関谷 隆夫

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.

[目的]Retained products of conception(RPOC)とは、妊娠終了後に妊娠に関連する組織が子宮内に残存した状態で、産褥晩期出血の一因となるが、本邦では診断基準や管理方法が確立されていないのが現状である。そこで、本疾患の病態解明と管理指針の確立をめざして、当施設で経験したRPOCとその対応の現状について臨床的検討を行った。[方法]院内倫理審査委員会の承認の下、2012年から2019年の8年間に当院で経験したRPOCの臨床所見と治療について、診療録を用いて後方視的に検討を行った。[成績]対象期間中のRPOCは20例で、平均年齢は34歳(27-40)、先行妊娠の転帰は、流産(人工妊娠中絶含む)が7例、分娩が13例(正期産12例、早産1例)で、分娩様式は、経腟分娩10例、帝王切開分娩3例であった。治療法は、子宮収縮剤投与による保存的治療が4例で、これら全てが自然退縮し、外科的治療を行ったのは16例(子宮内容除去術5例、子宮鏡下経頸管的切除術10例、腹式単純子宮全摘出術1例)であった。超音波ドプラ検査で豊富な腫瘤内血流を認めたのは20例中18例で、このうち15例に外科的治療を行なった。大量出血により緊急対応を必要とした4例中3例に子宮動脈塞栓術(UAE)を実施したが、これら4例の腫瘤最大径は、それ以外の16例に比して有意に大きかった(62.5mm vs 27.2mm)(p<0.05)。[結論]RPOCの病態は多彩であり、保存的治療も選択肢となり得るが、腫瘤の大きさと経腟超音波ドプラ検査による腫瘤内血流所見が外科的治療を考慮するポイントとなる。(著者抄録)

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.

分娩時異常出血への対応は分娩における母体管理の最重要項目の一つであり、我が国の分娩の約半数が1次医療施設で行われている現状の下で、母体の大量出血時における全身状態の評価と高次医療機関への搬送を行う為の適切な判断が求められている。このような背景の下「産科危機的出血への対応指針2017」では、母体出血に対する病態評価指標としてshock index(以下;SI)を用いた対応指針が示されている。今回我々は、当院で経腟分娩した4,932症例のうち、分娩時1,000ml以上の出血をきたした142症例を対象として、Shock index(以下;SI)・背景因子・原因疾患について検討した。さらに輸血例と搬送症例に関してはそれぞれの臨床経過についても検討を行った。全142例の分娩時最大SI値と、分娩時出血量の相関係数は0.419でありSI値と出血量の間には中程度の相関をみとめた。SI:1.0以上、未満でみるとSI:1.0以上の症例は21例、1.0未満の症例は121例であった。SI:1.0以上の群で有意に出血量が多く、分娩後最低ヘモグロビン値も有意に低値で、輸血とバルーンタンポナーデ法の実施率や、高次医療機関への搬送率も有意に高かった。原因疾患別に出血量とSI値を見ると、会陰裂傷・腟壁裂傷ではその他の疾患に比べSI値に対し出血量が少なかった。また、全体のうち輸血を実施した症例は16例、搬送症例は5例であった。1次医療施設においてSI値は分娩後の母体循環の迅速な把握に有用であり、産科危機的出血に対する迅速な評価指標になると考えられた。(著者抄録)

分娩時異常出血への対応は分娩における母体管理の最重要項目の一つであり、我が国の分娩の約半数が1次医療施設で行われている現状の下で、母体の大量出血時における全身状態の評価と高次医療機関への搬送を行う為の適切な判断が求められている。このような背景の下「産科危機的出血への対応指針2017」では、母体出血に対する病態評価指標としてshock index(以下;SI)を用いた対応指針が示されている。今回我々は、当院で経腟分娩した4,932症例のうち、分娩時1,000ml以上の出血をきたした142症例を対象として、Shock index(以下;SI)・背景因子・原因疾患について検討した。さらに輸血例と搬送症例に関してはそれぞれの臨床経過についても検討を行った。全142例の分娩時最大SI値と、分娩時出血量の相関係数は0.419でありSI値と出血量の間には中程度の相関をみとめた。SI:1.0以上、未満でみるとSI:1.0以上の症例は21例、1.0未満の症例は121例であった。SI:1.0以上の群で有意に出血量が多く、分娩後最低ヘモグロビン値も有意に低値で、輸血とバルーンタンポナーデ法の実施率や、高次医療機関への搬送率も有意に高かった。原因疾患別に出血量とSI値を見ると、会陰裂傷・腟壁裂傷ではその他の疾患に比べSI値に対し出血量が少なかった。また、全体のうち輸血を実施した症例は16例、搬送症例は5例であった。1次医療施設においてSI値は分娩後の母体循環の迅速な把握に有用であり、産科危機的出血に対する迅速な評価指標になると考えられた。(著者抄録)

＜回答のポイント＞1)ガイドラインに則った医療が実践され、病診・病病連携体制が整備された現在では、里帰り出産自体のリスクは低く、妊婦の社会的背景を念頭に積極的に対応する。2)里帰り出産の帰省時期は妊娠34週前後が一般的であり、その決定にあたっては、母体と胎児のリスク因子の評価を行うとともに妊婦の希望する産科医療施設の情報を収集し、帰省までの妊娠管理と診療情報提供を確実に行う。3)特にハイリスク妊娠・分娩においては、母体および胎児の状態によって対応可能な産科医療施設を選定し、当該施設と綿密な連携のもとで里帰りの時期と方法を決定するとともに家族のバックアップ体制の構築を促す。(著者抄録)

＜回答のポイント＞1)国内線と国際線では、妊婦の搭乗規定が異なる。2)国内線ではほとんどの航空会社の規約は共通しており、自由に搭乗できるのは分娩予定日の29日前までで、28日以内では医師の診断書、7日以内では医師の同伴が必要となる。3)国際線では航空会社によって基準が大きく異なる。自由に乗れるのは妊娠32週あるいは36週前までのことが多く、多胎妊娠では制限を設けている場合もある。稀に目的国が妊婦の入国を制限している場合もある。4)飛行機に搭乗すること自体の胎児への影響についてはエビデンスはないが、一般的な状況では問題はないと考えられている。5)飛行機に搭乗する際には、エコノミークラス症候群の発症を避けるために適度な運動と水分摂取が必要である。6)渡航先の特殊な感染症の情報を取得し、同時に自身の予防接種歴やウイルス感染症を把握しておく。(著者抄録)

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.

During next generation sequencing (NGS) analysis, many missense mutations were found in a well-known oncogene, many of which were variant of uncertain significance mutations. We recently treated an adult patient with pancreatoblastoma by chemotherapy. Using an NGS cancer panel, we found a previously unreported missense mutation in the 1835 codon of the adenomatous polyposis coli (APC) gene. We also found a heterogeneous mutation in the 1835 codon of the APC gene in the patient's germline by Sanger sequencing. Although this patient did not have a history of familial adenomatous polyposis, functional analysis suggested the R1835G mutant APC showed attenuated repression of Wnt/β-catenin signaling activity. This is the first report showing a novel APC missense mutation involved in the onset of adult pancreatoblastoma.

Aim: Patients with an ultra-short uterine cervix as a result of large conization, repeated conization or radical trachelectomy (RT), are at high risk of preterm premature rupture of the membrane, which leads to preterm birth. We have commenced performing transabdominal cerclage (TAC) of the uterine cervix for these patients. In this study, we examined the safety of TAC and its impact on pregnancy. Methods: We have performed TAC in 11 patients before pregnancy: in six after large cervical operations, such as repeated conization and in five for difficulties with cervical cerclage after RT. After laparotomy, a Teflon thread was placed in the avascular space between the uterine vessels and the uterine muscle, and tied. The clinical course of the patients after TAC and their pregnancy course were retrospectively reviewed. Results: TAC was performed safely without any complications. The mean operative duration was 53 ± 10 min, and the mean blood loss during the operation was 49 ± 64 mL. Seven women conceived within 2 years after TAC. Their pregnancy courses were favorable. Five of the women underwent scheduled cesarean sections, while two pregnancies are ongoing. Conclusions: Although there are risks of various complications as a result of the use of non-absorbable thread and the need for two extra laparotomies, TAC can be a safe and useful option for patients who show cervical incompetence after large uterine cervical operations, such as RT or large conization.

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.

Atrial natriuretic peptide is biologically activated by the atrial natriuretic peptide-converting enzyme, corin, and has an important role in regulating blood pressure. We detected elevated serum corin levels in women with pre-eclampsia. Interestingly, the serum corin levels were also found to be elevated in pregnancies with a related disorder, unexplained fetal growth restriction (FGR) without hypertension, suggesting that this phenomenon is not simply a response to maternal hypertension. CORIN mRNA levels were not elevated in placentas from pre-eclampsia or unexplained FGR cases. Likewise, similar signal intensities were found for corin in placental syncytiotrophoblast cells by immunostaining. In contrast, corin signals were higher in maternal decidua cells from pre-eclampsia and unexplained FGR cases. These data suggest that corin may be upregulated in maternal decidua in response to an etiologic pathway that is common to pre-eclampsia and FGR.