Publications by authors named "Kazuhiro Kawamura"

66 Publications

Profiling of volatile compounds in Japanese sake stored in sherry casks using solid phase microextraction/gas chromatography/mass spectrometry analysis.

J Biosci Bioeng 2021 May 1. Epub 2021 May 1.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Osaka University Shimadzu Analytical Innovation Research Laboratory, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Institute for Open and Transdisciplinary Research Initiatives, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

The combination of solid phase microextraction (SPME) and gas chromatograph-mass spectrometer (GC-MS) is frequently used for comprehensive analysis of aroma components in foods because it can be used to easily analyze volatile components, allowing saving of the amount of solvent used. In this study, SPME-GC-MS analysis of sake samples before and after sherry cask storage was performed to investigate the special flavor derived from sherry cask storage. A GC column with polyethylene glycol as the stationary phase, which is the first choice for volatile component analysis, was used. However, the peak of the acid having a carbonyl group was tailed due to its bond with the hydroxyl group of the stationary phase. In the analysis of sake samples, a large and tailing peak derived from the large amount of fatty acids in Japanese sake was observed. Additionally, it was not possible to analyze other co-eluting components. To overcome this problem, a novel extraction condition was examined using SPME and tris (hydroxymethyl) aminomethane (Tris). By adding Tris solution to sake, the fatty acid peak was removed successfully, thereby facilitating analysis of the peaks of compounds co-eluting with fatty acids and comprehensive analysis of the aroma components in sake. Furthermore, a comparative analysis of sake before and after storage in sherry cask showed that levels of fatty alcohols, organic acid esters, fatty acid esters, and terpenes increased significantly after storage in sherry cask, suggesting that these ingredients might constitute the special flavor of sherry cask-stored sake.
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http://dx.doi.org/10.1016/j.jbiosc.2021.03.014DOI Listing
May 2021

In Vitro Activation Early Follicles: From the Basic Science to the Clinical Perspectives.

Int J Mol Sci 2021 Apr 6;22(7). Epub 2021 Apr 6.

Department of Obstetrics and Gynecology, School of Medicine, International University of Health and Welfare, Narita, Chiba 286-8686, Japan.

Development of early follicles, especially the activation of primordial follicles, is strictly modulated by a network of signaling pathways. Recent advance in ovarian physiology has been allowed the development of several therapies to improve reproductive outcomes by manipulating early folliculogenesis. Among these, in vitro activation (IVA) has been recently developed to extend the possibility of achieving genetically related offspring for patients with premature ovarian insufficiency and ovarian dysfunction. This method was established based on basic science studies of the intraovarian signaling pathways: the phosphoinositide 3-kinase (PI3K)/Akt and the Hippo signaling pathways. These two pathways were found to play crucial roles in folliculogenesis from the primordial follicle to the early antral follicle. Following the results of rodent experiments, IVA was implemented in clinical practice. There have been multiple recorded live births and ongoing pregnancies. Further investigations are essential to confirm the efficacy and safety of IVA before used widely in clinics. This review aimed to summarize the published literature on IVA and provide future perspectives for its improvement.
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http://dx.doi.org/10.3390/ijms22073785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038686PMC
April 2021

Fertility Preservation in Patients with Severe Ovarian Dysfunction.

J Vis Exp 2021 03 25(169). Epub 2021 Mar 25.

Advanced Reproduction Medicine Research Center, Department of Obstetrics and Gynecology, International University of Health and Welfare School of Medicine;

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.
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http://dx.doi.org/10.3791/62098DOI Listing
March 2021

CXCL5-CXCR2 signaling is a senescence-associated secretory phenotype in preimplantation embryos.

Aging Cell 2020 10 22;19(10):e13240. Epub 2020 Sep 22.

Department of Obstetrics and Gynecology, Advanced Reproduction Research Center, International University of Health and Welfare School of Medicine, Narita, Japan.

Pregnancy rate of women decreases with age due to declining quality of oocytes and embryos. However, there is no established method to improve pregnancy rate in aging women. In this study, we identified a senescence-associated secretory phenotype (SASP) factor partially responsible for the decline in embryo implantation potential. Based on microarray analysis using young and aging human embryos at the same morphological grade, 702 genes showed >fivefold increases in aging human blastocysts. Among these genes, C-X-C motif chemokine 5 (CXCL5) showed 7.7-fold increases in aging human blastocysts. However, no-age-dependent changes in expression of the CXCR2, the cognate receptor for CXCL5, were found. In aging mice, Cxcl5 transcript levels were also increased in oocytes and embryos. Treatment of young mouse embryos with CXCL5 decreased implantation rates, together with increased expression of aging markers (P53, P21, Pai-1, and Il-6). Moreover, CXCL5 treatment suppressed trophoblast outgrowth in young mouse blastocysts. Conversely, suppression of CXCL5-CXCR2 signaling in aging mouse embryos using neutralizing antibodies and a receptor antagonist improved the implantation rate, leading to increases in pregnancy and delivery of normal pups. The gene expression pattern of these embryos was comparable to that in young mouse embryos showing enriched cell proliferation-related pathways. In conclusion, we identified CXCL5 as a SASP factor in human and mouse embryos and suppression of CXCL5-CXCR2 signaling during embryo culture improved pregnancy success in aging mice. Future analysis on CXCL5-CXCR2 signaling suppression in human embryos could be the basis to improve embryo development and pregnancy outcome in middle-aged infertile patients.
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http://dx.doi.org/10.1111/acel.13240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576282PMC
October 2020

Surgical approaches of drug-free in vitro activation and laparoscopic ovarian incision to treat patients with ovarian infertility.

Fertil Steril 2020 Dec 6;114(6):1355-1357. Epub 2020 Sep 6.

Department of Obstetrics and Gynecology, International University School of Medicine, Chiba, Japan; Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California. Electronic address:

Objective: To demonstrate our procedures of drug-free in vitro activation (IVA) for treating patients with premature ovarian insufficiency (POI) or diminished ovarian reserve (DOR), as well as the laparoscopic ovarian incision (LOI) procedure for treating patients with resistant ovary syndrome (ROS).

Design: Step-by-step video demonstration of the surgical procedures.

Setting: Fertility clinic and reproductive medicine department.

Patients: Women were diagnosed with POI based on recent amenorrhea before 40 years of age or with DOR according to the Bologna criteria, showing growth of a few antral follicles after ovarian stimulation. ROS patients were diagnosed based on amenorrhea with hypergonadotropic hypoestrogenism but showing age-appropriate number of antral follicles under transvaginal ultrasound.

Interventions: The drug-free IVA consists of the following 4 steps: removing a part of the cortex from one or both ovaries; cutting ovarian cortical pieces into small cubes in vitro; making pockets for ovarian tissue grafting; and grafting ovarian cortical cubes. The LOI procedure consisted of only one step: cutting ovarian cortex in situ. Both procedures were followed by ovarian hyperstimulation for at least 1 year. Informed consent was obtained from patients and approval was granted by the Biomedical Ethics Committee of the International University School of Medicine and the Rose Ladies Clinic. The present clinical trial was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Main Outcome Measure: Follicle growth.

Results: These procedures can be completed within 1 hour under laparoscopic surgery. There were no complications. In 13 of 15 patients treated with drug-free IVA, increases in antral follicle numbers were found, followed by a higher number of retrieved oocytes for in vitro fertilization. In addition to one spontaneous pregnancy, embryo transfer allowed four live births and one ongoing pregnancy. Five additional patients and one miscarriage patient have cryopreserved embryos for future transfer. We also found follicle growth to the preovulatory stage in seven of 11 ROS patients who have not responded to any endogenous and exogenous follicle-stimulating hormone stimulations for follicle growth prior to LOI treatment, allowing the retrieval of mature oocytes for in vitro fertilization. Four ROS patients became pregnant, followed by the delivery of three healthy infants and one ongoing pregnancy.

Conclusion: A drug-free IVA approach provided an infertility treatment for recent POI or DOR patients. This procedure promoted growth of residual ovarian follicles following ovarian tissue fragmentation in vitro, leading to Hippo signaling disruption. Although ROS patients exhibited symptoms of hypergonadotropic hypoestrogenism similar to that of POI patients, they still had multiple secondary follicles. Hippo signaling disruption in vivo based on cutting ovarian cortex using LOI could promote follicle growth.

Umin Clinical Trials Registration Number: UMIN000029807.
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http://dx.doi.org/10.1016/j.fertnstert.2020.07.029DOI Listing
December 2020

Hippo signaling disruption and ovarian follicle activation in infertile patients.

Fertil Steril 2020 09 8;114(3):458-464. Epub 2020 Aug 8.

Advanced Reproductive Medicine Research Center, Department of Obstetrics and Gynecology, International University of Health and Welfare School of Medicine, Chiba, Japan.

The Hippo signaling pathway, which is important in organ size regulation, is present in organisms from the fly to mammals. Disruption of the Hippo signaling pathway leads to increased nuclear translocation of the effector Yes-associated protein (YAP), resulting in the expression of cystein-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) growth factors and baculoviral inhibitors of apoptosis repeat containing (BIRC) apoptosis inhibitors to increase organ sizes. Furthermore, genome-wide knockdown of genes in insect cells demonstrated that actin polymerization promoted nuclear translocation of YAP. In the mammalian ovary, we demonstrated the expression of Hippo signaling pathway genes and showed that ovarian fragmentation increased actin polymerization, leading to YAP nuclear translocation and increased expression of cystein-rich 61, CCN growth factors and BIRC apoptosis inhibitors, followed by enhanced follicle growth. Here we summarize evidence suggesting the role of mechanical stress on follicle growth in the ovary and describe recent use of ovary-damaging procedures to treat ovarian infertility. Ovarian fragmentation, together with in vitro incubation with Akt-stimulating drugs, formed the basis of an in vitro activation (IVA) therapy to treat patients with premature ovarian insufficiency, whereas ovarian fragmentation alone (drug-free IVA) was successful in treating patients with premature ovarian insufficiency with recent menses cessation. For middle-aged women with poor ovarian responses and diminished ovarian reserve, drug-free IVA was also effective in promoting follicle growth for infertility treatment. In addition, an in vivo follicle activation approach based on laparoscopic ovarian incision showed promise for patients with resistant ovary syndrome. With initial success using mechanical disruption approaches, future investigation could evaluate possibilities to refine mechanical methods and to locally administer actin polymerization-enhancing drugs for ovarian infertility treatment.
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http://dx.doi.org/10.1016/j.fertnstert.2020.07.031DOI Listing
September 2020

Placensin is a glucogenic hormone secreted by human placenta.

EMBO Rep 2020 06 24;21(6):e49530. Epub 2020 Apr 24.

Henan Key Laboratory of Reproduction and Genetics, Center for Reproductive Medicine, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

FBN1 encodes asprosin, a glucogenic hormone, following furin cleavage of the C-terminus of profibrillin 1. Based on evolutionary conservation between FBN1 and FBN2, together with conserved furin cleavage sites, we identified a peptide hormone placensin encoded by FBN2 based on its high expression in trophoblasts of human placenta. In primary and immortalized murine hepatocytes, placensin stimulates cAMP production, protein kinase A (PKA) activity, and glucose secretion, accompanied by increased expression of gluconeogenesis enzymes. In situ perfusion of liver and in vivo injection with placensin also stimulate glucose secretion. Placensin is secreted by immortalized human trophoblastic HTR-8/SVneo cells, whereas placensin treatment stimulates cAMP-PKA signaling in these cells, accompanied by increases in MMP9 transcripts and activities, thereby promoting cell invasion. In pregnant women, levels of serum placensin increase in a stage-dependent manner. During third trimester, serum placensin levels of patients with gestational diabetes mellitus are increased to a bigger extent compared to healthy pregnant women. Thus, placensin represents a placenta-derived hormone, capable of stimulating glucose secretion and trophoblast invasion.
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http://dx.doi.org/10.15252/embr.201949530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271319PMC
June 2020

A case of desmoplastic fibroma of bone with CTNNB1 point mutation.

Oral Surg Oral Med Oral Pathol Oral Radiol 2020 Apr 16;129(4):e230-e233. Epub 2019 Sep 16.

Department of Diagnostic Pathology, Faculty of Medicine, Oita University, Oita, Japan.

Desmoplastic fibroma of bone (DFB), a bone tumor, is considered to be an osseous counterpart of desmoid-type fibromatosis (DF). Herein, we report a case of DFB with CTNNB1 point mutation. The 5-year-old male patient had complained of trismus and pain in the jaw. Magnetic resonance imaging revealed a mass in the left mandible. Radical treatment involved surgical resection. Microscopically, the lesion consisted of a bundle-like proliferation of uniform spindle-shaped cells with abundant collagenous stroma, which resembled DF. Immunohistochemical analysis revealed intranuclear accumulation of β-catenin in the tumor cells. Based on clinical and histologic analysis, we diagnosed the patient as having DFB. We examined the CTNNB1 and APC sequence and found an A-to-G transition at codon 41 of CTNNB1; i.e., Thr was substituted by Ala. Our findings suggest that the dysregulation of Wnt/β-catenin signaling pathway is related to the tumorigenesis of some cases of DFB. This hypothesis indicates that there are some cases of DFB in which nuclear positive expression of β-catenin is useful for diagnosis.
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http://dx.doi.org/10.1016/j.oooo.2019.09.007DOI Listing
April 2020

Rapamycin treatment maintains developmental potential of oocytes in mice and follicle reserve in human cortical fragments grafted into immune-deficient mice.

Mol Cell Endocrinol 2020 03 27;504:110694. Epub 2019 Dec 27.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, 216-8511, Japan. Electronic address:

The ovarian follicle pool size is limited; it decreases with age and following germ cell-damaging chemo- or radiation therapies. Due to a trend of delaying child-bearing age in the modern society, it is important to investigate the possibility to maintain the follicle reserve for middle-aged women and cancer-bearing patients subject to therapies. Earlier studies demonstrated the important role of the mammalian targets of the rapamycin (MTOR) signaling pathway in the activation of primordial follicles and suggested that treatment with the MTOR inhibitor rapamycin could maintain the follicle pool in rodents. Here, we confirmed the ability of rapamycin treatment for 3 weeks to suppress primordial follicle development and to maintain follicle pool size in mice. We further demonstrated that the developmental potential of oocytes was not affected by rapamycin treatment and the effect of rapamycin to decrease initial follicle recruitment is reversible. Using human ovarian cortical fragments grafted into immune-deficient mice, we demonstrated the ability of rapamycin to suppress follicle growth from the primordial stage. Our studies provide the basis for further studies on the possibility of using MTOR inhibitors to maintain follicle reserve in middle-aged women and cancer patients before/during germ cell-damaging therapies.
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http://dx.doi.org/10.1016/j.mce.2019.110694DOI Listing
March 2020

Drug-free in-vitro activation of follicles for infertility treatment in poor ovarian response patients with decreased ovarian reserve.

Reprod Biomed Online 2020 Feb 19;40(2):245-253. Epub 2019 Sep 19.

Department of Ob/Gyn, Stanford University School of Medicine, Stanford CA, USA.

Research Question: The recently developed in-vitro activation (IVA) approach provides a promising infertility treatment for patients with premature ovarian insufficiency. The IVA method promotes growth of residual ovarian follicles following ovarian tissue fragmentation leading to Hippo signalling disruption, together with in-vitro incubation with Akt stimulators. As poor ovarian response (POR) patients with decreased ovarian reserve (DOR) have multiple secondary follicles, this study tested whether Hippo signalling disruption alone using in-vitro ovarian cortical fragmentation, followed by autologous grafting, was sufficient to promote follicle growth.

Design: A case series study.

Results: In 9 out of 11 POR patients with DOR treated with a simplified IVA procedure, increases in antral follicle numbers in multiple growth waves were detected following FSH treatment. Subsequent injection with human chorionic gonadotrophin allowed retrieval of more mature oocytes for IVF (median antral follicle counts before and after IVA per ovarian stimulation: 1.0 versus 2.6) with 68.7% fertilization rates and 56.9% showing high-quality embryonic development. One natural conception and 16 embryo transfers in five patients resulted in one live birth, two ongoing pregnancies and one miscarriage. Three additional patients and the miscarriage patient have cryopreserved embryos for future transfer.

Conclusions: The present drug-free IVA approach may be suitable for POR patients with DOR, as it increased the number of antral follicles. The procedure also eliminated the need for 2-day incubation with drugs and required only one surgery. This approach could allow the retrieval of more oocytes in middle-aged women to achieve higher pregnancy rates and deserves proper evaluation in future randomized controlled trials.
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http://dx.doi.org/10.1016/j.rbmo.2019.09.007DOI Listing
February 2020

Seasonality in spatial distribution: Climate and land use have contrasting effects on the species richness of breeding and wintering birds.

Ecol Evol 2019 Jul 20;9(13):7549-7561. Epub 2019 Jun 20.

Department of Forest Science, Graduate School of Agriculture Hokkaido University Hokkaido Japan.

Aim: Many studies have examined large-scale distributions of various taxa and their drivers, emphasizing the importance of climate, topography, and land use. Most studies have dealt with distributions over a single season or annually without considering seasonality. However, animal distributions and their drivers can differ among seasons because many animals migrate to suitable climates and areas with abundant prey resources. We aim to clarify seasonality in bird distributions and their drivers.

Location: Japan.

Methods: We examined the effects of climate (annual mean temperature, snow depth), topography (elevation), and land use (extent of surrounding habitat) on bird species richness, in the breeding and wintering seasons separately, using nationwide data (254 forest and 43 grassland sites, respectively). We separately analyzed the species richness of all species, residents, short-, and long-distance migrants in forests and grasslands.

Results: In the breeding season, the annual mean temperature negatively affected all groups (except for forest and grassland residents), and the extent of surrounding habitat positively affected many groups. By contrast, in the wintering season, temperature positively affected all groups (except for forest residents), and the extent of surrounding habitat positively affected only grassland long-distance migrants. In both seasons, the species richness of forest and grassland residents was high in regions of moderate and high temperature, respectively. Moreover, snow depth negatively affected all forest groups in the wintering season. Mapping expected species richness suggested that regions with different climates served as habitats for different groups during different seasons.

Main Conclusions: All regions were important bird habitats depending on the season, reflecting the contrasting effects of temperature across seasons. In the breeding season, surrounding land use was also an important driver. To understand the seasonal role that each region and environment plays in maintaining species/communities, a large-scale study considering both environmental seasonality and species distribution is needed.
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http://dx.doi.org/10.1002/ece3.5286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636198PMC
July 2019

Decreased effector regulatory T cells and increased activated CD4 T cells in premature ovarian insufficiency.

Am J Reprod Immunol 2019 06 23;81(6):e13125. Epub 2019 Apr 23.

Department of Obstetrics and Gynecology, University of Toyama, Toyama, Japan.

Problem: Premature ovarian insufficiency (POI) is a clinical syndrome defined by the loss of ovarian activity before 40 years old. An autoimmune mechanism is suggested to be involved in the development of POI. Therefore, we examined the relationship between peripheral blood regulatory T (Treg) cells and autoantibodies in POI.

Method Of Study: Thirty POI patients and 23 control women were enrolled in the study. Using flow cytometry, we measured the abundance of CD4 T, CD4 CD69 T, CD8 T, CD8 CD69 T, naive Treg, effector Treg, and FOXP3 effector T cells in peripheral blood. Antinuclear and anti-thyroglobulin antibody (Tg-Ab) titers were measured in POI patients.

Results: The number of CD4 T or CD4 CD69 T cells was significantly higher in POI patients (P = 0.045, and P = 0.030), and there were significantly fewer effector Treg cells in POI patients (P = 0.016) than in the controls. There were significant negative correlations between effector Treg cells and Tg-Abs (r = -0.584, P = 0.0282), and between effector Treg cells and CD4 CD69 T cells (r = -0.415, P = 0.0226) in POI patients.

Conclusion: This is the first report of decreased numbers of effector Treg cells and increased CD4 CD69 activated T cells in peripheral blood in POI, suggesting that POI is an autoimmune disease.
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http://dx.doi.org/10.1111/aji.13125DOI Listing
June 2019

Distinctive subpopulations of the intestinal microbiota are present in women with unexplained chronic anovulation.

Reprod Biomed Online 2019 Apr 22;38(4):570-578. Epub 2018 Dec 22.

Department of Obstetrics and Gynecology, Keio University Graduate School of Medicine, 35 Shinanomachi, -Shinjyuku-ku, Tokyo 160-8582, Japan.

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.
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http://dx.doi.org/10.1016/j.rbmo.2018.12.026DOI Listing
April 2019

Heterosis extends the reproductive ability in aged female mice†.

Biol Reprod 2019 04;100(4):1082-1089

Department of Endocrinology, Hematology and Gerontology, Chiba University Graduate School of Medicine, Chiba, Japan.

Heterosis is the beneficial effect of genetical heterogeneity in animals and plants. Although heterosis induces changes in the cells and individual abilities, few reports have described the effect of heterosis on the female reproductive ability during aging. In this study, we investigated the reproductive capability of genetically heterogeneous (HET) mice established by the four-way crossing of C57BL/6N, BALB/c, C3H/He, and DBA/2. We found the HET females naturally and repeatedly produced offspring, even in old age (14-18 months of age). We also found that HET females showed a significantly enlarged body and organ sizes in both youth and old age. In histological analyses, the numbers of primordial follicles, primary follicles, secondary follicles, and corpora lutea were significantly increased in the old ovaries of HET females compared with those in inbred C57BL/6 mice of the same age. In vitro fertilization experiments revealed that aged HET oocytes showed identical rates of fertilization, early development, and birth compared to those of young and old C57BL/6 oocytes. We further found the significantly increased expression of sirtuin genes concomitant with the up-regulation of R-spondin2 in old HET ovaries. These results confirm the novel phenotype, characterized by fertility extension and follicular retention due to heterosis, in old HET females. The HET female will be a valuable model for clarifying the mechanism underlying the effect of heterosis in the field of reproduction.
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http://dx.doi.org/10.1093/biolre/ioy260DOI Listing
April 2019

Regulation of follicle growth through hormonal factors and mechanical cues mediated by Hippo signaling pathway.

Syst Biol Reprod Med 2018 Feb 9;64(1):3-11. Epub 2017 Dec 9.

a Department of Obstetrics and Gynecology , St. Marianna University School of Medicine , Kawasaki City , Kanagawa , Japan.

The ovary is an interesting organ that shows major structural changes within a short period of time during each reproductive cycle. Follicle development is controlled by local paracrine and systemic endocrine factors. Many hormonal and molecular analyses have been conducted to find the mechanisms underlying structural changes in ovaries, However, exact mechanisms still remain to be determined. Recent development of mechanobiology facilitates the understanding on the contribution of physical forces and changes in the mechanical properties of cells and tissues to physiology and pathophysiology. The Hippo signaling pathway is one of the key players in mechanotransduction, providing an understanding of the molecular mechanisms by which cells sense and respond to mechanical signals to regulate cell proliferation and apoptosis for maintaining optimal organ sizes. Our group recently demonstrated the involvement of the Hippo signaling pathway in the regulation of ovarian follicle development. Fragmentation of ovarian cortex into small cubes changed cytoskeletal actin dynamics and induced disruption of the Hippo signaling pathway, leading to the production of CCN growth factors and anti-apoptotic BIRC. These factors, in turn, stimulated secondary follicle growth in vitro and in vivo. In this review, we summarized hormonal regulation of follicular structural changes and further focused on the role of Hippo signaling in the regulation of follicle development. We also suggest a new strategy of infertility treatments in patients with polycystic ovary syndrome and primary ovarian insufficiency based on mechanobiology.
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http://dx.doi.org/10.1080/19396368.2017.1411990DOI Listing
February 2018

Determination of Follicular Localization in Human Ovarian Cortex for Vitrification.

J Adolesc Young Adult Oncol 2018 02 28;7(1):46-53. Epub 2017 Aug 28.

2 Department of Obstetrics and Gynecology, St. Marianna University School of Medicine , Kawasaki, Japan .

Purpose: To determine the optimal follicle localization for ovarian vitrification in adolescent and young adult (AYA)-aged (between 15 and 39 years of age) patients with cancer or primary ovarian insufficiency (POI).

Methods: In total, ovaries from 24 women were included in our study. These include women who received ovariectomy for fertility preservation before gonadotoxic treatments for cancer (n = 4), or for the treatment of POI by the in vitro activation method (n = 8), and other women and infants (0-3 years of age) whose ovaries were autopsied (n = 12). Before cryopreservation, a portion of the ovary sampled from cancer and POI patients was used for histological analysis. Depths of follicles from the surface of ovarian cortices were then measured by using digital imaging software. The locations of the follicles at different developmental stages in the ovarian cortex were noted.

Results: The mean depth at which the primordial and primary follicles were located was 271 μm in infants. This was deeper in women in their twenties, thirties, and forties (501, 462, and 493 μm, respectively). The majority of secondary follicles were located <1000 μm from the ovarian surface (mean depth, 639 μm). In regard to patients with POI, the mean depth of primordial and primary follicles was 566 μm, whereas 70% of secondary follicles were located >1000 μm deep.

Conclusion(s): These findings suggest that <1 mm is a potential optimum thickness of normal ovarian tissue for vitrification and a requirement that thicker ovarian cortices include secondary follicles in POI patients.
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http://dx.doi.org/10.1089/jayao.2017.0028DOI Listing
February 2018

Disorganization of the germ cell pool leads to primary ovarian insufficiency.

Reproduction 2017 06 13;153(6):R205-R213. Epub 2017 Mar 13.

Department of Advanced Reproductive MedicineSt. Marianna University School of Medicine, Kawasaki City, Kanagawa, Japan

The mammalian ovary is an organ that controls female germ cell development, storing them and releasing mature oocytes for transporting to the oviduct. During the fetal stage, female germ cells change from a proliferative state to meiosis before forming follicles with the potential for the growth of surrounding somatic cells. Understanding of molecular and physiological bases of germ cell development in the fetal ovary contributed not only to the elucidation of genetic disorders in primary ovarian insufficiency (POI), but also to the advancement of novel treatments for patients with POI. Accumulating evidence indicates that mutations in , and genes are associated with POI. In addition, cell biology studies revealed the important roles of these genes as essential translational factors for germ cell development. Recent insights into the role of the PI3K (phosphatidylinositol 3-kinase)-Akt signaling pathway in primordial follicle activation allowed the development of a new infertility treatment, IVA ( activation), leading to successful pregnancy/delivery in POI patients. Furthermore, elucidation of genetic dynamics underlying female germ cell development could allow regeneration of oocytes from ES (embryonic stem)/iPS (induced pluripotent stem) cells in mammals. The purpose of this review is to summarize basic findings related to female germ cell development and potential clinical implications, especially focusing on POI etiologies. We also summarize evolving new POI therapies based on IVA as well as oocyte regeneration.
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http://dx.doi.org/10.1530/REP-17-0015DOI Listing
June 2017

In Vitro Activation of Follicles and Fresh Tissue Auto-transplantation in Primary Ovarian Insufficiency Patients.

J Clin Endocrinol Metab 2016 11 29;101(11):4405-4412. Epub 2016 Aug 29.

Reproductive Medical Centre (J.Z., G.Y., F.D., Z.B., L.H., Y.Z., J.W., S.D., J.S., Y.Su), First Affiliated Hospital of Zhengzhou University, Zhengzhou, China; Department of Obstetrics and Gynecology (Y.C., A.J.H.), Stanford University School of Medicine, Stanford, California 94305; and Department of Obstetrics and Gynecology (Y.Sa., K.K.), St. Marianna University School of Medicine, Kawasaki, 216-8511 Kanagawa, Japan.

Context: Recently, two patients with primary ovarian insufficiency (POI) delivered healthy babies after in vitro activation (IVA) treatment followed by auto-transplantation of frozen-thawed ovarian tissues.

Objective: This study sought to report the first case of live birth after IVA treatment following fresh ovarian tissue grafting in patients with POI, together with monitoring of follicle development and serum hormonal changes.

Design: This was a prospective observational cohort study.

Setting: We performed IVA treatment in 14 patients with POI with mean age of 29 years, mean duration since last menses of 3.8 years, and average basal FSH level of 94.5 mIU/mL.

Interventions: Prior to IVA treatment, all patients received routine hormonal treatments with no follicle development. We removed one ovary from patients with POI and treated them with Akt stimulators. We improved upon early procedures by grafting back fresh tissues using a simplified protocol.

Main Outcome Measures: In six of the 14 patients (43%), a total of 15 follicle development waves were detected, and four patients had successful oocyte retrieval to yield six oocytes. For two patients showing no spontaneous follicle growth, human menopausal gonadotropin treatment induced follicle growth at 6-8 months after grafting. After vitro fertilization of oocyte retrieved, four early embryos were derived. Following embryo transfer, one patient became pregnant and delivered a healthy baby boy, with three other embryos under cryopreservation.

Conclusion: IVA technology can effectively activate residual follicles in some patients with POI and allow them to conceive their own genetic offspring. IVA may also be useful for treating patients with ovarian dysfunction including aging women and cancer survivors.
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http://dx.doi.org/10.1210/jc.2016-1589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095246PMC
November 2016

Activation of dormant follicles: a new treatment for premature ovarian failure?

Curr Opin Obstet Gynecol 2016 06;28(3):217-22

aDepartment of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan bDepartment of Obstetrics/Gynecology, Stanford University School of Medicine, Stanford, California, USA.

Purpose Of Review: Premature ovarian failure (POF) is diagnosed by amenorrhea before 40 years of age. Owing to exhaustion of follicles in POF ovaries, egg donation is the only option. Although menstrual cycles cease in POF patients, some of them still contain residual dormant follicles in ovaries. Recently, we developed a new infertility treatment and named it as in-vitro activation (IVA), which enables POF patients to conceive using their own eggs by activation of residual dormant follicles. Here, we summarize data showing the potential of IVA as a new infertility treatment for POF patients.

Recent Findings: Transgenic mouse studies revealed that the stimulation of phosphatidylinositol-3-kinase-AKT-forkhead box O3 pathway activated dormant primordial follicles. In murine and human ovaries, the phosphatase and tensin homolog inhibitors and phosphatidylinositol-3-kinase activators were demonstrated to activate dormant primordial follicles in in-vitro cultures. Subsequent studies showed that ovarian fragmentation suppressed Hippo signaling pathway, leading to ovarian follicle growth. Combining these two methods in an IVA approach followed by ovarian tissue autotransplantation, successful follicle growth, and pregnancies were reported in POF patients. Currently, two healthy babies were delivered, together with two additional pregnancies.

Summary: IVA treatment is a potential infertility therapy for POF patients who have residual follicles.
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http://dx.doi.org/10.1097/GCO.0000000000000268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536116PMC
June 2016

Ovary transplantation: to activate or not to activate.

Hum Reprod 2015 Nov 6;30(11):2457-60. Epub 2015 Sep 6.

Department of Ob/Gyn, Stanford University School of Medicine, Stanford, CA 94305-5317, USA

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http://dx.doi.org/10.1093/humrep/dev211DOI Listing
November 2015

Successful Pregnancy After Sacrectomy Combined With Chemotherapy and Radiation for Ewing Sarcoma: Case Report and Literature Review.

J Pediatr Adolesc Gynecol 2015 Jun 15;28(3):e79-81. Epub 2014 Jul 15.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, Japan.

Background: Ewing sarcoma is considered to have a poor prognosis. Recent advances in multidisciplinary management have resulted in a marked improvement in long-term survival.

Case: We describe a case of successful pregnancy in a patient who underwent sacrectomy combined with multi-agent chemotherapy and radiotherapy for Ewing sarcoma. The patient was diagnosed with Ewing sarcoma of the sacrum at the age of 16. The ovaries were transposed, the uterus was shielded, and a gonadotropin-releasing hormone agonist was used during treatment to protect ovarian function. The patient spontaneously conceived at the age of 27. After an uneventful pregnancy, the patient delivered a healthy neonate at term by cesarean section.

Summary And Conclusions: Successful pregnancy and delivery can be achieved after multi-modality treatment with pretreatment intervention for fertility preservation.
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http://dx.doi.org/10.1016/j.jpag.2014.06.009DOI Listing
June 2015

Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP.

FASEB J 2015 Jun 17;29(6):2423-30. Epub 2015 Feb 17.

*Department of Obstetrics and Gynecology and Department of Otolaryngology, Stanford University School of Medicine, Stanford, California, USA; Department of Obstetrics and Gynecology, St. Mariana University, Kawasaki, Japan; and Department of Obstetrics and Gynecology, Akita University Graduate School of Medicine, Akita, Japan

Hippo signaling pathway consists of conserved serine/threonine kinases to maintain optimal organ sizes. Studies have demonstrated that fragmentation of murine ovaries increases actin polymerization and disrupts Hippo signaling, leading to nuclear translocation of Hippo signaling effector Yes-associated protein (YAP) in ovarian follicles and follicle growth. For patients with polycystic ovarian syndrome showing follicle arrest, ovarian wedge resection and laser drilling promote follicle growth. Because these damaging procedures likely involve actin polymerization, we tested whether actin polymerization-promoting drugs could promote YAP translocation and stimulate follicle growth. Treatment of murine ovaries with μM Jasplakinolide (JASP), an actin polymerization-promoting cyclic peptide, or sphingosine-1-phosphate (S1P), a follicular fluid constituent known to promote actin polymerization, increased the conversion of globular actin to the filamentous form, followed by increased nuclear YAP and expression of downstream connective tissue growth factor (CCN2). After short-term treatments with JASP or S1P, in vitro cultured and in vivo grafted ovaries showed follicle growth. Furthermore, induction of constitutively active YAP in ovarian grafts of transgenic mice enhanced follicle development, whereas treatment of human ovarian cortices with JASP or S1P increased CCN2 expression. Thus, JASP and S1P stimulate follicle growth and are potential therapeutic agents for treating polycystic ovarian syndrome and other ovarian disorders.
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http://dx.doi.org/10.1096/fj.14-267856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447223PMC
June 2015

Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency.

Hum Reprod 2015 Mar 6;30(3):608-15. Epub 2015 Jan 6.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamaeku, Kawasaki, Kanagawa 216-8511, Japan

Study Question: Is ovarian tissue cryopreservation using vitrification followed by in vitro activation (IVA) of dormant follicles a potential approach for infertility treatment of patients with primary ovarian insufficiency (POI)?

Summary Answer: Our vitrification approach followed by IVA treatment is a potential infertility therapy for POI patients whose ovaries contain residual follicles.

What Is Known Already: Akt (protein kinase B) stimulators [PTEN (phosphatase with TENsin homology deleted in chromosome 10) inhibitor and phosphatidyinositol-3-kinase (PI3 kinase) stimulator] activate dormant primordial follicles in vitro and ovarian fragmentation disrupts the Hippo signaling pathway, leading to the promotion of follicle growth. We treated POI patients with a combination of ovarian vitrification, fragmentation and drug treatment, followed by auto-transplantation, and reported successful follicle growth and pregnancies.

Study Design, Size, Duration: Prospective clinical study of 37 infertile women with POI between 12 August 2011 and 1 November 2013. We enrolled 10 new patients since the previous publication.

Participants/materials, Setting, Methods: POI patients were originally selected based on a history of amenorrhea for more than 1 year and elevated serum FSH levels of >40 mIU/ml (n = 31) but this was later changed to >4 months, age <40 years and serum FSH levels of >35 mIU/ml (n = 6) (mean 71.8 ± 30.8, range 35.5-197.6) so as to include patients with a shorter duration of amenorrhea. Under laparoscopic surgery, ovariectomy was performed and ovarian cortices were dissected into strips for vitrification. Some pieces were examined histologically. After warming, two to three strips were fragmented into smaller cubes before culturing with Akt stimulators for 2 days. After washing, ovarian cubes were transplanted beneath the serosa of Fallopian tubes under laparoscopic surgery. Follicle growth was monitored by ultrasound and serum estrogen levels. After oocyte retrieval from mature follicles, IVF was performed.

Main Results And The Role Of Chance: Among 37 patients, 54% had residual follicles based on histology. Among patients with follicles, 9 out of 20 showed follicle growth in auto-grafts with 24 oocytes retrieved from six patients. Following IVF and embryo transfer into four patients, three pregnancies were detected based on serum hCG, followed by one miscarriage and two successful deliveries. For predicting IVA success, we found that routine histological analyses of ovarian cortices and shorter duration from initial POI diagnosis to ovariectomy are valid parameters.

Limitations, Reasons For Caution: Although our findings suggest that the present vitrification protocol is effective for ovarian tissue cryopreservation, we have not compared the potential of vitrification and slow freezing in follicle growth after grafting. We chose the serosa of Fallopian tubes as the auto-grating site due to its high vascularity and the ease to monitor follicle growth. Future studies are needed to evaluate the best auto-grafting sites for ovarian tissues. Also, future studies are needed to identify biological markers to indicate the presence of residual follicles in POI to predict IVA treatment outcome.

Wider Implications Of The Findings: In POI patients, ovarian reserve, namely the pool of residual follicles, continues to diminish with age. If one ovary is cryopreserved at an earlier stage of POI, patients could undergo additional non-invasive infertility treatments before the final decision for the IVA treatment. Furthermore, in the cases of unmarried POI patients, cryopreservation of ovarian tissues allows their fertility preservation until they desire to bear children.

Study Funding/competing Interests: This work was supported by Grant-In-Aid for Scientific Research (Research B: 24390376, Challenging Exploratory Research: 24659722, and Innovative Areas, Mechanisms regulating gamete formation in animals: 26114510) and by research funds from the Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest.

Trial Registration Number: UMIN000010828.
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http://dx.doi.org/10.1093/humrep/deu353DOI Listing
March 2015

The role of menstrual cycle phase and AMH levels in breast cancer patients whose ovarian tissue was cryopreserved for oncofertility treatment.

J Assist Reprod Genet 2015 Feb 3;32(2):305-12. Epub 2014 Dec 3.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki City, Kanagawa prefecture, Japan, 216-8511.

Purpose: To determine the factors that affect oocyte extraction efficiency when using the "combined procedure". In the present "combined procedure" ovarian tissue cryopreservation and oocyte extraction from an isolated ovary, later used in In Vitro Maturation (IVM), are performed concurrently.

Methods: Data were analyzed retrospectively and obtained from the clinical records of 27 young breast cancer patients referred for fertility preservation.

Results: The patients' mean age was 33.7 (±3.8) years, mean serum anti-Müllerian hormone (AMH) concentration was 3.5 (±2.1) ng/ml, and mean number of extracted oocytes was 8.3 (±6.1). The phase of menstruation (follicular or luteal) did not affect either the number of oocytes extracted (P = 0.99) nor oocyte survival or maturation rates. Likewise, the number of oocytes that could be extracted was not affected by the type of laparoscopic procedure (multiple-port or single-incision laparoscopy; P = 0.94) or the molecular subtype of breast cancer (either Luminal A or B; P = 0.52). Analysis revealed that the number of extracted oocytes was well-correlated with the patient's AMH serum level and age (coefficient of correlation: 0.60 and -0.48, respectively).

Conclusion: We conclude that the outcome of the "combined procedure" primarily depends upon the patient's serum AMH level and age. Importantly, the "combined procedure" may be used during any phase of the menstrual cycle to preserve the fertility of breast cancer patients.
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http://dx.doi.org/10.1007/s10815-014-0392-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354192PMC
February 2015

Intraovarian control of early folliculogenesis.

Endocr Rev 2015 Feb 9;36(1):1-24. Epub 2014 Sep 9.

Program of Reproductive and Stem Cell Biology (A.J.W.H., Y.C.), Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317; Department of Obstetrics and Gynecology (K.K.), St. Mariana University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan; Department of Reproductive Medicine & Gynecology (B.C.J.M.F.), University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

Although hormonal regulation of ovarian follicle development has been extensively investigated, most studies concentrate on the development of early antral follicles to the preovulatory stage, leading to the successful use of exogenous FSH for infertility treatment. Accumulating data indicate that preantral follicles are under stringent regulation by FSH and local intraovarian factors, thus providing the possibility to develop new therapeutic approaches. Granulosa cell-derived C-type natriuretic factor not only suppresses the final maturation of oocytes to undergo germinal vesicle breakdown before ovulation but also promotes preantral and antral follicle growth. In addition, several oocyte- and granulosa cell-derived factors stimulate preantral follicle growth by acting through wingless, receptor tyrosine kinase, receptor serine kinase, and other signaling pathways. In contrast, the ovarian Hippo signaling pathway constrains follicle growth and disruption of Hippo signaling promotes the secretion of downstream CCN growth factors capable of promoting follicle growth. Although the exact hormonal factors involved in primordial follicle activation has yet to be elucidated, the protein kinase B (AKT) and mammalian target of rapamycin signaling pathways are important for the activation of dormant primordial follicles. Hippo signaling disruption after ovarian fragmentation, combined with treating ovarian fragments with phosphatase and tensin homolog (PTEN) inhibitors and phosphoinositide-3-kinase stimulators to augment AKT signaling, promote the growth of preantral follicles in patients with primary ovarian insufficiency, leading to a new infertility intervention for such patients. Elucidation of intraovarian mechanisms underlying early folliculogenesis may allow the development of novel therapeutic strategies for patients diagnosed with primary ovarian insufficiency, polycystic ovary syndrome, and poor ovarian response to FSH stimulation, as well as for infertile women of advanced reproductive age.
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http://dx.doi.org/10.1210/er.2014-1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309737PMC
February 2015

Analysis of late-onset ovarian insufficiency after ovarian surgery: retrospective study with 75 patients of post-surgical ovarian insufficiency.

PLoS One 2014 23;9(5):e98174. Epub 2014 May 23.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kawasaki city, Kanagawa prefecture, Japan.

The primary objectives of the present study are to determine the period of onset of ovarian insufficiency after surgery and to confirm potential risk factors for ovarian insufficiency after surgery for the removal of benign ovarian cysts. Data were obtained from 75 patients who underwent surgery for benign ovarian cysts prior to the onset of ovarian insufficiency. Our analysis included 835 ovarian insufficiency patients who were referred to our institution from July 2003 to July 2013. Several epidemiological parameters of ovarian insufficiency after surgery (age at operation, period of onset of ovarian insufficiency, operation procedure, and pathological diagnosis) were investigated. Of the 835 patients who had ovarian insufficiency, 75 patients (9.0%) underwent ovarian surgery before the onset of ovarian insufficiency. Of those 75 patients, 66 patients (88.0%) underwent cystectomy. For the majority of the 75 patients the surgical indication was the presence of endometriotic cysts (57 patients; 76.0%). Twelve patients (16.0%) underwent multiple surgeries (all bilateral cystectomies). The mean age of the patients at the time of surgery was 27.8±5.5 years-old, and the mean period of onset of ovarian insufficiency was 5.8±3.8 years. In patients with cystectomy, the patient's age at the time of surgery and period of onset of ovarian insufficiency was well-correlated (coefficient of correlation; hemilateral endometriotic cystectomy: -0.64, bilateral endometriotic cystectomy: -0.61, and multiple endimetriotic cystectomy: -0.40). We found that cystectomy of endometriotic cysts is the potential risk factor for ovarian insufficiency after surgery, at times, the onset of ovarian insufficiency long after cystectomy. Therefore, it is important to monitor ovarian reserve for an extended period of time after ovarian surgery. It is particularly important to monitor ovarian reserve long-term for patients who wish to conceive in the future and to suggest a variety of infertility treatments appropriate for their ovarian reserve.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098174PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032277PMC
June 2015

Suppression of choriocarcinoma invasion and metastasis following blockade of BDNF/TrkB signaling.

Cancer Med 2013 Dec 7;2(6):849-61. Epub 2013 Nov 7.

Department of Obstetrics and Gynecology, St. Marianna University School of Medicine, Kanagawa, 216-8511, Japan; Department of Obstetrics and Gynecology, Akita University Graduate School of Medicine, Akita, 010-8543, Japan.

Brain-derived neurotrophic factor (BDNF) acts through its cognate receptor tyrosine kinase-B (TrkB) to regulate diverse physiological functions in reproductive and other tissues. In normal and malignant trophoblastic cells, the BDNF/TrkB signaling promotes cell growth. Due to the highly malignant nature of choriocarcinoma, we investigated possible involvement of this system in choriocarcinoma cell invasion and metastasis. We demonstrated that treatment of cultured choriocarcinoma cells, known to express both BDNF and TrkB, with a soluble TrkB ectodomain or a Trk receptor inhibitor K252a suppressed cell invasion accompanied with decreased expression of matrix metalloproteinase-2, a cell invasion marker. In vivo studies using a tumor xenograft model in athymic nude mice further showed inhibition of cell invasion from tumors to surrounding tissues following the suppression of endogenous TrkB signaling. For an in vivo model of choriocarcinoma metastasis, we performed intravenous injections of JAR cells expressing firefly luciferase into severe combined immunodeficiency (SCID) mice. Treatment with K252a inhibited metastasis of tumors to distant organs. In vivo K252a treatment also suppressed metastatic tumor growth as reflected by decreased cell proliferation and increased apoptosis and caspases-3/7 activities, together with reduced tissue levels of a tumor marker, human chorionic gonadotropin-β. In vivo suppression of TrkB signaling also led to decreased expression of angiogenic markers in metastatic tumor, including cluster of differentiation 31 and vascular endothelial growth factor A. Our findings suggested essential autocrine/paracrine roles of the BDNF/TrkB signaling system in choriocarcinoma invasion and metastasis. Inhibition of this signaling could serve as the basis to develop a novel therapy for patients with choriocarcinoma.
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http://dx.doi.org/10.1002/cam4.158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892389PMC
December 2013

Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment.

Proc Natl Acad Sci U S A 2013 Oct 30;110(43):17474-9. Epub 2013 Sep 30.

Department of Obstetrics and Gynecology and Department of Advanced Reproductive Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.
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http://dx.doi.org/10.1073/pnas.1312830110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808580PMC
October 2013

Solid variant of keratocystic odontogenic tumor of the mandible: report of a case with a clear cell component and review of the literature.

Oral Surg Oral Med Oral Pathol Oral Radiol 2013 Nov 3;116(5):e393-8. Epub 2013 May 3.

Professor and Head, Department of Dentistry and Oral-Maxillo-Facial Surgery, Faculty of Medicine, Oita University, Oita, Japan. Electronic address:

This is a case report on a solid variant of keratocystic odontogenic tumor arising in the mandible, which aggressively infiltrated into the cancelous spaces and involved the periosteal connective tissue of the mandible. The patient was a 57-year-old woman with an ill-defined radiolucent lesion having a moth-eaten pattern in the left molar region of the mandible. Computed tomography scans revealed that the tumor penetrated the buccal cortical plate of the mandible. Histologically, the lesion was characterized by multicystic spaces lined with a thin layer of keratinizing squamous epithelium, which contained basal cells with palisaded hyperchromatic nuclei. Lumina were filled with concentric layers of parakeratin. An additional feature was the appearance of a conspicuous clear cell component showing intraluminal papillary proliferation or forming small cord-like nests in the fibrous stroma. The patient underwent segmental mandibulectomy followed by reconstruction using a titanium plate. A 20-year follow-up revealed no recurrence of the tumor.
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http://dx.doi.org/10.1016/j.oooo.2013.02.020DOI Listing
November 2013