Inhibition of tumor cell proliferation in human uterine leiomyomas by vitamin D via Wnt/β-catenin pathway.

To assess the effect of vitamin D (VitD) on human uterine leiomyomas through Wnt/β-catenin pathway inhibition, apoptosis induction, and cell growth arrest.A prospective study comparing leiomyoma vs. myometrium tissues. Paired design study comparing human uterine leiomyoma primary (HULP) cells treated with or without VitD.University hospital.Human uterine leiomyoma and myometrium were collected from women (aged 35-52 years) without hormonal treatment.Samples were collected from women undergoing surgery due to symptomatic uterine leiomyoma pathology.Uterine leiomyoma and myometrium tissues were analyzed by western blot (WB) to determine proliferation, Wnt/β-catenin, and apoptosis pathways. HU…

Bioengineered endometrial hydrogels with growth factors promote tissue regeneration and restore fertility in murine models

Extracellular matrix (ECM) hydrogels obtained from decellularized tissues are promising biocompatible materials for tissue regeneration. These biomaterials may provide important options for endometrial pathologies such as Asherman's syndrome and endometrial atrophy, which lack effective therapies thus far. First, we performed a proteomic analysis of a decellularized endometrial porcine hydrogel (EndoECM) to describe the specific role of ECM proteins related to regenerative processes. Furthermore, we investigated the ability of a bioengineered system-EndoECM alone or supplemented with growth factors (GFs)-to repair the endometrium in a murine model of endometrial damage. For this model, the …

Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells.

Objective To isolate and characterize human leiomyoma stem cells by the side population (SP) method. Design Prospective experimental human and animal study. Setting University research laboratory-affiliated infertility clinic. Patient(s) Women undergoing laparoscopic myomectomy. Animal(s) Female non-obese diabetic severe combined immune deficiency (NOD-SCID) mutation mice. Intervention(s) Obtainment of human leiomyoma SP cells as candidate tumor-initiating cells and establishment of two leiomyoma SP lines. Main Outcome Measure(s) Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cDNA microarrays hybridization, cell culture, karyotype, molecular analysis, immu…

Long-term vitamin D treatment decreases human uterine leiomyoma size in a xenograft animal model

Objective To study the effects of short- and long-term vitamin D treatment on uterine leiomyomas in vivo through cell proliferation, extracellular matrix (ECM) degradation, and apoptosis. Design Preclinical study of human leiomyoma treatment with vitamin D in an nonhuman animal model. Setting Hospital and university laboratories. Patient(s)/Animal(s) Human leiomyomas were collected from patients and implanted in ovariectomized NOD-SCID mice. Intervention(s) Mice were treated with vitamin D (0.5 μg/kg/d or 1 μg/kg/d) or vehicle for 21 or 60 days. Main Outcome Measure(s) Vitamin D effect in xenograft tissue was assessed by monitoring tumor size (18F-FDG positron-emission tomography/computeriz…

Vitamin d long-term treatment decreases human uterine leiomyoma size through specific molecular mechanisms in a xenograft animal model

Undetectable viral RNA from SARS-CoV-2 in endometrial biopsies from women with COVID-19: a preliminary study.

Stem cell paracrine actions in tissue regeneration and its potential therapeutic effect in human endometrium: a retrospective study.

Objective: Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's Syndrome and Endometrial Atrophy (AS/EA) patients after autologous CD133+ bone marrow-derived stem cells (CD133+BMDSCs) transplantation. Design: Retrospective study using human endometrial biopsies and mouse models. Setting: Fundación-IVI, IIS-La Fe, Valencia, Spain. Samples: Endometrial biopsies collected before and after CD133+BMDSCs therapy, from 8 women with AS/EA (NCT02144987). And uterus from 5 mice, with only left horns receiving CD133+BMDSCs therapy. Methods: In human samples, hematoxylin and eosin (H&E) staining, RNA arrays, PCR validation and neutrophil elastase (NE) immunohistoch…

P–543 Inhibition of cell proliferation and extracellular matrix formation in human uterine leiomyomas by 5-aza–2’-deoxycitidine via Wnt/ β-catenin pathway

Abstract Study question Is DNA methylation reversion through DNA methyltransferases (DNMT) inhibitors, such as 5-aza–2’-deoxycitidine, a potential therapeutic option for treatment of patients with uterine leiomyomas (UL)? Summary answer 5-aza–2’-deoxycitidine reduces proliferation and extracellular matrix (ECM) formation by inhibition of Wnt/ β-catenin pathway on UL cells, suggesting DNMT inhibitors as an option to treat UL. What is known already: UL is a multifactorial disease with an unclear pathogenesis and inaccurate treatment. Aberrant DNA methylation have been found in UL compared to myometrium (MM) tissue, showing hypermethylation of tumor suppressor genes, which contributes to the d…

Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

Abstract Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonall…

Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoEC…

O-142 COVID19-free endometrium: Undetectable viral RNA in endometrial biopsies from positive symptomatic SARS-CoV-2 women

Abstract Study question Does SARS-CoV-2 infect the endometrial tissue in women with coronavirus disease 2019 (COVID-19)? Summary answer Symptomatic women with COVID-19 report no presence, in the short term, of viral RNA from SARS-CoV-2 in the endometrium. What is known already The recent emergence of COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has not allowed yet to establish putative relations between this disorder and other existing pathologies. It is the case with fertility problems and the reproductive organs, including a possible endometrial disorder caused by the virus. Thus, an important step is to elucidate the viral presence in different hu…

A Natural Xenogeneic Endometrial Extracellular Matrix Hydrogel Toward Improving Current Human in vitro Models and Future in vivo Applications

Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in t…

DEVELOPMENT OF SECRETOR AND GESTATIONAL PATIENT-DERIVED ORGANOIDS FROM ADENOMYOSIS DISEASE AS AN IN VITRO MODEL TO STUDY ADENOMYOSIS-RELATED INFERTILITY AND PERSONALIZED DRUG SCREENING

Vitamin D as an effective treatment in human uterine leiomyomas independent of mediator complex subunit 12 mutation.

Objective To study whether vitamin D (VitD) inhibits cell proliferation and Wnt/β-catenin and transforming growth factor−β (TGFβ) signaling pathways in uterine leiomyomas independent of mediator complex subunit 12 (MED12) mutation status. Design Prospective study comparing leiomyoma vs. myometrial tissues and human uterine leiomyoma primary (HULP) cells treated with or without VitD and analyzed by MED12 mutation status. Setting Hospital and university laboratories. Patient(s) Women with uterine leiomyoma without any treatment (n = 37). Intervention(s) Uterine leiomyoma and myometrium samples were collected from women undergoing surgery because of symptomatic leiomyoma pathology. Main Outcom…

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) as a putative human endometrial stem cell marker†

The endometrium is recognized for its remarkable regenerative and remodeling capacity. Every month this hormonally regu- lated organ undergoes cycles of growth (from 0.5-2 to 7 mm), regression and shedding of two-third of the tissue, leading to its monthly renewal that occurs � 400 times in a woman's reproductive lifetime. Several groups have suggested the existence of a human endometrial somatic stem cell (SSC) population located around the spiral arterioles of the basalis. Different groups have isolated, identified and charac- terized putative endometrial SSC populations in human endometrium based on the general features of undifferentiated cells, such as slow cycling detected using the 5…

Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines

Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1-7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12-15 passages (20 weeks) and cryopreserved.…

Comparison of different sources of platelet-rich plasma as treatment option for infertility-causing endometrial pathologies

Objective To study the effect of human plasma from different sources, namely, umbilical cord blood and adult blood platelet-rich plasma (PRP), on the regeneration of endometrial damage. Design Composition analysis, in vitro approaches, and a preclinical murine model using plasma to promote endometrial regeneration. Setting Hospital and university laboratories. Patient(s)/Animal(s) Adult plasma from four Asherman syndrome/endometrial atrophy patients and one fertile woman, commercial umbilical cord plasma, and uterine-damaged NOD/SCID mice model were used. Intervention(s) Endometrial stromal cells from primary culture and an endometrial stem cell line were cultured in vitro, and uterine-dama…

Overexpression of the truncated form of high mobility group a proteins (HMGA2) in human myometrial cells induces leiomyoma-like tissue formation

The pathogenesis of uterine leiomyomas, the most common benign tumor in women, is still unknown. This lack of basic knowledge limits the development of novel non-invasive therapies. Our group has previously demonstrated that leiomyoma side population (SP) cells are present in tumor lesions and act like putative tumor-initiating stemcells in human leiomyoma. Moreover, accumulated evidence demonstrates that these benign tumors of mesenchymal origin are characterized by rearrangements of the High Mobility Group A proteins (HMGA). In this work, we tested the hypothesis that leiomyoma development may be due to overexpression of HMGA2 (encoding high mobility group AT-hook2) in myometrial stem cel…

Dysregulated genes and their functional pathways in luteinized granulosa cells from PCOS patients after cabergoline treatment

Polycystic ovarian syndrome (PCOS) is a common reproductive disorder frequently associated with a substantial risk factor for ovarian hyperstimulation syndrome (OHSS). Dopamine receptor 2 (D2) agonists, like cabergoline (Cb2), have been used to reduce the OHSS risk. However, lutein granulosa cells (LGCs) from PCOS patients treated with Cb2 still show a deregulated dopaminergic tone (decreased D2 expression and low dopamine production) and increased vascularization compared to non-PCOS LGCs. Therefore, to understand the PCOS ovarian physiology, it is important to explore the mechanisms that underlie syndrome based on the therapeutic effects of Cb2. Here, LGCs from non-PCOS and PCOS patients …

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma.

Objective To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells in vitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor β3 (TGF-β3) signaling. Design Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. Setting Hospital and university laboratories. Patient(s) Women with UL without any hormone treatment. Intervention(s) Myomectomy or hysterectomy surgery in women for leiomyoma disease. Main Outcome Measure(s) HDAC activity was assessed by enzyme-li…

Bone Marrow-Derived Cells from Male Donors Do Not Contribute to the Endometrial Side Population of the Recipient

Accumulated evidence demonstrates the existence of bone marrow-derived cells origin in the endometria of women undergoing bone marrow transplantation (BMT). In these reports, cells of a bone marrow (BM) origin are able to differentiate into endometrial cells, although their contribution to endometrial regeneration is not yet clear. We have previously demonstrated the functional relevance of side population (SP) cells as the endogenous source of somatic stem cells (SSC) in the human endometrium. The present work aims to understand the presence and contribution of bone marrow-derived cells to the endometrium and the endometrial SP population of women who received BMT from male donors. Five fe…

Contribution of different bone marrow-derived cell types in endometrial regeneration using an irradiated murine model.

Objective To study the involvement of seven types of bone marrow-derived cells (BMDCs) in the endometrial regeneration in mice after total body irradiation. Design Prospective experimental animal study. Setting University research laboratories. Animal(s) β-Actin-green fluorescent protein (GFP) transgenic C57BL/6-Tg (CAG-EGFP) and C57BL/6J female mice. Intervention(s) The BMDCs were isolated from CAG-EGFP mice: unfractionated bone marrow cells, hematopoietic progenitor cells, endothelial progenitor cells (EPCs), and mesenchymal stem cells (MSCs). In addition three murine GFP + cell lines were used: mouse Oct4 negative BMDC multipotent adult progenitor cells (mOct4 − BM-MAPCs), BMDC hypoblast…