IVF Namba Clinic

Nishi-Tokyo-shi, Japan

IVF Namba Clinic

Nishi-Tokyo-shi, Japan
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PubMed | IVF Namba Clinic and HORAC Grand Front Osaka Clinic
Type: Journal Article | Journal: Zygote (Cambridge, England) | Year: 2016

Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.

Allahbadia G.N.,Rotunda The Center for Human Reproduction | Morimoto Y.,IVF Namba Clinic
Ovarian Stimulation Protocols | Year: 2015

Ovarian Stimulation Protocols is a concise handbook that aims to deliver everything the reader needs to know for performing a risk-free ovarian stimulation for assisted reproductive technique (ART) and get a favorable outcome. Review of crucial issues such as the significance of monitoring ovarian stimulation, advantages and disadvantages of ovarian hyperstimulation versus minimal stimulation, and the use of various drug regimens and stimulation protocols for various patient sub-sets, will help clinicians in selecting the better or more appropriate protocols. The contributors of this book have leading scientific and clinical backgrounds, with years of experience to support their views. The book serves as a handy practical guide, targeting and settling clinical dilemma that ART practitioners commonly experience in their clinics, while providing a window to the newer developments. © Springer India 2016.

Hashimoto S.,IVF Namba Clinic | Kato N.,Kinki University | Saeki K.,Kinki University | Morimoto Y.,IVF Namba Clinic
Fertility and Sterility | Year: 2012

Objective: To assess the developmental kinetics of human embryos and their ability to develop to morphologically normal blastocysts. Design: Experimental study on human embryos donated for research using a time-lapse imaging system based on individual embryo culture in poly(dimethylsiloxane) microwells and monitored using a microscope inside the incubator. Setting: Private fertility clinic. Patient(s): Surplus embryos donated by couples after undergoing fertility treatment. Intervention(s): None. Main Outcome Measure(s): Blastocyst score and times required from beginning to completion of the second and third mitotic divisions. Result(s): The time required for completion of the second division (the three- to four-cell stage) was shorter in embryos that developed to high-scoring blastocysts (0.7 hours, n = 17) than in those forming low-scoring blastocysts (3.7 hours, n = 24). Similarly, the mean time required to completion of the third division (five- to eight-cell stage) was also significantly shorter in embryos forming high-scoring blastocysts (5.7 hours) than among those forming low-scoring blastocysts (16.9 hours). Conclusion(s): Individual embryos with the potential to develop to high-scoring blastocysts could be selected at 2-3 days of culture using this system by examining the times required to complete the second and third mitotic divisions. © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.

Hashimoto S.,IVF NAMBA CLINIC | Suzuki N.,Kanagawa University | Yamanaka M.,IVF NAMBA CLINIC | Hosoi Y.,Kinki University | And 2 more authors.
Reproductive BioMedicine Online | Year: 2010

This study assessed the effects of vitrification solutions and equilibration times on morphology of cynomolgus ovarian tissues. Ovarian cortical sections (0.1-0.2 cm thickness) of seven cynomolgus monkeys were randomly allocated to either a control group or one of six vitrification groups. Ovarian tissue sections were vitrified ultra-rapidly by placing them directly into liquid nitrogen using two different vitrification solutions (VSEGP: 5.64 mol/l ethylene glycol + 5% (w/v) polyvinylpyrrolidone + 0.5 mol/l sucrose; and VSED: 3.22 mol/l ethylene glycol + 2.56 mol/l dimethylsulphoxide + 0.5 mol/l sucrose) after three different exposure times (5-20 min). After warming, follicle morphology was analysed using light and transmission electron microscopy. The proportion of morphologically normal follicles vitrified using VSED after a 5-min exposure was lower (P < 0.05) than those vitrified by other conditions. The proportion of normally structured mitochondria in oocytes of preantral follicles vitrified after a 5-min exposure to VSED (56%) was lower (P < 0.01) than those vitrified by other conditions (78-88%). Following tissue vitrification with VSED, the surface ratio of lysosome was increased compared with non-vitrified oocytes (1.64% versus 1.11%; P < 0.05). These results indicate that VSEGP can support the morphology of vitrified preantral follicles and oocytes. © 2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Hashimoto S.,IVF Namba Clinic | Amo A.,IVF Namba Clinic | Hama S.,IVF Namba Clinic | Ito K.,IVF Namba Clinic | And 2 more authors.
Human Reproduction | Year: 2013

STUDY QUESTION Does the human embryo growth rate affect the outcome of vitrified-warmed blastocyst transfer? SUMMARY ANSWER Following vitrification, the incidence of abnormal spindle morphology was increased and the implantation competence was decreased in growth-retarded embryos compared with normally developing embryos. WHAT IS KNOWN ALREADY Various types of spindle abnormality occur in human cleavage- and blastocyst-stage embryos. However, the incidence of abnormal spindle morphology in growth-retarded blastocysts is not known. Furthermore, there is conflicting data about the implantation potential of such blastocysts. STUDY DESIGN, SIZE, DURATION This was a retrospective cohort study including 878 single vitrified-warmed blastocyst transfers between 9 January 2010 and 10 July 2012, and an experimental study using 121 vitrified-warmed blastocysts donated to research. A comparison on the implantation potential and spindle shape of vitrified-warmed blastocysts was made between normally developing and growth-retarded blastocysts.PARTICIPANTS/ MATERIALS, SETTING, METHODS In the clinical study, we compared the implantation rates of vitrified-warmed embryos that developed to the blastocyst stage on Day 5 after insemination (normally developing embryos) with those that required culture to Day 6 (growth-retarded embryo). In the experimental study, donated vitrified-warmed blastocysts were immunostained with an anti-α-tubulin antibody to visualize microtubules, an anti-γ-tubulin antibody to image centrosomes and Hoechst 33342 or 4,6-diamidino-2-phenylindole to visualize DNA. Confocal image analysis captured a z-series stack of 0.5-μm-thick optical sections encompassing the entire blastocyst. Only spindles with fusiform poles and with chromosomes aligned at the equator were classified as normal. MAIN RESULTS AND THE ROLE OF CHANCE The implantation rate of growth-retarded embryos (47%, n = 270) was significantly lower (P < 0.05) than that of normally developing embryos (57%, n = 608). A total of 533 spindles were analyzed in Day 5 and 6 vitrified-warmed blastocysts. The incidence of abnormal spindles in the growth-retarded embryos (47%, n = 274) was significantly higher (P < 0.01) than in the normally developing embryos (30%, n = 259). LIMITATIONS, REASONS FOR CAUTION Further studies are required to clarify the link between an increase in abnormal spindle formation and a decrease in embryonic implantation potential. WIDER IMPLICATIONS OF THE FINDINGS This study provided new insights into the possible implications of abnormalities in spindle formation in growth-retarded human blastocysts. STUDY FUNDING/COMPETING INTEREST(S) Part of this work was supported by a grant from the Japan Society for the Promotion of Science (JPS-RFTF 23 580 397 to S.H.). No other competing interests are declared. © 2013 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Suzuki N.,St. Marianna University School of Medicine | Hashimoto S.,IVF Namba Clinic | Igarashi S.,St. Marianna University School of Medicine | Takae S.,St. Marianna University School of Medicine | And 6 more authors.
Human Reproduction | Year: 2012

background: Ovarian tissue cryopreservation by rapid cooling (vitrification) is a convenient fertility preservation option. However, the progress of vitrified ovarian tissue after transplantation is not well understood in primates. methods: For tissues from cynomolgus monkeys, we used closed straw vitrification and open cryosupport vitrification in which tissues are immersed directly into liquid nitrogen. Following warming, ovarian cortical pieces were autotransplanted and their function was monitored by computed tomography (CT), hormone assays and oocyte recovery, ICSI and embryo transfers (ETs). results: Hormone cycles were restored in 6 of 7 animals in a mean of 126 days with no significant difference between the two vitrification regimens. The presence of new blood vessels supplying the grafted ovarian tissue was confirmed by contrast-enhanced CT. Oocyte retrieval from two monkeys after transplantation of the ovarian cortex vitrified by cryosupport vitrification yielded a total of nine oocytes of which six fertilized after ICSI, but ETs did not lead to any pregnancies. conclusions: This work shows that CT can give insight into ovarian function after heterotopic transplantation, and that heterotopic autografts of vitrified ovarian cortex can give rise to long-term ovarian function and embryos in a primate model. It remains to be established how outcomes following rapid vitrification compared with outcomes following conventional slow cooling procedures. © The Author 2012.

Suzuki N.,St. Marianna University School of Medicine | Yoshioka N.,St. Marianna University School of Medicine | Takae S.,St. Marianna University School of Medicine | Sugishita Y.,St. Marianna University School of Medicine | And 4 more authors.
Human Reproduction | Year: 2015

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)? summaryanswer: 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 1November 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, ovariectomywas 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 interest(s): 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 fromthe Smoking Research Foundation, and the Takeda Science Foundation. None of the authors has a conflict of interest. trial registration number: UMIN000010828. © 2015 The Author.

Yamanaka M.,IVF Namba Clinic | Hashimoto S.,IVF Namba Clinic | Amo A.,IVF Namba Clinic | Ito-Sasaki T.,Clino Corporation | And 2 more authors.
Human Reproduction | Year: 2011

Background: In this study, we aimed to develop a model for embryo selection based on oxygen consumption following cryopreservation, the relationship between the developmental competence of blastocysts and their oxygen consumption was assessed.Methods: Oxygen consumption of vitrified-warmed human blastocysts was measured at 0, 1.5, 3, 4.5, 6, 7.5, 9 and 24 h after warming using scanning electrochemical microscopy. On the basis of their developmental stage at 24 h, blastocysts were classified into four groups (hatched, hatching, arrested and degenerated). Moreover, cytochrome c oxidase (CCO) activity in vitrified-warmed blastocysts was assessed at 0 and 24 h.Results: The oxygen consumption rate of blastocysts just after warming was significantly lower than that of non-vitrified blastocysts (P< 0.05). The oxygen consumption rate of blastocysts was significantly higher in the hatched group than in the arrested and the degenerated groups after 1.5 h (P< 0.05) and than in the hatching group (P< 0.05) at 7.5 and 9 h. Moreover, CCO activity was absent in vitrified-warmed blastocysts at 0 h, but was detected at 24 h. Conclusions: The respiratory rate of vitrified blastocysts after warming was significantly lower than non-cryopreserved blastocysts. Oxygen consumption of blastocysts with high developmental potential was restored earlier than that of blastocysts with low developmental potential. The Results: of this study suggest that it is possible to select vitrified-warmed blastocysts with high developmental potential based on their respiratory activity. © 2011 The Author. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Hashimoto S.,IVF Namba Clinic | Amo A.,IVF Namba Clinic | Hama S.,IVF Namba Clinic | Ohsumi K.,IVF Namba Clinic | And 2 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2013

Purpose: Closed-system vitrification may enable the risk of contamination to be minimised. We performed three studies to compare the developmental competence of human embryos vitrified using either a closed vitrification system (CVS; Rapid-i®) or an open vitrification system (OVS; Cryo-top®). Methods: The first study was performed in vitro using 66 zygotes previously vitrified at pronuclear stage. These were warmed and randomised 1:1 to revitrification using either the OVS or the CVS. After re-warming, embryo development and blastocyst cell number were assessed. For the second study, also performed in vitro, 60 vitrified-warmed blastocysts were randomised 1:1:1 into three groups (OVS or CVS revitrification, or no revitrification). The proportion of dead cells was assessed by staining. The third study was performed in vivo, using 263 high-grade blastocysts randomly assigned to vitrification using either the CVS (n = 100) or the OVS (n = 163). After warming, single blastocyst transfer was performed. Results: There were no differences between the CVS and the OVS in survival rate (100 % vs. 97 %), blastulation rate (96 h: 50 % vs. 50 %; 120 h: 68 % vs. 56 %), proportion of good blastocysts (96 h: 32 % vs. 22 %, 120 h: 47 % vs. 41 %), or mean number of cells (137 vs. 138). The proportion of dead cells in blastocysts re-vitrified by CVS (31 %) was similar to that for OVS (38 %) and non-revitrification (32 %). In vivo, the implantation rate for blastocysts vitrified using the CVS (54 %) was similar to that with the OVS (53 %). Conclusion: Our studies consistently indicate that human embryos may be vitrified using a CVS without impairment of developmental competence. © 2013 Springer Science+Business Media New York.

Sakakibara Y.,RIKEN | Hashimoto S.,IVF Namba Clinic | Nakaoka Y.,IVF Namba Clinic | Kouznetsova A.,Karolinska Institutet | And 2 more authors.
Nature Communications | Year: 2015

The frequency of chromosome segregation errors during meiosis I (MI) in oocytes increases with age. The two-hit model suggests that errors are caused by the combination of a first hit that creates susceptible crossover configurations and a second hit comprising an age-related reduction in chromosome cohesion. This model predicts an age-related increase in univalents, but direct evidence of this phenomenon as a major cause of segregation errors has been lacking. Here, we provide the first live analysis of single chromosomes undergoing segregation errors during MI in the oocytes of naturally aged mice. Chromosome tracking reveals that 80% of the errors are preceded by bivalent separation into univalents. The set of the univalents is biased towards balanced and unbalanced predivision of sister chromatids during MI. Moreover, we find univalents predisposed to predivision in human oocytes. This study defines premature bivalent separation into univalents as the primary defect responsible for age-related aneuploidy. © 2015 Macmillan Publishers Limited. All rights reserved.

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