Publishing type：Research paper (scientific journal)   Publisher：Medknow  

DOI： 10.4103/aja202152

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Cancer therapy has priority over fertility preservation. The time available for fertility preservation in patients with cancer is often very limited and depends on the condition of the underlying disease. This case report presents the results of two rounds of controlled ovarian stimulations (COSs) performed after an induced abortion. The patient had mixed phenotype acute leukemia diagnosed during early pregnancy and underwent a surgical abortion, followed by ovarian stimulation using urinary follicle-stimulating hormone (uFSH) and gonadotropin-releasing hormone (GnRH) agonists. Oocyte retrieval was subsequently performed for oocyte cryopreservation. Despite good hormonal and ultrasonic follicular growth, no oocytes were obtained. During a second COS performed at a low human chorionic gonadotropin (hCG) level (less than 100 IU/L), several mature oocytes were obtained, suggesting that higher hCG levels during COS induce the absence of mature oocytes during normal follicular growth. It is recommended to start COS post-abortion after confirming a low hCG level while considering the timing of cancer treatment.

DOI： 10.1080/09513590.2021.1950135

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Heritable mitochondrial DNA (mtDNA) mutations are common, yet only a few recurring pathogenic mtDNA variants account for the majority of known familial cases in humans. Purifying selection in the female germline is thought to be responsible for the elimination of most harmful mtDNA mutations during oogenesis. Here we show that deleterious mtDNA mutations are abundant in ovulated mature mouse oocytes and preimplantation embryos recovered from PolG mutator females but not in their live offspring. This implies that purifying selection acts not in the maternal germline per se, but during post-implantation development. We further show that oocyte mtDNA mutations can be captured and stably maintained in embryonic stem cells and then reintroduced into chimeras, thereby allowing examination of the effects of specific mutations on fetal and postnatal development.

DOI： 10.1093/biolre/ioz202

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Language：English  

Change history In this Letter, there are several errors regarding the assignments of mtDNA haplotypes for a subset of egg donors from our study. These errors have not been corrected online.

DOI： 10.1038/s41586-019-0876-1

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Language：English   Publishing type：Research paper (scientific journal)  

In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5-9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived.

DOI： 10.1016/j.stemcr.2018.08.015

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DOI： 10.1038/s41586-018-0381-y

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The accumulation of acquired mitochondrial genome (mtDNA) mutations with aging in somatic cells has been implicated in mitochondrial dysfunction and linked to age-onset diseases in humans. Here, we asked if somatic mtDNA mutations are also associated with aging in the mouse. MtDNA integrity in multiple organs and tissues in young and old (2-34 months) wild type (wt) mice was investigated by whole genome sequencing. Remarkably, no acquired somatic mutations were detected in tested tissues. However, we identified several non-synonymous germline mtDNA variants whose heteroplasmy levels (ratio of normal to mutant mtDNA) increased significantly with aging suggesting clonal expansion of inherited mtDNA mutations. Polg mutator mice, a model for premature aging, exhibited both germline and somatic mtDNA mutations whose numbers and heteroplasmy levels increased significantly with age implicating involvement in premature aging. Our results suggest that, in contrast to humans, acquired somatic mtDNA mutations do not accompany the aging process in wt mice.

DOI： 10.1371/journal.pone.0201304

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Mitochondria, the ubiquitous power packs in nearly every eukaryotic cell, contain their own DNA, known as mtDNA, which is inherited exclusively from the mother. The number of mitochondrial genomes varies depending on the cell's energy needs. The mature oocyte contains the highest number of mitochondria of any cell type, although there is little if any mtDNA replication after fertilization until the embryo implants. This has potential repercussions for mitochondrial replacement therapy (MRT; see description of currently employed methods below) used to prevent the transmission of mtDNA-based disorders. If only a few mitochondria with defective mtDNA are left in the embryo and undergo extensive replication, it might therefore thwart the purpose of MRT. In order to improve the safety and efficacy of this experimental therapy, we need a better understanding of how and which mtDNA is tagged for replication versus transcription after fertilization of the oocyte.

DOI： 10.15252/embj.201797843

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

DOI： 10.1038/nature23305

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Mitochondria, the ubiquitous power packs in nearly every eukaryotic cell, contain their own DNA, known as mtDNA, which is inherited exclusively from the mother. The number of mitochondrial genomes varies depending on the cell's energy needs. The mature oocyte contains the highest number of mitochondria of any cell type, although there is little if any mtDNA replication after fertilization until the embryo implants. This has potential repercussions for mitochondrial replacement therapy (MRT; see description of currently employed methods below) used to prevent the transmission of mtDNA-based disorders. If only a few mitochondria with defective mtDNA are left in the embryo and undergo extensive replication, it might therefore thwart the purpose of MRT In order to improve the safety and efficacy of this experimental therapy, we need a better understanding of how and which mtDNA is tagged for replication versus transcription after fertilization of the oocyte.

DOI： 10.15252/embj.201797606

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

DOI： 10.1016/j.stem.2016.10.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT-ESCs) and transcription factor-mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT-ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT-ESCs-the ability to replace mutant mitochondrial DNA in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT-ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from fertilized embryos. Thus, although NT-ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT-ESCs and bona fide ESCs may make NT-ESCs superior for future applications in regenerative medicine. Stem Cells 2017;35:26-34.

DOI： 10.1002/stem.2496

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

DOI： 10.1038/nature20592

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Vertebrate cells carry two different genomes, nuclear (nDNA) and mitochondrial (mtDNA), both encoding proteins involved in oxidative phosphorylation. Because of the extensive interactions, adaptive coevolution of the two genomes must occur to ensure normal mitochondrial function. To investigate whether incompatibilities between these two genomes could contribute to interspecies reproductive barriers, we performed reciprocal mtDNA replacement (MR) in zygotes between widely divergent Mus m. domesticus (B6) and conplastic Mus m. musculus (PWD) mice. Transfer of MR1 cybrid embryos (B6nDNA-PWDmtDNA) supported normal development of F1 offspring with reduced male fertility but unaffected reproductive fitness in females. Furthermore, donor PWD mtDNA was faithfully transmitted through the germline into F2 and F3 generations. In contrast, reciprocal MR2 (PWDnDNA-B6mtDNA) produced high embryonic loss and stillborn rates, suggesting an association between mitochondrial function and infertility. These results strongly suggest that functional incompatibility between nuclear and mitochondrial genomes contributes to interspecies reproductive isolation in mammals.

DOI： 10.1016/j.cmet.2016.06.012

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.

DOI： 10.1002/mrd.22644

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

DOI： 10.1016/j.stem.2016.02.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

DOI： 10.1016/j.stemcr.2015.07.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

DOI： 10.1038/nature14546

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of β-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/β-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of β-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs.

DOI： 10.1016/j.stemcr.2014.12.003

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

DOI： 10.1095/biolreprod.114.121640

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

DOI： 10.1371/journal.pone.0041007

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：(株)メディカルレビュー社  

マウスを主にした研究において胚性幹細胞(ES細胞)で蓄積された生殖細胞誘導技術、生殖補助技術などの研究は2008年のYamanakaらによって報告された人工多能性幹細胞(iPS細胞)によって飛躍的に進む可能性がある。iPS細胞はES細胞と同等の能力(多分化能、自己複製能)をもち、患者の体細胞から樹立可能であることから、ES細胞で問題となっていた移植における拒絶反応、作製において初期胚を破壊する必要があるという倫理的問題などを解決することができるからである。本稿ではES細胞、iPS細胞について概説し、また多能性幹細胞からの生殖細胞分化についても紹介したい。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPAN ENDOCRINE SOC  

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Language：Japanese   Publisher：日本産科婦人科学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=444898

Language：Japanese   Publisher：日本産科婦人科学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=444805

Language：Japanese   Publisher：日本産科婦人科学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=444897

Language：Japanese   Publisher：日本産科婦人科学会  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=444887

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=442842

Language：Japanese   Publisher：日本産科婦人科学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=442835

Language：Japanese   Publisher：日本産科婦人科学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=442586

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=442848

Language：Japanese   Publisher：神奈川県医師会  

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Language：Japanese   Publisher：神奈川産科婦人科学会  

46歳0経産婦.患者は不正性器出血を主訴に近医を受診,絨毛性疾患を疑われ,著者らの施設へ紹介入院となった.入院時,尿中hCG,血中hCG-βの上昇を認め,子宮頸部/内膜細胞診はclassI/IIIであった.経腟超音波では子宮内に中〜高輝度の腫瘍を認めた.絨毛癌診断スコアは1点であったが,絨毛癌に移行する可能性が高かったため,手術を予定した.入院6日目に下腹部痛,出血とともに内容物が一部排出され,排出物は胞状奇胎と診断された.high risk群の侵入奇胎が疑われ,入院7日目に腹式単純子宮全摘術および両側付属器摘出術を行った.その結果,術後病理診断は侵入奇胎で,遺伝子診断では雄性発生2精子受精疑いであった.術後経過は良好であった

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Language：Japanese   Publisher：日本産科婦人科学会  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=438210

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CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=438211

Language：Japanese   Publisher：神奈川県精神医学会  

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Event date： 2020.11

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