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.

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.

Initial viral load in cases of single human papillomavirus 16 or 52 persistent infection is associated with progression of later cytopathological findings in the uterine cervix.

The aim of this study was to investigate the relationship between viral load in single human papillomavirus (HPV) 16 or 52 persistent infection and the progression of later cytopathological findings in the uterine cervix. Cervical cytology and HPV genotyping tests were repeated within 3-6 months in 305 women with oncogenic HPV. Twenty-four cases of single HPV 52 persistent infection and 24 cases of single HPV 16 persistent infection were identified. Cases with later cytopathological findings showing progression were defined as the progression group, while those with no change or regression were the non-progression group. Relative HPV DNA loads were determined by quantitative real-time polymerase chain reaction and expressed relative to human albumin (ALB) DNA. Differences between the two groups were evaluated. The median relative HPV 52 DNA load was 2.211 in the progression group and 0.022 in the non-progression group (Mann-Whitney U-test, P = 0.003). The median relative HPV 16 DNA load was 4.206 in the progression group and 0.103 in the non-progression group (P = 0.001). HPV 52 and 16 DNA loads assessed by quantitative real-time methods may be useful short-term markers for identifying women at high risk for progression of cervical cytological pathology.

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.

Pre-vaccination epidemiology of human papillomavirus infections in Japanese women with abnormal cytology.

AIM: To investigate the pre-vaccination epidemiology of genital human papillomavirus (HPV) infections and genotypes in women with abnormal cytology in Nagasaki, Japan. MATERIAL AND METHODS: We performed Pap smear tests, biopsies and HPV genotype testing in Nagasaki Prefecture from August 2007 through November 2009. RESULTS: During the study period, serial samples of uterine cervical specimens were obtained from 539 subjects with abnormal cytology and/or squamous intraepithelial lesions (SIL) confirmed previously, or with clinically suspected invasive cervical cancer. In 119 HPV-positive subjects with low-grade SIL, the three most prevalent high-risk HPV genotypes were HPV52 (21.8%; 26/119), HPV16 (20.2%; 24/119) and HPV56 (17.6%; 21/119). In 199 women, 127 HPV-positive subjects with high-grade SIL and 67 HPV-positive subjects with squamous cell carcinoma (SCC), the three most prevalent high-risk HPV genotypes were HPV16 (44.3%; 86/194), HPV52 (20.6%; 40/194) and HPV58 (16.0%; 31/194). CONCLUSION: Compared with the distribution of high-risk HPV genotypes in other countries, HPV52 was a more common genotype in Nagasaki. With disease progression to SCC, the distribution of high-risk HPV56 belonging to the A6 HPV family decreased, while HPV16 and HPV52 belonging to the A9 HPV family persisted. Our data provide an important resource to address the case for vaccination against HPV genotypes other than HPV16 and HPV18 in Japan.

Epidemiology of human papillomavirus genotypes in pregnant Japanese women.

To investigate the pre-vaccination epidemiology of genital human papillomavirus (HPV) infections and genotypes in pregnant Japanese women, we performed Pap smear tests and HPV genotype testing in patients attending Nagasaki University Hospital and collaborating hospitals from August 2007 to July 2010. Serial uterine cervical specimens were obtained from 151 pregnant women. The HPV test was positive on the first visit in 54 women (35.8%; 54/151, average age 30). A total of 49 women (32.5%; 49/151) were infected by at least one high-risk HPV and 5 women were infected by only low-risk HPV. The three most prevalent high-risk HPV genotypes were HPV 52 (31.5%; 17/54), HPV 16 (29.6%; 16/51) and HPV 31 (13.0%; 7/51). The HPV infection pattern (negative, single infection and multiple infection) differed significantly according to the pregnancy trimester (χ(2)-test; P<0.01(Pearson)). Among HPV-infected pregnant Japanese women, HPV52 was the most common genotype. The second most common genotype was HPV16, and these two genotypes accounted for ∼60% of HPV-positive pregnant women. Infection with multiple HPV genotypes was observed more frequently in the first trimester of pregnancy and the pattern of infection changed significantly depending on pregnancy stage.

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.

The possibility of microarray-based analysis using cell-free placental mRNA in maternal plasma.

OBJECTIVE: The purpose of this study is to investigate a possibility of overall assessment of cell-free (CF) placental mRNAs in maternal plasma. METHODS: First, placenta-predominantly expressed transcripts were selected by the analysis of GeneChip using three sets of placental tissues and corresponding maternal blood cells. Subsequently, a custom cDNA array panel of placenta-predominantly expressed transcripts was designed and used to compare the RNA profiles of maternal plasma collected from 12 preeclamptic and 12 uncomplicated pregnancies. Scatter plots for the signal intensities of the comparative cDNA hybridization revealed either unchanged or aberrant patterns. RESULTS: We selected top 50 placenta-predominantly expressed transcripts that were > 2500 times higher in placental tissues than in corresponding whole blood samples. A custom cDNA array analysis detected the aberrant pattern in five preeclamptic women with severe hypertension but not in seven preeclamptic women with mild hypertension (P < 0.05, Fisher's direct method). The aberrant pattern of above RNA transcripts in maternal plasma was validated by quantitative real-time reverse transcription-polymerase chain reaction. The mean (range) value of coefficient of variations in this custom array quantification was 9.4% (3.0-16.2%). CONCLUSION: Our custom cDNA array is expected to be useful for overall assessment of CF placental mRNAs in maternal plasma in a single experiment.

Human papillomavirus DNA in plasma of patients with HPV16 DNA-positive uterine cervical cancer.

OBJECTIVES: The squamous cell carcinoma antigen is considered the most accurate serologic tumor marker for uterine cervical carcinoma. However, serum squamous cell carcinoma antigen levels were found to correlate significantly with clinical severity of atopic dermatitis and chronic renal failure. The present study was conducted in patients with human papillomavirus 16 DNA-positive uterine cervical cancer to determine the plasma level of human papillomavirus 16 DNA and the diagnostic values of plasma human papillomavirus DNA in these patients. METHODS: Forty-three human papillomavirus 16-positive patients with cervical intraepithelial neoplasia or uterine cervical squamous cell carcinoma were recruited in this study. The diagnosis was cervical cancer in 20 patients, high-grade squamous intraepithelial lesions in 21, low-grade squamous intraepithelial lesions in 1 and negative for intraepithelial lesion or malignancy in 3 patients. Before any treatment, blood samples were collected from all patients. For analysis of human papillomavirus DNA in plasma of patients with cervical cancer, quantitative polymerase chain reaction fluorescent assay for human papillomavirus 16 was performed using human papillomavirus 16 primers and SYBR Green dye using the LightCycler 480 SW1.5 apparatus. RESULTS: Plasma human papillomavirus 16 DNA was detected in only 30.0% of the patients with human papillomavirus 16-positive cervical cancer and in none of normal controls. The copy number of plasma human papillomavirus 16 DNA was higher in patients with invasive cancer than in those with cervical intraepithelial neoplasia (CIN3), micro-invasive cancer and in normal individuals. CONCLUSIONS: These results indicated that the plasma human papillomavirus DNA level could be potentially used as a marker of low-invasive cervical cancer tumors in patients with normal squamous cell carcinoma antigen levels before treatment.

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

OBJECTIVE: 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. METHODS: Twenty-eight singleton pregnant women with placenta previa were classified into the following four groups: 16 women with placenta located at a posterior part of the uterine wall (Group A); 4 each with placenta located at the anterior wall without (Group B) or with (Group C) previous cesarean section; and the other 4 with a history of previous cesarean section and who had the placenta located at an anterior part of uterine wall and underwent a cesarean hysterectomy (Group D). The maternal plasma concentrations of the cell-free placental lactogen (PL) mRNA measured by real-time reverse-transcription polymerase chain reaction (PCR) were converted into multiples of the median (MoM). RESULTS: The MoM (range) values of cell-free PL mRNA in the control group and Groups A to D were 1.00 (0.39-2.35), 2.04 (0.91-3.93), 2.58 (1.90-3.90), 3.50 (1.20-4.30), and 6.28 (5.24-7.63), respectively. The concentration of cell-free PL mRNA was significantly higher in Group D than in Group A, B, or C (Mann-Whitney's U-test, P < 0.05). CONCLUSION: The cell-free PL mRNA concentration in maternal plasma has the potential to predict a subgroup of placenta accreta resulting in hysterectomy.

Does increased nuchal translucency indicate a fetal abnormality? A retrospective study to clarify the clinical significance of nuchal translucency in Japan.

The results of a chromosomal test by genetic amniocentesis in 58 cases with an increased nuchal translucency (NT; > or =3 mm thickness) revealed 47 cases showing a normal karyotype (81%) and 11 cases (19%) showing an abnormal karyotype. However, the cases of a normal karyotype with increased NT also included those with fetal abnormalities. Among the 49 cases in which NT was observed during the first trimester and then subsequently disappeared, chromosomal abnormalities were observed in five, and fetal abnormalities other than chromosomal abnormalities were observed in two. Meanwhile, all nine cases in which an increased NT remained or in which NT continued to increase in size during the second trimester were diagnosed as having cystic hygroma, and chromosomal abnormalities were found in six cases (67%). It should be noted that the shape of increased NT includes NT with a notch (notched NT) and NT without a notch (smooth NT). Among the 20 cases of notched NT, chromosomal abnormalities were observed in eight (40%), and cystic hygroma was observed in nine (45%). On the other hand, among the 38 cases of smooth NT, chromosomal abnormalities were observed in three (7.9%), but no cystic hygroma was observed. Our results confirm that increased NT does not always indicate a fetal abnormality. Whether NT thickness should be measured as a screening tool for fetal abnormalities remains controversial. However, increased NT may be detected by chance, because a maternal-fetal medical examination using ultrasonography is usually performed in Japan. It is therefore considered to be extremely important to establish a system in which cases are referred to obstetricians who are licensed clinical genetic specialists to obtain appropriate genetic counseling whenever increased NT is clinically observed.

A strong association between human earwax-type and apocrine colostrum secretion from the mammary gland.

Here we provided the first genetic evidence for an association between the degree of apocrine colostrum secretion and human earwax type. Genotyping at the earwax-type locus, rs17822931 within the ABCC11 gene, revealed that 155 of 225 Japanese women were dry-type and 70 wet-type. Frequency of women without colostrum among dry-type women was significantly higher than that among wet-type women (P<0.0002), and the measurable colostrum volume in dry-type women was significantly smaller than in wet-type women (P=0.0341).

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.

Imprinting analysis of 10 genes and/or transcripts in a 1.5-Mb MEST-flanking region at human chromosome 7q32.

MEST is one of the imprinted genes in human. With the assistance of our integration map and the complete sequence in the registry, we mapped a total of 16 genes/transcripts at the 1.5-Mb MEST-flanking region at 7q32. This region has been suggested to form an imprinted gene cluster, because MEST and its three flanking genes/transcripts (MESTIT1, CPA4, and COPG2IT1) were reported to be imprinted, although two (TSGA14 and COPG2) were shown to escape imprinting. In this study, 10 other genes/transcripts were examined for their imprinting status in human fetal tissues. The results indicated that 8 genes/transcripts (NRF1, UBE2H, HSPC216, KIAA0265, FLJ14803, CPA2, CPA1, and DKFZp667F0312) were expressed biallelically. The imprinting status of two (TSGA13 and CPA5) was not conclusive, because of their weak and/or tissue-specific expression and inconstant results. These findings provided evidence that only 4 of the 16 genes/transcripts located to the region show monoallelic expression, while others are not involved in imprinting. Therefore, it is less likely that the MEST-flanking 7q32 region forms a large imprinted domain.