Impaired early-phase suppression of glucagon secretion after glucose load is associated with insulin requirement during pregnancy in gestational diabetes.

AIMS/INTRODUCTION: The role of glucagon abnormality has recently been reported in type 2 diabetes; however, its role in gestational diabetes mellitus (GDM) is still unknown. The glucose intolerance in GDM is heterogeneous, and not all patients require insulin treatment during pregnancy. Here, we investigated whether glucagon abnormality is associated with the requirement for insulin treatment during pregnancy. MATERIALS AND METHODS: A total of 49 pregnant women diagnosed with GDM were enrolled. They underwent a 75-g oral glucose tolerance test during mid-gestation, and we measured their plasma glucagon levels (by a new sandwich enzyme-linked immunosorbent assay) at fasting (0 min), and at 30, 60 and 120 min after glucose load in addition to the levels of plasma glucose and serum insulin. All participants underwent another oral glucose tolerance test at postpartum. RESULTS: Of the 49 patients, 15 required insulin treatment (Insulin group) and 34 were treated with diet therapy alone until delivery (Diet group). The early-phase glucagon secretion after glucose load, as determined by the changes in glucagon from the baseline to 30 min, was paradoxically augmented during mid-gestation in the Insulin group, but not in the Diet group. The impaired glucagon suppression during mid-gestation in the Insulin group was not associated with insulin secretory/sensitivity indexes studied, and was ameliorated postpartum, although the plasma glucose levels remained higher in the Insulin group versus the Diet group. CONCLUSIONS: Impaired early-phase suppression of glucagon could be associated with the requirement for insulin treatment during pregnancy in patients with GDM.

Malignant transformation from mature cystic teratoma of the ovary.

We present a case of malignant change of an ovarian mature cystic teratoma. Our patient was a 48-year-old woman and she visited a primary care doctor presenting with abdominal pain. At her first visit, her pelvic tumor measured 70 × 50 mm by ultrasonography. She was diagnosed as rupture of the malignant tumor occurred secondary to mature cystic teratoma and she took the surgery (abdominal total hysterectomy, bilateral oophorectomy and partial omentectomy). Pathologic diagnosis was squamous cell carcinoma (SCC) occurred secondary to mature cystic teratoma. Treatment with paclitaxel/carboplatin (TC chemotherapy) and gemcitabine hydrochloride/carboplatin (GC chemotherapy) after operation was not effective, and the refractory ileus resulting from rapid progression of the disease continued. She was died of disease progression 7 months after the diagnosis of ovarian cancer. We discuss about the clinical characteristics of malignant transformation of mature cystic teratoma and considered about the treatment of the ovarian SCC.

Reference values for circulating pregnancy-associated microRNAs in maternal plasma and their clinical usefulness in uncomplicated pregnancy and hypertensive disorder of pregnancy.

AIM: The aim of this study was to establish the reference values for circulating pregnancy-associated placental microRNAs in maternal plasma and clarify their clinical significance in patients with hypertensive disorder of pregnancy (HDP). METHODS: Blood samples were collected from 145 women with uncomplicated pregnancies (24, 26, 31 and 32 women at 12, 23, 30 and 36 weeks of gestation, respectively, and 32 women 1 day after delivery). Plasma concentrations of pregnancy-associated placental microRNAs (miR-515-3p, miR-517a, miR-517c and miR-518b) were measured by quantitative real-time reverse-transcription polymerase chain reaction. Reference values for each microRNA were determined by the line of best fit and 95% prediction interval and are expressed as logarithmic transformation. To clarify the clinical significance of these reference values, we measured the plasma concentrations of pregnancy-associated microRNAs in a different population comprising 33 pregnant women with HDP and 44 women with uncomplicated pregnancies. RESULTS: Reference values for circulating pregnancy-associated placental microRNAs on chromosome 19 miRNA clusters showed an increasing tendency as pregnancy progressed and decreased significantly 1 day after delivery (P < 0.05). The sensitivity and specificity of each reference value were 57.6% and 93.2% for miR-515-3p, 63.6% and 75.0% for miR-517a, 75.8% and 79.5% for miR-517c and 63.6% and 75.0% for miR-518b, respectively. The positive and negative predictive values of each reference value were 86.4% and 74.5% for miR-515-3p, 65.6% and 73.3% for miR-517a, 73.5% and 81.4% for miR-517c and 65.6% and 73.3% for miR-518b, respectively. CONCLUSION: Establishing the reference values for circulating pregnancy-associated placental microRNAs in maternal plasma could be useful for the evaluation of HDP.

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.

A significant association between rs8067378 at 17q12 and invasive cervical cancer originally identified by a genome-wide association study in Han Chinese is replicated in a Japanese population.

In this study, associations between invasive cervical cancer and four cervical cancer susceptibility loci (rs13117307 at 4q12, rs8067378 at 17q12, and rs4282438 and rs9277952 at 6p21.32) in the Han Chinese population were investigated in a Japanese population. Human leukocyte antigen (HLA)-DPB1 alleles were also investigated for their association with cervical cancer risk in the Japanese population. After receiving written informed consent, 214 unrelated Japanese women with invasive cervical cancer and 288 cancer-free Japanese women were recruited, and DNA samples were obtained (study protocol approved by Institutional Review Board of Nagasaki University). Of the four single-nucleotide polymorphisms, rs8067378 showed a significant association with invasive cervical cancer (P=0.0071). Under a recessive model, the minor allele G of rs8067378 contributed to the risk of invasive cervical cancer (odds ratio=2.92, 95% confidence interval=1.40-6.36; P=0.0021). No association was detected between HLA-DPB1 alleles and cervical cancer risk in the Japanese population. In conclusion, we show for the first time, to the best of our knowledge, that an association between increased risk of invasive cervical cancer and rs8067378 in the Han Chinese population is replicated in a Japanese population. In addition, Japanese women with the GG genotype of rs8067378 are a candidate high-risk group for invasive cervical carcinoma.

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

AIM: To clarify the association between circulating chromosome 19 miRNA cluster (C19MC) microRNAs in maternal plasma and severe pre-eclampsia. METHOD: Maternal blood samples (7 mL) at 27-34 weeks of gestation were obtained from 20 pregnant women with severe pre-eclampsia (sPE group) and from 20 uncomplicated pregnant women (NP group). Twenty cases of severe pre-eclampsia were classified into late onset (sPELO group; n = 14) and early onset (sPEEO group; n = 6). Plasma concentration of C19MC microRNAs (miR-518b, -1323, -516b, -516a-5p, -525-5p, -515-5p, -520 h, -520a-5p, -519d and -526b) was measured on quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: The circulating levels of all 10 C19MC microRNAs in maternal plasma were significantly increased in the sPE group compared with the NP group. Plasma concentration of all 10 C19MC microRNAs tested was significantly increased in the sPEEO group compared with the NP group, while plasma concentration of nine miRNAs, except for miR-519d, was significantly increased in the sPELO group compared with the NP group. Of the 10 C19MC microRNAs measured, plasma concentration of eight miRNAs, except for miR-518b and miR-519d, was significantly increased in the sPEEO group compared with the sPELO group. CONCLUSIONS: Increased levels of C19MC microRNAs in maternal plasma are a characteristic phenomenon of established severe pre-eclampsia, and it has been shown for the first time that the upregulation of C19MC miRNAs occurred as a consequence of, not in advance of, the onset of pre-eclampsia.

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.

Pregnancy-associated microRNAs in plasma as potential molecular markers of ectopic pregnancy.

OBJECTIVE: To investigate cell-free pregnancy-associated microRNAs as molecular markers for the diagnosis of ectopic pregnancy. DESIGN: Laboratory study using human plasma samples. SETTING: Research unit in a university hospital. PATIENT(S): Plasma samples from 18 women with ectopic pregnancies (EP group), 12 women with spontaneous abortion (SA group), and 26 normal women with singleton pregnancies (NP group). INTERVENTION(S): Total RNAs containing small RNA molecules extracted from 1.2 mL of plasma. MAIN OUTCOME MEASURE(S): Plasma concentrations of cell-free microRNAs measured by quantitative real-time reverse-transcriptase polymerase chain reaction. RESULT(S): Plasma concentrations of cell-free pregnancy-associated microRNAs (miR-323-3p, miR-515-3p, miR-517a, miR-517c, and miR-518b) and serum concentration of human chorionic gonadotropin (hCG) were confirmed to have statistically significantly different plasma or serum concentrations in women with EP, SA, or NP. There was no statistically significant difference in the plasma concentrations of cell-free miR-21 between the three groups. By correlation coefficient analysis, no relationship was detected between serum hCG levels and plasma cell-free miR-517c, miR-515-3p, miR-517a, miR-518b, miR-323-3p, or miR-21 levels. Plasma concentrations of cell-free miR-517a could distinguish EP/SA from NP, yielding an area under the curve of 0.9654 (95% confidence interval, 0.9172-1.0). Plasma concentrations of cell-free miR-323-3p could distinguish EP from SA, yielding an area under the curve of 0.7454 (95% confidence interval, 0.5558-0.9349). CONCLUSION(S): Cell-free pregnancy-associated microRNAs have potential as molecular markers of ectopic pregnancy.

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

OBJECTIVE: 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. METHOD: In the non-labor group (32 women), cesarean section (C/S) was performed before the beginning of labor. In the labor group (32 women), C/S was performed after the beginning of labor. Plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs (miR-515-3p, miR-517a, miR-517c, and miR-518b) were measured by real-time quantitative PCR. Each miRNA concentration was compared between the non-labor and labor groups. RESULTS: Before C/S, plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs in the labor group were significantly higher than those in the non-labor group (P = 0.001 for 515-3p, P = 0.002 for 517a, P = 0.001 for 517c, and P = 0.003 for 518b). Twenty-four hours after delivery, plasma concentrations of cell-free, pregnancy-associated, placenta-specific miRNAs in the labor group were significantly higher than those in the non-labor group (P = 0.002 for 515-3p, P = 0.017 for 517a, P = 0.043 for 517c, and P = 0.009 for 518b). CONCLUSION: The presence of labor affects cell-free, pregnancy-associated, placenta-specific miRNA levels in maternal plasma. Labor also affects postpartum clearance of these miRNAs 24 h after delivery.

Single human papillomavirus 16 or 52 infection and later cytological findings in Japanese women with NILM or ASC-US.

The relationship between oncogenic human papillomavirus (HPV) infection and later cytological findings in the uterine cervix is unknown in women who were negative for intraepithelial lesion and malignancy (NILM) or atypical squamous cells of undetermined significance (ASC-US). This was investigated in this study in a Japanese population to determine the clinical utility of oncogenic (HPV) genotyping. The relative risk of progressive cytological findings 2 years after identification of oncogenic HPV infection was higher than in cases of non-oncogenic HPV infection (relative risk 3.827; 95% confidence interval (CI): 1.282-11.422), as well as in cases of negative HPV infection (relative risk 2.124; 95% CI: 1.451-3.110). Moreover, the relative risk of progression of cytological findings 2 years later in cases of HPV-16 infection was higher than in cases of HPV-52 infection (relative risk 2.094; 95% CI: 1.005-3.935). Therefore, the initial HPV-DNA genotype may be a potential predictive marker of later progression of cytological findings in the uterine cervix in cases of NILM or ASC-US.

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.

Predominantly placenta-expressed mRNAs in maternal plasma as predictive markers for twin-twin transfusion syndrome.

OBJECTIVE: This study aimed to identify a set of predominantly placental (PP) mRNAs, which are associated with later-developing twin-to-twin transfusion syndrome (TTTS). METHOD: First, out of 50 PP mRNAs we previously reported, we select target mRNAs that are ordinarily detectable in maternal plasma. Plasma concentrations of these PP mRNAs were measured in monochorionic diamniotic twin (MCDA-T) pregnancies complicated by TTTS later (n = 11) and in uncomplicated MCDA-T pregnancies (n = 17). Finally, the diagnostic values of the PP mRNAs in plasma were evaluated. RESULTS: From 50 PP mRNAs, nine [human placental lactogen (hPL); pregnancy-specific glycoproteins 2 (PSG2); human pregnancy-specific glycoproteins 3 (PSG3); syncytin; syncytin 2; retinoic acid-induced 14; A disintegrin and metalloproteinase domain-containing protein 12 (ADAM12); chorionic glycoprotein hormones, alpha polypeptide; and chorionic glycoprotein hormones, and beta polypeptide] were selected as target mRNAs. Changes in six PP mRNAs [increased hPL, PSG2, and PSG3 and decreased syncytin, syncytin2, and ADAM12] in maternal plasma were detected in MCDA-T pregnant women who subsequently developed TTTS. Finally, mRNA signatures gave elevated AUCs (hPL/PSG2: 0.8717; hPL/PSG3: 0.8449; hPL/ADAM12: 0.8396) compared with single hPL mRNA. CONCLUSION: Quantitative aberration of plural cell-free PP mRNAs in maternal plasma precedes the appearance of clinically apparent TTTS. This suggests that pathophysiological changes in the placenta are associated with morbid conditions of TTTS.

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 ((ΔΔ)CT) 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.

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

OBJECTIVE: The aim of this study was to characterize placenta-specific microRNAs in fetal growth restriction (FGR) pregnancy. METHOD: Placenta-specific miRNAs were identified by next-generation sequencing analysis. Subsequently, quantitative real-time reverse-transcription polymerase chain reaction was used to identify FGR placenta-specific miRNAs whose level of expression was significantly decreased in FGR placenta (n = 45) compared with uncomplicated placenta (n = 50). FGR pregnancy-associated, placenta-specific microRNAs were identified in maternal plasma after delivery at significantly decreased concentrations, and their circulating levels in maternal plasma was compared between FGR pregnancies (n = 10) and uncomplicated pregnancies (n = 10). RESULTS: Out of the ten placenta-specific microRNAs that we identified, seven placenta-specific microRNAs (hsa-miR-518b, hsa-miR-1323, hsa-miR-516b, hsa-miR-515-5p, hsa-miR-520h, hsa-miR-519d, and hsa-miR-526b) from the chromosome 19 microRNA cluster were identified as FGR placenta-specific microRNAs. Four FGR placenta-specific microRNAs (hsa-miR-518b, hsa-miR-1323, hsa-miR-520h, and hsa-miR-519d) were confirmed as FGR pregnancy-associated, placenta-specific miRNAs, but their circulating levels in maternal plasma showed no significant differences between FGR pregnancy and uncomplicated pregnancy. CONCLUSION: Our data suggest that reduced expression in placenta of certain FGR placenta-specific miRNAs is associated with FGR and that the discrepancy between expression in FGR placenta and their circulating levels in maternal plasma will be crucial to understanding how placenta-specific microRNAs are released into the maternal circulation.

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.

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.