A Decellularized Uterine Endometrial Scaffold Enhances Regeneration of the Endometrium in Rats.

Partial or whole regeneration of the uterine endometrium using extracellular matrix (ECM)-based scaffolds is a therapeutic strategy for uterine infertility due to functional and/or structural endometrial defects. Here, we examined whether the entire endometrium can be regenerated circumferentially using an acellular ECM scaffold (decellularized endometrial scaffold, DES) prepared from rat endometrium. We placed a silicone tube alone to prevent adhesions or a DES loaded with a silicone tube into a recipient uterus in which the endometrium had been surgically removed circumferentially. Histological and immunofluorescent analyses of the uteri one month after tube placement revealed more abundant regenerated endometrial stroma in the uterine horns treated with tube-loaded DES compared to those treated with a tube alone. Luminal and glandular epithelia, however, were not fully recapitulated. These results suggest that DES can enhance the regeneration of endometrial stroma but additional intervention(s) are needed to induce epithelization. Furthermore, the prevention of adhesions alone allowed the endometrial stroma to regenerate circumferentially even without a DES, but to a lesser degree than that with a DES. The use of a DES together with the prevention of adhesions may be beneficial for efficient endometrial regeneration in the uterus that is largely deficient of endometrium.

A mediator complex subunit 12 gain-of-function mutation induces partial leiomyoma cell properties in human uterine smooth muscle cells.

OBJECTIVE: To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. MAIN OUTCOME MEASURE(S): Cell proliferation, collagen production, and in vivo tumorigenicity of USMCs with vs. without the MED12 mutation. RESULT(S): Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC in vitro, and this production was likely enhanced by EP and TGFB3. CONCLUSION(S): The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrial stem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.

Sorafenib targets and inhibits the oncogenic properties of endometrial cancer stem cells via the RAF/ERK pathway.

BACKGROUND: Distinct subsets of cancer stem cells (CSCs) drive the initiation and progression of malignant tumors via enhanced self-renewal and development of treatment/apoptosis resistance. Endometrial CSC-selective drugs have not been successfully developed because most endometrial cell lines do not contain a sufficient proportion of stable CSCs. Here, we aimed to identify endometrial CSC-containing cell lines and to search for endometrial CSC-selective drugs. METHODS: We first assessed the presence of CSCs by identifying side populations (SPs) in several endometrial cancer cell lines. We then characterized cell viability, colony-formation, transwell invasion and xenotransplantion capability using the isolated SP cells. We also conducted real-time RT-PCR, immunoblot and immunofluorescence analyses of the cells' expression of CSC-associated markers. Focusing on 14 putative CSC-selective drugs, we characterized their effects on the proliferation and apoptosis of endometrial cancer cell lines, examining cell viability and annexin V staining. We further examined the inhibitory effects of the selected drugs, focusing on proliferation, invasion, expression of CSC-associated markers and tumor formation. RESULTS: We focused on HHUA cells, an endometrial cancer cell line derived from a well-differentiated endometrial adenocarcinoma. HHUA cells contained a sufficient proportion of stable CSCs with an SP phenotype (HHUA-SP). HHUA-SP showed greater proliferation, colony-formation, and invasive capabilities compared with the main population of HHUA cells (HHUA-MP). HHUA-SP generated larger tumors with higher expression of proliferation-related markers, Ki67, c-MYC and phosphorylated ERK compared with HHUA-MP when transplanted into immunodeficient mice. Among the 14 candidate drugs, sorafenib, an inhibitor of RAF pathways and multiple kinase receptors, inhibited cell proliferation and invasion in both HHUA-SP and -MP, but more profoundly in HHUA-SP. In vivo treatment with sorafenib for 4 weeks reduced the weights of HHUA-SP-derived tumors and decreased the expression of Ki67, ZEB1, and RAF1. CONCLUSIONS: Our results suggest that HHUA is a useful cell line for discovery and identification of endometrial CSC-selective drugs, and that sorafenib may be an effective anti-endometrial cancer drug targeting endometrial CSCs.

A Revised Stem Cell Theory for the Pathogenesis of Endometriosis.

During the past decade, a stem cell-based hypothesis has emerged (among many others) to explain the pathogenesis of endometriosis. The initial hypothesis proposed that endometriosis arose from a single or a few specific cells with stem cell properties, including self-renewal and multi-lineage cell differentiation. The origins of the endometriosis-initiating stem cells were thought to be the bone marrow, uterine endometrium, and other tissues. Based on the implantation or metastatic theory in combination with the initial stem cell theory, one or a few multipotent stem/progenitor cells present in the eutopic endometrium or bone marrow translocate to ectopic sites via fallopian tubes during menstruation, vasculolymphatic routes, or through direct migration and invasion. Subsequently, they give rise to endometriotic lesions followed by differentiation into various cell components of endometriosis, including glandular and stromal cells. Recent somatic mutation analyses of deep infiltrating endometriosis, endometrioma, and eutopic normal endometrium using next-generation sequencing techniques have redefined the stem cell theory. It is now proposed that stem/progenitor cells of at least two different origins-epithelium and stroma-sequentially, differentially, but coordinately contribute to the genesis of endometriosis. The dual stem cell theory on how two (or more) stem/progenitor cells differentially and coordinately participate in the establishment of endometriotic lesions remains to be elucidated. Furthermore, the stem/progenitor cells involved in this theory also remain to be identified. Given that the origin of endometriosis is eutopic endometrium, the candidate cells for endometriotic epithelium-initiating cells are likely to be endometrial epithelial cells positive for either N-cadherin or SSEA-1 or both. The candidate cells for endometriotic stroma-initiating cells may be endometrial mesenchymal stem cells positive for SUSD2. Endometrial side population cells are also a possible candidate because they contain unipotent or multipotent cells capable of behaving as endometrial epithelial and stromal stem/progenitor cells.

Bioengineering of the Uterus.

Impairment of uterine structure and function causes infertility, pregnancy loss, and perinatal complications in humans. Some types of uterine impairments such as Asherman's syndrome, also known as uterine synechiae, can be treated medically and surgically in a standard clinical setting, but absolute defects of uterine function or structure cannot be cured by conventional approaches. To overcome such hurdles, partial or whole regeneration and reconstruction of the uterus have recently emerged as new therapeutic strategies. Transplantation of the whole uterus into patients with uterine agenesis results in the successful birth of children. However, it remains an experimental treatment with numerous difficulties such as the need for continuous and long-term use of immunosuppressive drugs until a live birth is achieved. Thus, the generation of the uterus by tissue engineering technologies has become an alternative but indispensable therapeutic strategy to treat patients without a functional or well-structured uterus. For the past 20 years, the bioengineering of the uterus has been studied intensively in animal models, providing the basis for clinical applications. A variety of templates and scaffolds made from natural biomaterials, synthetic materials, or decellularized matrices have been characterized to efficiently generate the uterus in a manner similar to the bioengineering of other organs and tissues. The goal of this review is to provide a comprehensive overview and perspectives of uterine bioengineering focusing on the type, preparation, and characteristics of the currently available scaffolds.

Cigarette Smoke Extract Activates Hypoxia-Inducible Factors in a Reactive Oxygen Species-Dependent Manner in Stroma Cells from Human Endometrium.

Cigarette smoking (CS) is a major contributing factor in the development of a large number of fatal and debilitating disorders, including degenerative diseases and cancers. Smoking and passive smoking also affect the establishment and maintenance of pregnancy. However, to the best of our knowledge, the effects of smoking on the human endometrium remain poorly understood. In this study, we investigated the regulatory mechanism underlying CS-induced hypoxia-inducible factor (HIF)-1α activation using primary human endometrial stromal cells and an immortalized cell line (KC02-44D). We found that the CS extract (CSE) increased reactive oxygen species levels and stimulated HIF-1α protein stabilization in endometrial stromal cells, and that CS-induced HIF-1α-dependent gene expression under non-hypoxic conditions in a concentration- and time-dependent manner. Additionally, we revealed the upregulated expression of a hypoxia-induced gene set following the CSE treatment, even under normoxic conditions. These results indicated that HIF-1α might play an important role in CS-exposure-induced cellular stress, inflammation, and endometrial remodeling.

Optogenetic regulation of embryo implantation in mice using photoactivatable CRISPR-Cas9.

Embryo implantation is achieved upon successful interaction between a fertilized egg and receptive endometrium and is mediated by spatiotemporal expression of implantation-associated molecules including leukemia inhibitory factor (LIF). Here we demonstrate, in mice, that LIF knockdown via a photoactivatable CRISPR-Cas9 gene editing system and illumination with a light-emitting diode can spatiotemporally disrupt fertility. This system enables dissection of spatiotemporal molecular mechanisms associated with embryo implantation and provides a therapeutic strategy for temporal control of reproductive functions in vivo.

Visualization of spatiotemporal dynamics of human glioma stem cell invasion.

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

Guidelines for office gynecology in Japan: Japan Society of Obstetrics and Gynecology (JSOG) and Japan Association of Obstetricians and Gynecologists (JAOG) 2017 edition.

Six years after the first edition of The Guideline for Gynecological Practice, which was jointly edited by The Japan Society of Obstetrics and Gynecology and The Japan Association of Obstetricians and Gynecologists, the third revised edition was published in 2017. The 2017 Guidelines includes 10 additional clinical questions (CQ), which brings the total to 95 CQ (12 on infectious disease, 28 on oncology and benign tumors, 27 on endocrinology and infertility and 28 on healthcare for women). Currently a consensus has been reached on the Guidelines and therefore the objective of this report is to present the general policies regarding diagnostic and treatment methods used in standard gynecological outpatient care that are considered appropriate. At the end of each answer, the corresponding recommendation level (A, B, C) is indicated.

Generation of Progesterone-Responsive Endometrial Stromal Fibroblasts from Human Induced Pluripotent Stem Cells: Role of the WNT/CTNNB1 Pathway.

Defective endometrial stromal fibroblasts (EMSFs) contribute to uterine factor infertility, endometriosis, and endometrial cancer. Induced pluripotent stem cells (iPSCs) derived from skin or bone marrow biopsies provide a patient-specific source that can be differentiated to various cells types. Replacement of abnormal EMSFs is a potential novel therapeutic approach for endometrial disease; however, the methodology or mechanism for differentiating iPSCs to EMSFs is unknown. The uterus differentiates from the intermediate mesoderm (IM) to form coelomic epithelium (CE) followed by the Müllerian duct (MD). Here, we successfully directed the differentiation of human iPSCs (hiPSCs) through IM, CE, and MD to EMSFs under molecularly defined embryoid body culture conditions using specific hormonal treatments. Activation of CTNNB1 was essential for expression of progesterone receptor that mediated the final differentiation step of EMSFs before implantation. These hiPSC-derived tissues illustrate the potential for iPSC-based endometrial regeneration for future cell-based treatments.

ZEB1 expression is a potential indicator of invasive endometriosis.

INTRODUCTION: Although endometriosis is a benign disease, it shares some features with cancers, such as invasiveness and the potential to metastasize. This study sought to investigate the epithelial-mesenchymal transition status in human endometriotic lesions. MATERIAL AND METHODS: Thirteen endometriosis patients and 10 control women without endometriosis undergoing surgery for benign indications were recruited. We examined the expression of E-cadherin, vimentin, and epithelial-mesenchymal transition-induced transcriptional factors, such as Snail and ZEB1, by immunohistochemistry. We evaluated the expression of each marker in epithelial cells of both endometriotic lesions (ovarian endometrioma, deep infiltrating endometriosis, adenomyosis) and normal endometria. The correlation between ZEB1 expression and serum level of CA125 was also investigated. RESULTS: Immunohistochemical analysis revealed that although E-cadherin, vimentin, and Snail were expressed in epithelia of normal endometria and endometriotic lesions, ZEB1 expression was only expressed in epithelia of endometriotic lesions. Additionally, ZEB1 was most frequently observed in epithelial cells of invasive endometriosis. The endometriosis patients with high serum CA125 level were more likely to have ZEB1-positive lesions. CONCLUSIONS: This is the first observation of ZEB1 expression in epithelial cells of benign disease. The preferential expression of ZEB1 in epithelial cells of endometriotic lesions suggests that these cells may have, at least in part, a higher level of mesenchymal features possibly via ZEB1-driven epithelial-mesenchymal transition than normal endometria and that ZEB1 can be a potential indicator of invasiveness or severity of endometriosis.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10 mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5 mM L-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20 mM unlabeled MeAIB. L-Arginine (5 mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10 mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

Bioengineered uterine tissue supports pregnancy in a rat model.

OBJECTIVE: To create a bioengineered uterine patch for uterine repair of a partially defect uterus. DESIGN: Three different decellularized uterine scaffolds were recellularized in vitro with primary uterine cells and green fluorescent protein- (GPF-) labeled bone marrow-derived mesenchymal stem cells (GFP-MSCs). The patches were transplanted in vivo to investigate their tissue adaptation and supporting capacity during pregnancy. SETTING: Research laboratory. ANIMAL(S): Female Lewis rats (n = 9) as donors to generate whole-uterus scaffolds using three different protocols (n = 3 per protocol); Sprague Dawley rats (n = 40) for primary uterus cell isolation procedures (n = 10) and for transplantation/pregnancy studies (n = 30); and male Sprague Dawley rats (n = 12) for mating. INTERVENTION(S): Decellularization was achieved by whole-uterus perfusion with buffered or nonbuffered Triton-X100 and dimethyl sulfoxide (DMSO; group P1/P2) or with sodium deoxycholate (group P3). Primary uterine cells and GFP-MSCs were used to develop uterine tissue constructs, which were grafted to uteri with partial tissue defects. MAIN OUTCOME MEASURE(S): Recellularization efficiency and graft quality were analyzed morphologically, immunohistochemically, and by real-time quantitative polymerase chain reaction (PCR). The location and number of fetuses were documented during pregnancy days 16-20. RESULT(S): Pregnancy and fetal development were normal in groups P1 and P2, with fetal development over patched areas. Group P3 showed significant reduction of fetal numbers, and embryos were not seen in the grafted area. Quantitative PCR and immunohistochemistry revealed uterus-like tissue in the patches, which had been further reconstructed by infiltrating host cells after transplantation. CONCLUSION(S): Primary uterine cells and MSCs can be used to reconstruct decellularized uterine tissue. The bioengineered patches made from triton-X100+DMSO-generated scaffolds were supportive during pregnancy. These protocols should be explored further to develop suitable grafting material to repair partially defect uteri and possibly to create a complete bioengineered uterus.

How to Create an Embryo Penetration Route.

Numerous past investigations into human implantation have tended to pay attention to the mechanism of embryo adhesion to endometrial cells or embryo invasion into endometrial stromal tissue. For successful pregnancy, however, embryo penetration through the endometrial epithelial cell (EEC) sheet is also absolutely required. To improve the performance of assisted reproductive technology, implantation studies should also focus on EEC dynamics, in particular, the action of EECs during embryo penetration. Although only EEC apoptosis has been proposed as a mechanism for the formation of the embryo penetration route, we have also recently reported that characteristic EEC collective motion, regulated by the epithelial to mesenchymal transition, also plays a key role in permitting embryo penetration. We review here how EECs form embryo penetration routes in light of our findings.

Successful treatment of atypical cesarean scar defect using endoscopic surgery.

BACKGROUND: Cesarean scar syndrome results from a postoperative defect of the uterine isthmus, also known as an isthmocele. Patients present with gynecological symptoms, such as abnormal genital bleeding or infertility, after cesarean delivery. Although the cesarean rate is increasing worldwide, this syndrome is not widely known. CASE PRESENTATION: A 43-year-old G2P1 Japanese woman with atypical cesarean scar syndrome had a 3-year history of secondary infertility and postmenstrual brown discharge. Laparoscopic and hysteroscopic exploration revealed a cesarean scar defect connected to a small cavity in the myometrium: this was not an endometrial cavity or a uterine diverticulum. After endoscopic excision of the cavity, the brown discharge resolved, and the patient achieved ongoing pregnancy on her third attempt at intrauterine insemination. CONCLUSION: Consensus is still lacking regarding the diagnosis and treatment of cesarean scar defect. However, the gynecologists should be aware that cesarean scar syndrome can have scar defects forming cavities of unusual shapes and features. Surgical correction of these defects will often improve postmenstrual bleeding and subfertility in these cases.

Uterine Leiomyoma Stem Cells: Linking Progesterone to Growth.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/ß-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

Stem Cells in Endometrial Physiology.

Human endometrial mucosa is a dynamically remodeling tissue, undergoing cyclical morphologic and functional changes in response to fluctuating sex steroid hormones each menstrual cycle during a woman's reproductive life. Postmenopausal endometrium responds similarly to exogenous estrogen. Cyclical endometrial regeneration also occurs in nonmenstruating rodents, although to a lesser extent. The recent identification of rare populations of endogenous epithelial progenitor cells, mesenchymal stem/stromal cells (MSCs), the side population (SP) cells, and label-retaining cells (LRCs) suggests these stem/progenitor cell populations may play a key role in endometrial regeneration during menstrual and estrus cycles. This review summarizes the identification of epithelial progenitors, MSC, SP, and LRC, and discusses their contribution to endometrial tissue regeneration, maintaining tissue homeostasis, decidualization, and placentation. Markers for human endometrial MSC have been identified, revealing their perivascular location in both the functionalis and basalis layers. These markers also allow their purification from biopsy tissue and menstrual blood. These findings have advanced our understanding of normal endometrial physiology and will provide new insight into endometrial proliferative disorders (endometriosis, endometrial cancer). The ability to prospectively isolate endometrial MSC will enable their utilization in cell-based therapies for reproductive tract pathologies.

Cell Therapy and Tissue Engineering from and toward the Uterus.

Regenerative medicine offers the potential for replacement or repair of different types of cells within damaged tissues or the tissues themselves, typically through cell therapy or tissue engineering. Stem cells are critical to these approaches; indeed, the involvement of bone marrow in the differentiation of stem cells to nonhematopoietic cells is well demonstrated. Further, the contribution of bone marrow-derived stem cells in promoting neoangiogenesis has been demonstrated not only in animal models, but also in human clinical trials with an excellent safety profile. Recent evidence indicates that the endometrium is a tissue with the potential for regeneration through such approaches. The presence of donor cells in the endometrium of women receiving bone marrow transplantation suggests a hematopoietic source with the ability to renew this tissue. Here we describe the role of cell therapy with bone marrow-derived stem cells in treating endometrial dysfunction in Asherman syndrome and/or endometrial atrophy in human and murine models. Additionally, the emerging field of tissue engineering has recently been applied in the reproductive tissues-beyond the endometrium-with elegant studies involving humans and animal models.