関谷 隆夫

Objectives: We previously reported a high prevalence of hypovitaminosis D (25OHD < 20 ng/mL) in Japanese pregnant women with threatened premature delivery. This study aimed to assess nutritional status and its relationship with bone-related markers and microarchitecture, as measured using quantitative ultrasonography (QUS), in Japanese women during the perinatal period. Methods: We recruited Japanese women who had just delivered at Fujita Health University Hospital (n = 103, cesarean/vaginal delivery = 50/53, age 33.9 ± 4.9 years). On the third day postpartum, their calcaneal QUS was measured, and fasting blood samples were collected. Results: The mean total energy intake (1720 ± 298 kcal/day) was lower than the normal range for Japanese women (2100 kcal/day). Their calcium intake (446 ± 130 mg/day) was significantly below the recommended daily intake (RDI) in Japan (660 mg/day), with 95% of participants consuming less than the RDI. Although the average vitamin D intake (8.7 ± 1.8 μg/day) met the Japanese RDI (8.5 μg/day), 36% of participants consumed less than the RDI. Calcium intake was positively associated with the intake of lipids, protein, and vitamins A, D, and K. Additionally, calcium intake but not vitamin D intake tended to correlate with serum 25-hydroxyvitamin D (25OHD) levels. The QUS indices showed no significant association with calcium or vitamin D intake. Conclusions: During the perinatal period, Japanese women had low calcium intake and relatively low vitamin D intake, accompanied by reduced 25OHD levels. These findings highlight the need for public health recommendations and policies to promote adequate calcium and vitamin D intake during pregnancy.

OBJECTIVES: Nectin-4 is a cell adhesion molecule with vital functions at adherens and tight junctions. Cumulative evidence now indicates that the NECTIN4 gene is overexpressed in a variety of cancers, and that the nectin-4 protein is both a disease marker and therapeutic target in a subset of these cancers. We previously demonstrated that NECTIN4 is overexpressed in placenta during pre-eclamptic pregnancy, which is one of the most serious obstetric disorders. METHODS: Nectin-4 protein levels were measured in maternal sera from pregnant women with pre-eclampsia and its related disorder, unexplained fetal growth retardation. RESULTS: Maternal serum concentrations of nectin-4 were significantly elevated in pre-eclamptic women compared with those with an uncomplicated normotensive pregnancy. However, no increase was observed in pregnancies with unexplained fetal growth retardation. Serum nectin-4 levels were higher in cases with early-onset pre-eclampsia that generally showed more severe clinical symptoms, but levels were not correlated to other clinical indicators of disease severity. CONCLUSIONS: Nectin-4 is a potential new diagnostic and predictive biomarker for severe pre-eclampsia.

BACKGROUND: FLT1 is one of the significantly overexpressed genes found in a pre-eclamptic placenta and is involved with the etiology of this disease. METHODS: We conducted genome-wide expression profiling by RNA-seq of placentas from women with pre-eclampsia and those with normotensive pregnancy. RESULTS: We identified a lncRNA gene, MG828507, located ~80 kb upstream of the FLT1 gene in a head-to-head orientation, which was overexpressed in the pre-eclamptic placenta. MG828507 and FLT1 are located within the same topologically associated domain in the genome. The MG828507 mRNA level correlated with that of the FLT1 in placentas from pre-eclamptic women as well as in samples from uncomplicated pregnancies. However, neither the overexpression nor knockdown of MG828507 affected the expression of FLT1. Analysis of pre-eclampsia-linking genetic variants at this locus suggested that the placental genotype of one variant was associated with the expression of MG828507. The MG828507 transcript level was not found to be associated with maternal blood pressure, but showed a relationship with birth and placental weights, suggesting that this lncRNA might be one of the pivotal placental factors in pre-eclampsia. CONCLUSION: Further characterization of the MG828507 gene may elucidate the etiological roles of the MG828507 and FLT1 genes in pre-eclampsia in a genomic context.

Objectives: Alterations in the vaginal bacterial flora reflect the status of various obstetric conditions and are associated with mechanisms that underlie certain pregnancy-associated complications. These changes are also a predictive biomarker for clinical outcomes of these adverse events. Methods: We examined the vaginal microbiome in samples from pregnant Japanese women with preterm labor. Results: The microbiota composition in preterm delivery (PD) samples differed from those of control or threatened preterm delivery (TPD) samples in principal component analysis. An increase in Firmicutes and a decrease in Actinobacteria were significantly associated with PD only (both P<0.01). In the Firmicutes phylum, Lactobacillus tended to be abundant, and the abundance of L. iners and L. crispatus was especially high, whereas the L. gasseri population was low in PD samples. Longitudinal analysis showed that the abundance of L. iners decreased after commencing tocolytic treatment in TPD samples compared with before treatment, but it remained high in PD samples. Conclusions: The vaginal microbiome may be a useful prognostic indicator of preterm labor and a monitoring tool for tocolytic treatment to prevent preterm birth.

OBJECTIVE: The proprotein convertase furin is known to be involved in the processing of pro-B-type natriuretic peptide (proBNP) and prorenin receptor (PRR), suggesting that it has a potential function in blood pressure regulation. We investigated the role of furin in the etiology of pre-eclampsia and its related disorder, unexplained fetal growth restriction (FGR) without hypertension. METHODS: We evaluated serum and placental furin levels in pre-eclampsia, FGR and uncomplicated pregnancy. Additionally, we investigated the correlation between the serum furin levels and products of furin enzymatic activity or clinical parameters. RESULTS: We demonstrated that the maternal circulation in cases of pre-eclampsia and FGR had lower levels of soluble furin than uncomplicated pregnancies. Both NT-proBNP and soluble PRR were elevated in pre-eclampsia, whereas only soluble PRR was at higher levels in unexplained FGR. Linear regression analysis revealed a negative correlation between the serum furin level and that of NT-proBNP or soluble PRR. While we observed that the serum furin or soluble PRR level correlated with blood pressure, a stronger correlation was observed with birth and placental weights. Further to this, the FURIN mRNA levels were significantly reduced in placental pre-eclamptic placentas as well as in FGR cases. CONCLUSION: These data suggest the possibility that reduced levels of furin may be the result of a negative feedback from the activation of the renin-angiotensin pathway that leads to feto-placental dysfunction with or without maternal hypertension. This may represent an etiologic pathway of pre-eclampsia and unexplained FGR.

BACKGROUND: Soluble fms-like tyrosine kinase 1 (sFlt-1) is believed to be a prominent component in the pathogenesis of pre-eclampsia, although the precise etiology has remained elusive. In this study, the etiological role of FLT1 variant was further validated in pre-eclampsia by examining this association in a Japanese sample population. METHODS: The genotypes of three variants (rs4769613, rs12050029 and rs149427560) were examined in the upstream region of the FLT1 gene in placentas from pre-eclamptic (n=47) or normotensive control (n=49) pregnancy samples. Additionally, FLT1 mRNA levels in placenta were determined by qRT-PCR. ELISA was further used to detect circulating sFlt-1 levels in maternal sera. The intergroup comparisons were made using the Mann-Whitney U test or one way analysis of variance and P values of less than 0.05 were considered statistically significant. RESULTS: First, the rs4769613 (C>T) and rs12050029 (G>A) genotypes were examined in placentas but no significant differences were found in the genotype or allele-type frequencies. Next, nearby short tandem repeat, rs149427560, was examined which manifested four size variants. In the genotypewise analysis, the frequency of the 474/476 heterozygote was significantly lower in pre-eclampsia (p<0.05). As expected, the FLT1 mRNA levels were significantly elevated in the pre-eclamptic placentas and sFlt-1 was higher in pre-eclamptic maternal sera. However, the genotype of these variants did not affect the FLT1 mRNA or serum sFlt-1 levels. CONCLUSION: Our findings did not support the hypothesis that genetic variations around the FLT1 gene affect the subtle expression changes underlying the etiologic pathway of pre-eclampsia. The hypothesis deserves further investigation through a larger sample size.

Examination of maternal plasma cell-free DNA (cfDNA) for noninvasive prenatal testing for fetal trisomy is a highly effective method for pregnant women at high risk. This can be also applied to fetal gender determination in female carriers of severe X-linked disease. Polymerase chain reaction (PCR) analysis is a relatively simpler and less expensive method of detecting Y chromosome-specific repeats (Y-specific PCR; YSP), but is limited by the risk of false-negative results. To address this, we have developed a combined strategy incorporating YSP and an estimation of the fetal DNA fraction. Multiplex PCR for 30 single nucleotide polymorphism (SNP) loci selected by high heterozygosity enables the robust detection of the fetal DNA fraction in cfDNA. The cfDNA sample is first subjected to YSP. When the YSP result is positive, the fetus is male and invasive testing for an X-linked mutation is then required. When the YSP result is negative, the cfDNA sample is analyzed using multiplex PCR. If fetal DNA is then found in the cfDNA, invasive testing is not then required. If the multiplex PCR analysis of cfDNA is negative for fetal DNA, the fetal gender cannot be determined and invasive testing is still required. Our technique provides a potentially effective procedure that can help to avoid unnecessary invasive prenatal testing in some female carriers of severe X-linked disease.

BACKGROUND: Nectins are cell adhesion molecules that play a pivotal role in adherens junctions and tight junctions. Our previous study using whole-genome oligonucleotide microarrays revealed that nectin-4 was upregulated in pre-eclamptic placentas. We investigated the role of nectin-4 in the etiology of pre-eclampsia. METHODS: We investigated the expression of nectin-4 using real-time RT-PCR, western blot and immunostaining. Additionally, we performed matrigel invasion assay and cytotoxicity assay using cells overexpressing the nectin-4. RESULTS: NECTIN4 transcripts were elevated in pre-eclamptic placentas relative to uncomplicated pregnancies. Nectin-4 protein levels in pre-eclamptic placentas were higher on a semi-quantitative western blot. Nectin-4 was localized at the apical cell membrane in syncytiotrophoblast cells and not at the adherens junctions. Nectin-4 was also detected in cytotrophoblasts and a subset of cells in the decidua. Nectin-4 overexpressing trophoblast cells migrated normally in the matrix. However, Natural killer (NK) cells showed a strong cytotoxic effect against nectin-4 overexpressing trophoblast cells. No causative genetic variation was evident in the NECTIN4 gene from a pre-eclamptic placenta. CONCLUSIONS: There are as yet unknown factors that induce nectin-4 overexpression in trophoblast cells that may contribute to abnormal placentation via an aberrant immune response and the onset of a pre-eclamptic pregnancy.

Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.