruption group was 21 672.2 copies/mL, whereas that in the uncomplicated pregnancy group was 13 452.0 copies/mL (Mann-Whitney U test, P = .047). Receiver operating characteristic curve analysis revealed that plasma cell-free miR-517c levels discriminated placental abruption from uncomplicated pregnancy with an area under the curve of 0.692. When a cutoff negative/positive value of 15 669.6 copies/mL was selected, the sensitivity and specificity were 73.3% and 62.5%, respectively. In addition, the positive and negative predictive values were 55.0% and 78.9%, respectively. Plasma cell-free miR-517a and miR-517c levels in the large abruption (degree of abruption ≥50% of placenta) group were significantly higher than in the small abruption (<50%) group (P = .03 for both miRNAs). In conclusion, the circulating level of cell-free miR-517c in maternal plasma was increased as a consequence of placental abruption and may be a potential biomedical marker for placental abruption.

Predominantly placenta‐expressed mRNAs in maternal plasma as predictive markers for twin–twin transfusion syndrome

This study aimed to identify a set of predominantly placental (PP) mRNAs, which are associated with later‐developing twin‐to‐twin transfusion syndrome (TTTS).

Circulating levels of maternal plasma cell-free miR-21 are associated with maternal body mass index and neonatal birth weight.

Circulating cell-free microRNA (miRNA) levels in maternal plasma are involved in, or associatedwith, pregnancy-associated disorders, such as preeclampsia, fetal growth restriction, and preterm delivery. Therefore, they have a strong potential for use as sensitive and specific biomarkers. Recently, aberrant expression of miR-21 was reported to be associated with fetal hypoxia, fetal growth restriction, and macrosomia, suggesting that plasma miR-21 levels may be a candidate biomarker for fetal status. MiRNA-21 is expressed in maternal fetal and placenta tissues. Therefore, maternal miR-21, fetal miR-21, and placental miR-21 are circulating in maternal plasma. However, which clinical variables (maternal, fetal, or placental factors) affect the circulating levels of total miRNAs in maternal plasma remains unknown. In this study, to clarify the factors affecting the circulating levels of total miRNAs in maternal plasma, we measured plasma cell-free miR-21 levels and investigated their association with maternal body mass index (BMI) as a maternal factor, neonatal birth weight (BW) and fetal gender as a fetal factor, and placental weight (PW) as a placental factor. All samples were obtained after receiving written informed consent, and the Institutional Review Board of Nagasaki University approved the study protocol. Women who smoked; those who had multiple gestations, placenta previa, invasive placentation, preterm labor, preeclampsia, or infection; and those with the presence of fetal anomalies or aneuploidy or fetal growth restriction were excluded. Finally, we obtained maternal blood from 52 uncomplicated pregnant women with a female singleton fetus and 30 uncomplicated pregnant women with a male singleton fetus at 37–38weeks’ gestation to exclude the possibility of preterm labor. Gestational age was assessed using ultrasonography. All of the women had nothing to eat or drink for 8 h prior to blood collection. Maternal blood samples (7mL) were collected within 3h of elective cesarean section. At the time of blood sampling, they had no signs of labor. Preparation and extraction of total RNA containing small RNA molecules were performed as described previously. All specific primers and TaqMan probe of miR-21 were purchased fromTaqManMicroRNA Assays (Applied Biosystems, Warrington, UK). Absolute qRT-PCR of miRNAs in plasma samples was performed as described previously. For each miRNA assay, we prepared a calibration curve by tenfold serial dilution of single-stranded cDNA oligonucleotides corresponding to each miRNA sequence from 1.0 × 10 to 1.0 × 10 copies/mL. Each sample and each calibration dilution were analyzed in triplicate. Each assay could detect down to 300 RNA copies/mL [9,10]. Every batch of amplifications included three water blanks as negative controls for each of the reverse transcription and PCR steps. All of the data were collected and analyzed using the LightCycler® 480 real-time PCR system (Roche, Pleasanton, CA, USA). Pearson product–moment correlation coefficients between plasma cell-free miR-21 levels and clinical variables (BMI, BW, and PW) were analyzed with SPSS version 19 (IBM Japan, Tokyo, Japan). Significance was defined asP< 0.05. To eliminate spurious correlations between circulating cell-free miR-21 levels and BMI, BW, or PW, partial correction coefficient analysis was performed. In 52 pregnant women with a female fetus, the median (minimum–maximum) cell-freemiR-21 level inmaternal plasma was 3.23 × 10 copies/mL (6.41 × 10–4.98 × 10 copies/mL). In 30 pregnant women with a male fetus, the median cell-free miR-21 level in maternal plasma was 1.73 × 10 copies/mL (1.14 × 10–4.75 × 10 copies/mL). There was no significant difference in plasma cell-free miR-21 levels between pregnant women bearingmale and female fetuses (Mann–WhitneyU-test, P> 0.05). In 52 pregnant womenwith a female fetus, themedian (minimum–maximum) BMI, BW, and PWwere 20.3 kg/m (14.7– 37.0 kg/m), 2849 g (2274–4040 g), and 595 g (400–870 g), respectively. No relationship was detected between BMI and PW (r= 0.130 and P = 0.358). However, significant associations