Shoko Miura

Identification of Pregnancy-Associated MicroRNAs in Maternal Plasma

BACKGROUND Several placental microRNAs (miRNAs) have been identified as pregnancy-associated molecules with the potential for use in estimating the condition of the placenta. Our understanding of these novel molecules is still limited, however. The aim of this study was to isolate and characterize pregnancy-associated miRNAs in maternal plasma. METHODS By microarray-based screening of 723 human miRNAs, we selected miRNAs that exhibited signal intensities >100 times higher in placental tissues than in the corresponding whole blood samples. Subsequent quantitative real-time reverse-transcription PCR revealed miRNAs produced predominantly in the placenta that showed significantly decreased concentrations in maternal plasma after delivery. These miRNAs were identified as pregnancy-associated miRNAs. RESULTS We selected 82 miRNAs produced predominantly in the placenta and identified 24 as pregnancy-associated miRNAs. The genes encoding these miRNAs included 16 that are clustered on 19q13.42 and 5 clustered on 14q32. As the pregnancy progressed into the third trimester, the plasma concentrations of cell-free chromosome 19-derived miRNAs (has-miR-515-3p, has-miR-517a, has-miR-517c, has-miR-518b, and has-miR-526b) increased significantly (P = 0.0284, 0.0069, 0.0125, 0.0284, and 0.0093, respectively, Wilcoxon signed rank test), whereas that of cell-free has-miR-323-3p on chromosome 14q32.31 showed no change (P = 0.2026). CONCLUSIONS In addition to the known pregnancy-associated miRNAs, we identified new pregnancy-associated miRNAs with our microarray-based approach. Most of the genes encoding these miRNAs were clustered on 19q13.42 or 14q32, which are critical regions for placental and embryonic development. These new pregnancy-associated miRNAs may be useful molecular markers for monitoring pregnancy-associated diseases.

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.

Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid

AbstractCell-free fetal DNA (cffDNA) in the supernatant of amniotic fluid, which is usually discarded, can be used as a sample for prenatal diagnosis. For rapid prenatal diagnosis of frequent chromosome abnormalities, for example trisomies 13, 18, and 21, and monosomy X, using cffDNA, we have developed a targeted microarray-based comparative genomic hybridization (CGH) panel on which BAC clones from chromosomes 13, 18, 21, X, and Y were spotted. Microarray-CGH analysis was performed for a total of 13 fetuses with congenital anomalies using cffDNA from their uncultured amniotic fluid. Microarray CGH with cffDNA led to successful molecular karyotyping for 12 of 13 fetuses within 5 days. Karyotypes of the 12 fetuses (one case of trisomy 13, two of trisomy 18, two of trisomy 21, one of monosomy X, and six of normal karyotype) were later confirmed by conventional chromosome analysis using cultured amniocytes. The one fetus whose molecular-karyotype was indicated as normal by microarray CGH actually had a balanced translocation, 45,XY,der(14;21)(q10;q10). The results indicated that microarray CGH with cffDNA is a useful rapid prenatal diagnostic method at late gestation for chromosome abnormalities with copy-number changes, especially when combined with conventional karyotyping of cultured amniocytes.

Identification of endometrioid endometrial carcinoma-associated microRNAs in tissue and plasma

OBJECTIVE This study aimed to identify a set of endometrioid endometrial carcinoma EEC-associated microRNAs (miRNAs) in tissue and plasma, and evaluate their clinical significance. METHODS A set of EEC-associated miRNAs in tissue and plasma was identified by next-generation sequencing (NGS), which could enable in-depth characterization of the global repertoire of miRNAs. RESULTS NGS identified 11 candidate EEC-associated miRNAs. Quantitative reverse-transcriptase PCR identified 8 EEC-associated miRNAs in tissue (upregulated: miR-499, miR-135b, miR-205, downregulated: miR-10b, miR-195, miR-30a-5p, miR-30a-3p and miR-21). Expression of hsa-miR-499 in International Federation of Gynecology and Obstetrics (FIGO) Stage IA and Grade 1 tissues was significantly lower than in others (FIGO Stage IB or more advanced, and Grade 2 or 3). By receiver operating characteristic (ROC) curve analysis, compared with single EEC-associated miRNA, two miRNA signatures (miR135b/miR195 and miR135b/miR30a-3p) could distinguish between EEC and normal endometrial tissue samples yielding a high area under the curve (AUC) of 0.9835 [95% confidence interval (CI): 0.9677-1.0], and 0.9898 (95% CI: 0.9677-1.0), respectively. As possible non-invasive markers for EEC, four EEC-associated miRNAs (increased level: miR-135b and miR-205, decreased-level: miR-30a-3p and miR-21) in plasma were identified. Circulating levels of three EEC-associated miRNAs (miR-135b, miR-205 and miR-30a-3p) in plasma were significantly decreased after hysterectomy. ROC curves analysis revealed that miR-135b and miR-205 levels in plasma yielded AUCs of 0.9722 (95% CI: 0.913-1.0) and 1.0 (95% CI: 1.0-1.0), respectively. CONCLUSION Measurement of tissue and plasma EEC-associated miRNAs may be useful for early detection, diagnostic, and follow-up tests for EEC.

Increased level of cell-free placental mRNA in a subgroup of placenta previa that needs hysterectomy.

The purpose of this study was to investigate whether cell‐free placental mRNA levels have the potential to predict a placenta previa resulting in hysterectomy.

Origin and mechanisms of formation of fetus-in-fetu: two cases with genotype and methylation analyses.

Fetus‐in‐fetu (FIF) is a condition in which a host infant has a fetus‐like mass(es) within its body. We describe here results of molecular genetic analysis in two cases of FIF. In FIF‐1, a male host had two retroperitoneal fetiform masses each with a vertebral column, and in FIF‐2, a fetiform mass with vertebral column was present in the cranial cavity of a male host. Genotyping of each case using microsatellite markers showed that the host infant and its fetus(es) inherited one copy each of parental alleles and shared identical genotypes. These findings were confirmed by single nucleotide polymorphism (SNP) analysis using Affymetrix GeneChip Human Mapping 50K Array, and supported a monozygotic twin theory of FIF. Analysis of the methylation status was done in both cases at the differentially methylated region (DMR) within the human IGF2‐H19 locus after bisulfite treatment, methylation‐specific PCR, and cloning of PCR products. Normally, only the paternal allele is methylated and the maternal allele unmethylated in DMR. However, in FIF‐1, 7 (46.7%) of 15 clones from a fetiform mass and 6 (66.7%) of 9 clones from the other mass showed an unmethylated paternal allele, while the methylation status of a host infant and its fetiform mass in FIF‐2 was the same in all clones examined with normal patterns. These data suggest that in FIF‐1, two isolated blastocysts originated from one zygote, one of the two was implanted into (or included by) the other blastocyst during the process of methylation, and such abnormal implantation may have occurred in FIF‐2 after the establishment of methylation. This is the first case of FIF showing different methylation patterns between a host infant and fetiform mass.

Clinical application of fetal sex determination using cell-free fetal DNA in pregnant carriers of X-linked genetic disorders

As the first step in prenatal diagnosis of X-linked genetic disorders, chorionic villus sampling (CVS) for fetal sex determination is generally performed at 11–13 weeks of gestation. However, as the procedure-related miscarriage rate of CVS is 0.5–1.0%, non-invasive methods such as PCR of cell-free fetal DNA (cff-DNA) in maternal plasma are preferable. Here, we determined fetal sex at 9–12 weeks of gestation using PCR of cff-DNA in three pregnant carriers of Duchenne muscular dystrophy. The fetal sex was accurately determined in all three cases, as confirmed by ultrasound and amniocentesis at 16 weeks (for the two female fetuses) and CVS at 12 weeks (for the one male fetus). This procedure could avoid unnecessary CVS in female fetuses.

Clinical outcome of infants with confined placental mosaicism and intrauterine growth restriction of unknown cause.

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<−2.0 SD) from 3,257 deliveries in a regional medical center during the past 10‐year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non‐CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non‐CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell–Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<−2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

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.