Published papers

© 2020 The Authors Introduction: P2Y14, one of the P2Y purinergic G-protein coupled receptors, is expressed in a variety of cells and tissues. Its ligand, UDP-glucose (UDPG), is released from damaged and stress-stimulated cells and acts as a danger signal via P2Y14. Thus, P2Y14 plays an important role in immunological defense systems. Here, we aimed to elucidate the expression, localization, and role of P2Y14 in human trophoblasts and the placenta. Methods: Human chorionic villus and placental tissues were subjected to immunostaining for P2Y14 protein and an extravillous trophoblast (EVT) marker, HLA-G. We examined the expression of P2Y14 and the effect of UDPG on cell proliferation and invasion in an EVT cell line, HTR-8/SVneo, using an MTS assay and a Transwell assay, respectively. We tested the effect of UDPG on cell invasion in P2Y14-underexpressing HTR-8/SVneo clones established by the lentiviral introduction of shRNA for P2RY14 mRNA. Results: Immunostaining revealed that P2Y14 was exclusively expressed by EVTs. P2RY14 mRNA and P2Y14 protein were expressed in HTR-8/SVneo cells. UDPG did not affect cell proliferation but it did enhance invasion. Inhibition of P2Y14 and decreasing the expression of P2Y14 suppressed UDPG-mediated invasive activity. Conclusions: These results showed that EVT selectively expressed P2Y14 and that P2Y14 was positively involved in UDPG-enhanced EVT invasion. It suggests the possible existence of a danger signal-mediated physiological system at the fetomaternal interface where UDPG released from maternal tissues through destruction by EVT invasion may accelerate EVT invasion, allowing EVTs to undergo successful placentation and vascular remodeling.

© 2019 The Authors Zygotic genome activation (ZGA) begins after fertilization and is essential for establishing pluripotency and genome stability. However, it is unclear how ZGA genes prevent mitotic errors. Here we show that knockout of the ZGA gene Zscan5b, which encodes a SCAN domain with C2H2 zinc fingers, causes a high incidence of chromosomal abnormalities in embryonic stem cells (ESCs), and leads to the development of early-stage cancers. After irradiation, Zscan5b-deficient ESCs displayed significantly increased levels of γ-H2AX despite increased expression of the DNA repair genes Rad51l3 and Bard. Re-expression of Zscan5b reduced γ-H2AX content, implying a role for Zscan5b in DNA damage repair processes. A co-immunoprecipitation analysis showed that Zscan5b bound to the linker histone H1, suggesting that Zscan5b may protect chromosomal architecture. Our report demonstrates that the ZGA gene Zscan5b is involved in genomic integrity and acts to promote DNA damage repair and regulate chromatin dynamics during mitosis. In this article, Yamada and colleagues show that Zscan5b deficiency increases DNA stress, compromises chromosomal structure during mitosis, and leads to the development of early-stage cancers. Zscan5b deficiency may offer a murine model of human chromosomal breakage syndromes.

© 2021 Elsevier Ltd Even in the context of modern medicine, infants with fetal and neonatal neurological diseases such as cerebral palsy and myelomeningocele suffer serious long-lasting impairment due to the irreversible neuronal damage. The promotion of neurologically intact survival in patients with perinatal intractable neurological diseases requires the development of novel strategies. One promising strategy involves the use of human amniotic fluid stem cells (hAFSCs), which have attracted much attention in recent years and are known to exert anti-inflammatory and neuroprotective effects. In recent years, the therapeutic effects of hAFSCs on fetal-neonatal neurological diseases have become evident as per intense research efforts by our group and others. Specifically, hAFSCs administered into the nasal cavity migrated to the brain and controlled local inflammation in a rodent model of neonatal hypoxic-ischemic encephalopathy. In contrast, hAFSCs administered intraperitoneally did not migrate to the brain; they rather formed spheroids in the abdominal cavity, resulting in the suppression of systemic inflammation (including in the brain) via the secretion of anti-inflammatory cytokines in concert with peritoneal macrophages in a rodent model of periventricular leukomalacia. Moreover, studies in a rat model of myelomeningocele suggested that hAFSCs administered in utero secreted hepatocyte growth factor and protected the exposed spinal cord during pregnancy. Importantly, autologous hAFSCs, whose use for fetal-neonatal treatment does not raise ethical issues, can be collected during pregnancy and prepared in sufficient numbers for therapeutic use. This article outlines the results of preclinical research on fetal stem cell therapy, mainly involving hAFSCs, in the context of perinatal neurological diseases.

© 2019, The Author(s). Calcium and nutrients are transferred from mothers to fetuses or infants during pregnancy or lactation, respectively, promoting metabolic changes in the mother, many uncharacterized. To evaluate these changes, we undertook two parallel studies. In one we analyzed fourteen clinical cases of vertebral fragility fractures, at or before three months after partum, in mothers who breastfed their infants. In the other, we enrolled 79 additional pregnant subjects, some who chose to breastfeed and others who did not, and analyzed changes in bone metabolic status starting between 34 and 36 weeks of gestation and ending one month after partum. In the larger group, bone-resorbing and bone-forming parameters such as serum TRACP5b and osteocalcin, respectively, significantly increased after partum. Among parameters that changed after partum, serum PTH and the bone-resorbing markers serum TRACP5b and urine NTX were significantly higher in mothers who only breastfed infants compared to mothers who fed infants formula or a mix of both. However, bone-forming parameters were comparable between breastfeeding and non-breast-feeding groups after partum, suggesting that elevated bone-resorption occurs only in the breastfeeding group. Radiographic analysis after partum demonstrated that no subject among the 79 analyzed showed vertebral fractures, even those who breastfed exclusively. Among fracture cases analyzed, subjects exhibited significantly lower bone mineral density than did non-fracture cases in breastfeeding-only subjects. We conclude that bone metabolic status significantly changes over the period between pregnancy and post-partum lactation, and that low bone mineral density seen in a small subset of breastfeeding-only cases likely causes post-partum vertebral fragility fractures.

© 2018 The Authors Mitochondria are the powerhouses of eukaryotic cells and their positioning contributes to fertilization and early developmental processes. We report that sperm fusion triggers Ca2+ oscillations and mitochondrial movement toward fused sperm (mitochondrial chemotaxis) in mouse eggs. Mitochondria functioned in Ca2+ storage and were colocalized with endoplasmic reticulum (ER) during Ca2+ oscillations. Mitochondria then moved toward the fused sperm. Sperm extracts lacking nuclei induced Ca2+ oscillations, but did not promote mitochondrial chemotaxis. Our results suggest that sperm fusion motivates Ca2+ oscillation-independent mitochondrial chemotaxis. This phenomenon indicates that egg mitochondria interact with sperm materials, presumably nuclear substances, and their network tethers egg and sperm nuclei at the early stage of zygote formation.

© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd Aims/Introduction: Risk factors of type 2 diabetes mellitus in Japanese women with recent gestational diabetes mellitus are unknown. The objective of the present study was to investigate the clinical and genetic characteristics associated with postpartum abnormal glucose tolerance in Japanese women with gestational diabetes mellitus. Materials and Methods: A total of 213 Japanese women with recent gestational diabetes mellitus who underwent a postpartum 2-h oral glucose tolerance test were investigated. The association between antepartum clinical characteristics and postpartum abnormal glucose tolerance (diabetes or prediabetes based on the Japan Diabetes Society criteria) was examined. Frequencies of 45 known type 2 diabetes mellitus-associated genetic variants were also compared between women with and without postpartum abnormal glucose tolerance. Results: A total of 59 women showed postpartum abnormal glucose tolerance (prediabetes, n = 51; diabetes, n = 8). Plasma glucose levels at 1 or 2 h, the insulinogenic index and the insulin secretion-sensitivity index-2 of the antepartum oral glucose tolerance test were independent of postpartum abnormal glucose tolerance risk factors (P = 0.006, P = 0.00002, P = 0.01 and P = 0.006, respectively). Four genetic variants (rs266729 [ADIPOQ], rs6017317 [HNF4A], rs5215 [KCNJ11] and rs7177055 [HMG20A]) showed a nominally significant association with postpartum abnormal glucose tolerance (P < 0.05, respectively). Among these, three were related to insulin secretion. Postpartum abnormal glucose tolerance risk significantly increased with increasing risk-allele number (P = 0.0005; odds ratio 1.91). Conclusions: Clinical features and genetic variants related to impaired insulin secretion are risk factors of postpartum abnormal glucose tolerance in Japanese women with recent gestational diabetes mellitus.

© 2017 Wiley Periodicals, Inc. The cover image, by Satoru Ikenoue et al., is based on the Clinical Report Discordant fetal phenotype of hypophosphatasia in two siblings, DOI: 10.1002/ajmg.a.38531.

© 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a “molecular fossil” due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

© 2020 Royal College of Obstetricians and Gynaecologists Objective: Fetal fractional limb volume has been proposed as a useful measure for quantifying fetal soft tissue development. The aim of this study was to investigate the growth of fractional arm volume (AVol) and fractional thigh volume (TVol) of fetuses with maternal gestational diabetes (GDM) compared with those of fetuses with normal glucose tolerance (NGT). We hypothesised fetal fractional limb volume would be larger in the GDM group than in the NGT group in late gestation. Design: Exploratory observational study. Setting: Saitama Municipal Hospital. Sample: A total of 165 (125 NGT and 40 GDM) singleton Japanese pregnant women. Methods: AVol and TVol were assessed between 20 and 37 weeks’ gestation as cylindrical limb volumes based on 50% of the fetal humeral or femoral diaphysis length. Women were diagnosed as GDM based on the criteria of the Japan Society of Obstetrics and Gynecology. Main outcome measures: AVol and TVol were compared between women with NGT and those with GDM at each gestational age period (2-week intervals from 20 to 37 weeks’ gestation). Results: Overall, 287 ultrasound scans were performed (NGT group, 205 scans; GDM group, 82 scans). There was no significant difference of AVol between the groups before 32 weeks’ gestation. AVol was significantly larger in the GDM group than in the NGT group after 32 weeks’ gestation (P < 0.05). TVol was not statistically different between the groups across gestation. Conclusions: Detection of variations in fetal AVol may provide greater insight into understanding the origins of altered fetal body proportion in GDM. Tweetable abstract: AVol, but not TVol, is significantly larger in fetuses with GDM than in those with NGT after 32 weeks’ gestation.

© 2017 Wiley Periodicals, Inc. Hypophosphatasia (HPP) is an autosomal recessive metabolic disorder with impaired bone mineralization due to mutations in the ALPL gene. The genotype-phenotype correlation of this disorder has been widely described. Here, we present two affected siblings, whose fetal phenotypes were discordant. A 31-year-old Japanese woman, G0P0, was referred to our institution because of fetal micromelia. After obstetric counseling, the pregnancy was terminated at 21 weeks’ gestation. Post-mortem radiographs demonstrated severely defective mineralization of the skeleton. The calvarial, spinal, and tubular bones were mostly missing. Only the occipital bones, mandible, clavicles, ribs, one thoracic vertebra, ilia, and tibia were relatively well ossified. The radiological findings suggested lethal HPP. Genetic testing for genomic DNA extracted from the umbilical cord identified compound heterozygous mutations in the ALPL gene (c.532T>C, p.Y178H; c.1559delT, p.Leu520Argfs*86). c.532T>C was a novel variant showing no residual activity of the protein by the functional analysis. The parents were heterozygous carriers. In the next pregnancy, biometric values on fetal ultrasonography at 20 and 26 weeks’ gestation were normal. At 34 weeks, however, a small chest and shortening of distal long bones came to attention. The neonate delivered at 41 weeks showed serum ALP of <5U/L. Radiological examination showed only mild thoracic hypoplasia and metaphyseal mineralization defects of the long bones. ALP replacement therapy was introduced shortly after birth, and the neonate was discharged at day 22 without respiratory distress. Awareness of discordant fetal phenotypes in siblings with HPP precludes a diagnostic error, and enables early medical intervention to mildly affected neonates.

© 2018 The Authors Research question: Do gut microbiota associate with the ovulatory cycle in women showing normogonadotrophic anovulation? In humans, the gut microbiota affects diverse physiological functions and dysbiosis (microbial imbalance) may lead to pathological syndromes. However, there is comparatively little information on the relevance of gut microbiota to reproductive functions in women. Here, a group of women with idiopathic chronic anovulation were examined, who do not exhibit any apparent endocrinological disorder, as they are suitable for investigating the relationship between intestinal bacteria and ovulatory disorders. Design: A prospective observational cohort study was performed on two groups of women who did not exhibit apparent endocrinological disorders but showed either irregular menstrual cycles (IMC group) or normal menstrual cycles (controls). The bacterial composition of faeces from rectal swabs from the women was analysed using next-generation sequencing based on bacterial 16SrRNA genes. Results: A metagenomic analysis indicated that the two groups of women had significant differences in 28 bacterial taxa in their faeces. Prevotella-enriched microbiomes were more abundant in the IMC group, whereas Clostridiales, Ruminococcus and Lachnospiraceae (butyrate-producing bacteria) were present at lower levels in the IMC group. Conclusions: Distinctive subpopulations of intestinal microbiota were identified in women with unexplained chronic anovulation. The results indicate that gut microbiota could be associated with ovarian functions.

© 2020 Japan Society of Obstetrics and Gynecology Aim: We performed a birth cohort study involving 124 mother–infant pairs to investigate whether placental DNA methylation is associated with maternal choline status and fetal development. Methods: Plasma choline concentration was assayed longitudinally in the 1st and 3rd trimesters and at term-pregnancy in mothers and cord blood. Placental DNA methylation was measured for 12 target candidate genes that are related to fetal growth, adipogenesis, lipid and energy metabolism, or long interspersed nuclear elements. Results: Higher maternal plasma and cord blood choline levels at term tended to associate with lower birthweight (r = −0.246, P < 0.013; r = −0.290, P < 0.002) and body mass index (BMI) at birth (r = 0.344, P < 1E−3; r = −0.360, P < 1E−3). The correlation between maternal plasma choline level and cord blood choline level was relatively modest (r = 0.049, P = 0.639). There was an inverse correlation between placental DNA methylation at the retinoid X receptor alpha (RXRA) gene and maternal plasma choline level (r = −0.188 to r = −0.452, P = 0.043 to P < 1E−3 at three points). RXRA methylation level was positively associated with birthweight and BMI at birth (r = 0.306, P = 0.001; r = 0.390, P < 1E−3). Further, RXRA methylation was inversely correlated with RXRA gene expression level (r = 0.333, P < 1E−3). Conclusion: Our results suggest that the association between maternal choline status and placental RXRA methylation represents a potential fetal programing mechanism contributing to fetal growth.

© 2018, Japan Human Cell Society and Springer Japan KK, part of Springer Nature. Adult wound healing can result in fibrotic scarring (FS) characterized by excess expression of myofibroblasts and increased type I/type III collagen expression. In contrast, fetal wound healing results in complete regeneration without FS, and the mechanism remains unclear. Amniotic fluid cells could contribute to scar-free wound healing, but the effects of human amniotic fluid cells are not well characterized. Here, we determined the effect of human amniotic fluid stem cells (hAFS) on FS during wound healing. Human amniotic fluid was obtained by amniocentesis at 15–17 weeks of gestation. CD117-positive cells were isolated and defined as hAFS. hAFS (1 × 10 6 ) suspended in PBS or cell-free PBS were injected around wounds created in the dorsal region of BALB/c mice. Wound size was macroscopically measured, and re-epithelialization in the epidermis, granulation tissue area in the dermis and collagen contents in the regenerated wound were histologically analyzed. The ability of hAFS to engraft in the wound was assessed by tracking hAFS labeled with PKH-26. hAFS fulfilled the minimal criteria for mesenchymal stem cells. hAFS injection into the wound accelerated wound closure via enhancement of re-epithelialization with less FS. The process was characterized by lower numbers of myofibroblasts and higher expression of type III collagen. Finally, transplanted hAFS were clearly observed in the dermis until day 7 implying that hAFS worked in a paracrine manner. hAFS can function in a paracrine manner to accelerate cutaneous wound healing, producing less FS, a process resembling fetal wound healing.

© 2018 by The Keio Journal of Medicine. Mesenchymal stem cells (MSCs) have generated great interest in the fields of regenerative medicine and immunotherapy because of their unique biological properties. Among MSCs, amniotic fluid stem cells (AFS) have a number of characteristics that make them attractive candidates for tissue engineering and cell replacement strategies, particularly for perinatal medicine. If various neonatal conditions, including birth asphyxia, preterm birth, and congenital abnormalities, which result in long-lasting severe impairments, could be predicted during pregnancy, it would allow collection of small samples of amniotic fluid cells by amniocentesis. In vitro culture of these autologous AFS during pregnancy would make them available for use soon after birth. Hypoxic-ischemic encephalopathy (HIE) and myelomeningocele (MMC) are neonatal conditions that cause permanent neurological disability, for which the treatment options are extremely limited. Experiments using animal models of HIE and MMC and human clinical trials have demonstrated that MSCs, including AFS, have beneficial effects on the central nervous system through paracrine influences, indicating that autologous AFS treatment may be applicable for intractable neurological diseases, including HIE and MMC, during the perinatal period. In this review, we focus on recent research related to the therapeutic potential of AFS for perinatal neurological diseases such as HIE and MMC.

© Copyright © 2019 Sasaki, Hamatani, Kamijo, Iwai, Kobanawa, Ogawa, Miyado and Tanaka. Reproductive capacity in women starts to decline beyond their mid-30s and pregnancies in older women result in higher rates of miscarriage with aneuploidy. Age-related decline in fertility is strongly attributed to ovarian aging, diminished ovarian reserves, and decreased developmental competence of oocytes. In this review, we discuss the underlying mechanisms of age-related decline in oocyte quality, focusing on oxidative stress (OS) in oocytes. The primary cause is the accumulation of spontaneous damage to the mitochondria arising from increased reactive oxygen species (ROS) in oocytes, generated by the mitochondria themselves during daily biological metabolism. Mitochondrial dysfunction reduces ATP synthesis and influences the meiotic spindle assembly responsible for chromosomal segregation. Moreover, reproductively aged oocytes produce a decline in the fidelity of the protective mechanisms against ROS, namely the ROS-scavenging metabolism, repair of ROS-damaged DNA, and the proteasome and autophagy system for ROS-damaged proteins. Accordingly, increased ROS and increased vulnerability of oocytes to ROS lead to spindle instability, chromosomal abnormalities, telomere shortening, and reduced developmental competence of aged oocytes.

© 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group. Background: No reports have focused on the clinical and metabolic characteristics of gestational diabetes (GDM) in underweight women. The aim of this study is to investigate the clinical and metabolic features of underweight GDM (pregravid BMI, <18.5 kg/m2: U-GDM). Materials and methods: Women diagnosed with GDM were categorized based on their pre-pregnancy BMI as either underweight (n = 49) or normal weight (pregravid BMI, 18.5–25.0 kg/m2: n = 271: N-GDM). During the study period, GDM was diagnosed using the International Association of Diabetes in Pregnancy Study Group criteria. Women with multi-fetal pregnancies, fetal congenital anomalies, overt diabetes in pregnancy, and pre-gestational diabetes mellitus were excluded. Results: There were no notable differences in maternal age at delivery and the rate of nulliparous between the U-GDM and N-GDM groups. Regarding antepartum oral glucose tolerance test profiles, women with U-GDM exhibited significantly lower fasting plasma glucose (FPG) levels than those with N-GDM (p <.01). The Insulinogenic Index of women with U-GDM was significantly lower than that of women with N-GDM (p <.05). Conclusion: Impaired early phase insulin secretion was associated with GDM onset in underweight women.

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. Introduction: The average birth weight in Japan has gradually decreased and the number of low birth weight infant has increased over the last 30 years. Japanese pregnant women are characterized with lower prevalence of obesity than those in other developed countries, and maternal underweight before pregnancy is independently associated with low birth weight. However, the association between maternal inadequate gestational weight gain (GWG) and the risk of small for gestational age (SGA) in Japanese uncomplicated pregnant women has not been fully understood. This study aimed to examine the effect of maternal inadequate GWG on the risk of SGA in Japanese uncomplicated pregnancies. Methods: We retrospectively analyzed uncomplicated Japanese singleton pregnancies that delivered at term gestation in our institution from 2006 to 2016. The association between GWG and birth weight was analyzed by multiple linear regression. Potential confounding factors included maternal age, parity, prepregnancy BMI, and neonatal sex. The association between inadequate GWG and SGA was also examined by logistic regression. Results: A total of 3837 mother–neonate dyads were analyzed. Maternal GWG was 10.1 ± 3.7 kg (mean ± SD), and 2529 (66%) had inadequate GWG. After adjusting for confounding factors, GWG significantly correlated with birth weight (standardized β = 0.199, p <.001). Inadequate GWG increased the risk of delivering SGA neonate (adjusted odds ratio (aOR) = 1.97 [1.45–2.68], p <.001). This association was particularly pronounced in underweight (aOR = 2.95 [1.38–6.29], p =.005) and normal weight mothers (aOR = 1.79 [1.27–2.52], p =.001), and not in overweight/obese mothers (p =.115). Conclusion: Maternal GWG is associated with birth weight in Japanese women with uncomplicated singleton pregnancies. Inadequate GWG is an important risk factor of SGA, particularly in non-obese women. The present finding would potentially provide further evidence that promoting adequate gestational weight gain in Japanese underweight and normal weight mothers would reduce SGA, as well as metabolic dysfunction in later life.