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Background: Women with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing. Methods: Endometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student's t-test or Wilcoxon test. Results: We identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia. Conclusions: Identifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Are the extracellular vesicles (EVs) secreted by the maternal endometrium uptaken by human embryos and is their miRNA cargo involved in implantation and embryo development? SUMMARY ANSWER Data suggest that EVs secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. WHAT IS KNOWN ALREADY Successful implantation is dependent on coordination between maternal endometrium and embryo, and EVs role in the required cell-to-cell crosstalk has recently been established. In this regard, our group previously showed that protein cargo of EVs secreted by primary human endometrial epithelial cells (pHEECs) is implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development. However, little is known about the regulation of these biological processes through EVs secreted by the endometrium at a transcriptomic level. STUDY DESIGN, SIZE, DURATION A prospective descriptive study was performed. Endometrial biopsies were collected from healthy oocyte donors with confirmed fertility on the day of oocyte retrieval, 36 h after the LH surge. pHEECs were isolated from endometrial biopsies (n   =   8 in each pool) and cultured in vitro. Subsequently, conditioned medium was collected and EVs were isolated and characterized. Uptake of EVs by human blastocysts and miRNA cargo of these EVs (n   =   3 pools) was analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS EVs were isolated from the conditioned culture media using ultracentrifugation, and characterization was performed using western blotting, nanoparticle tracking analysis, and transmission electron microscopy. EVs were fluorescently labeled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of EVs was performed using miRNA sequencing, target genes of the most expressed miRNA were annotated, and functional enrichment analysis was performed. MAIN RESULTS AND THE ROLE OF CHANCE EVs measured 100–300 nm in diameter, a concentration of 1.78 × 1011 ± 4.12 × 1010 (SD) particles/ml and expressed intraluminal protein markers Heat shock protein 70 (HSP70) and Tumor Susceptibility Gene 101 (TSG101), in addition to CD9 and CD81 transmembrane proteins. Human blastocysts efficiently internalized fluorescent EVs within 1–2 h, and more pronounced internalization was observed in the hatched pole of the embryos. miRNA-seq analysis featured 149 annotated miRNAs, of which 37 were deemed most relevant. The latter had 6592 reported gene targets, that in turn, have functional implications in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization, and gene expression. Among the relevant miRNAs contained in these EVs, we highlight hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p, and hsa-let-7a-5p as master regulators of the biological processes. LIMITATIONS, REASONS FOR CAUTION This is an in vitro study in which conditions of endometrial cell culture could not mimic the intrauterine environment. WIDER IMPLICATIONS OF THE FINDINGS This study defines potential biomarkers of endometrial receptivity and embryo competence that could be useful diagnostic and therapeutic targets for implantation success, as well as open insight further investigations to elucidate the molecular mechanisms implicated in a successful implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Spanish Ministry of Education through FPU awarded to M.S.-B. (FPU18/03735), the Health Institute Carlos III awarded to E.J.-B. (FI19/00110) and awarded to H.F. by the Miguel Servet Program 'Fondo Social Europeo «El FSE invierte en tu futuro»' (CP20/00120), and Generalitat Valenciana through VALi+d Programme awarded to M.C.C.-G. (ACIF/2019/139). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Background: Uterine leiomyomas (UL) are the most common benign tumor in women of reproductive age. Their pathology remains unclear, which hampers the development of safe and effective treatments. Raising evidence suggests epigenetics as a main mechanism involved in tumor development. Histone modification is a key component in the epigenetic regulation of gene expression. Specifically, the histone mark H3K4me3, which promotes gene expression, is altered in many tumors. In this study, we aimed to identify if the histone modification H3K4me3 regulates the expression of genes involved in uterine leiomyoma pathogenesis. Methods: Prospective study integrating RNA-seq (n = 48) and H3K4me3 CHIP-seq (n = 19) data of uterine leiomyomas versus their adjacent myometrium. Differentially expressed genes (FDR < 0.01, log2FC > 1 or < − 1) were selected following DESeq2, edgeR, and limma analysis. Their differential methylation and functional enrichment (FDR < 0.05) were respectively analyzed with limma and ShinyGO. Results: CHIP-seq data showed a global suppression of H3K4me3 in uterine leiomyomas versus their adjacent myometrial tissue (p-value< 2.2e-16). Integrating CHIP-seq and RNA-seq data highlighted that transcription of 696/922 uterine leiomyoma-related differentially expressed genes (DEG) (FDR < 0.01, log2FC > 1 or < − 1) was epigenetically mediated by H3K4me3. Further, 50 genes were differentially trimethylated (FDR < 0.05), including 33 hypertrimethylated/upregulated, and 17 hypotrimethylated/downregulated genes. Functional enrichment analysis of the latter showed dysregulation of neuron-related processes and synapsis-related cellular components in uterine leiomyomas, and a literature review study of these DEG found additional implications with tumorigenesis (i.e. aberrant proliferation, invasion, and dysregulation of Wnt/β-catenin, and TGF-β pathways). Finally, SATB2, DCX, SHOX2, ST8SIA2, CAPN6, and NPTX2 proto-oncogenes were identified among the hypertrimethylated/upregulated DEG, while KRT19, ABCA8, and HOXB4 tumor suppressor genes were identified among hypotrimethylated/downregulated DEG. Conclusions: H3K4me3 instabilities alter the expression of oncogenes and tumor suppressor genes, inducing aberrant proliferation, and dysregulated Wnt/β-catenin, and TGF-β pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can human umbilical cord platelet-rich plasma (hUC-PRP) efficiently treat endometrial damage and restore fertility in a preclinical murine model? SUMMARY ANSWER Local application of hUC-PRP promotes tissue regeneration and fertility restoration in a murine model of Asherman syndrome and endometrial atrophy (AS/EA). WHAT IS KNOWN ALREADY AS/EA are well-described endometrial pathologies that cause infertility; however, there are currently no gold-standard treatments available. Recent reports have described the successful use of human platelet-rich plasma in reproductive medicine, and its regenerative potential is further enhanced using hUC-PRP, due to the ample growth factors and reduced pro-inflammatory cytokines in the latter. STUDY DESIGN, SIZE, DURATION hUC-PRP (n = 3) was processed, characterized and delivered locally to endometrial damage in a murine model (n = 50). The hUC-PRP was either used alone or loaded into a decellularized porcine endometrium-derived extracellular matrix (EndoECM) hydrogel; endometrial regeneration, fertility outcomes and immunocompatibility were evaluated 2 weeks following treatment administration. PARTICIPANTS/MATERIALS, SETTING, METHODS Umbilical cord blood was obtained from women in childbirth. Endometrial damage (mimicking AS/EA) was induced using ethanol in 8-week-old C57BL/6 mice, and treated with the most concentrated hUC-PRP sample 4 days later. Characterization of hUC-PRP and immunotolerance was carried out with multiplex technology, while uterine samples were analyzed by immunohistochemistry and quantitative PCR. The number of embryos and their morphology was determined visually. MAIN RESULTS AND THE ROLE OF CHANCE Platelet density was enhanced 3-fold in hUC-PRP compared to that in hUC blood (P  < 0.05). hUC-PRP was enriched with growth factors related to tissue regeneration (i.e. hepatocyte growth factor, platelet-derived growth factor-BB and epidermal growth factor), which were released constantly (in vitro) when hUC-PRP was loaded into EndoECM. Both treatments (hUC-PRP alone and hUC-PRP with EndoECM) were immunotolerated and caused significantly regeneration of the damaged endometrium, evidenced by increased endometrial area, neoangiogenesis, cell proliferation and gland density and lower collagen deposition with respect to non-treated uterine horns (P  < 0.05). Additionally, we detected augmented gene expression of Akt1 , VEGF and Ang , which are involved in regenerative and proliferation pathways. Finally, hUC-PRP treatment restored pregnancy rates in the mouse model. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This proof-of-concept pilot study was based on a murine model of endometrial damage and the use of EndoECM requires further validation prior to clinical implementation for women affected by AS/EA. WIDER IMPLICATIONS OF THE FINDINGS The local administration of hUC-PRP has high impact and is immunotolerated in a murine model of AS/EA, as has been reported in other tissues, making it a promising candidate for heterologous treatment of these endometrial pathologies. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Ministerio de Ciencia, Innovación y Universidades; Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana; and Instituto de Salud Carlos III. The authors do not have any conflicts of interest to declare. [ABSTRACT FROM AUTHOR]

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 hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results: Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions: EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success. [ABSTRACT FROM AUTHOR]

Background: Uterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2'-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option. Methods: Prospective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR. Results: DNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza- CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577). Conclusions: 5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma. [ABSTRACT FROM AUTHOR]

Background: Uterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option. Methods: Prospective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR. Results: DNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577). Conclusions: 5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma. [ABSTRACT FROM AUTHOR]

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism. [ABSTRACT FROM AUTHOR]

Endometrial regeneration has long been proposed to be mediated by stem cells, but the isolation of endometrial stem cells has been hampered by a lack of validated markers. Specific markers would enable isolation of these stem cells, thereby promoting advancements in regenerative medicine for the treatment of endometrial diseases and dysfunctions. We sought to investigate the regenerative ability of human endometrial positive for sushi domain containing 2/intercellular adhesion molecule 1 (SUSD2+/ICAM1+) cells and Side Population cell lines in a xenograft mice model. The injection of total endometrial cell suspensions and Side Population cell lines under kidney capsules induced neoformation of human endometrium verified by the presence of typical endometrial markers (vimentin, cytokeratin 18, and progesterone receptor) by immunofluorescence. Total endometrial cell types promoted a better reconstitution in comparison to injecting ICAM1+ and SUSD2+ cells alone. The endometrial fraction is probably acting as a niche, resulting in increased reconstruction efficiency of pure fractions. Human engrafted cells were localized near blood vessels and induced the proliferation of surrounding cells. Our results suggest that human endometrial Side Population, a heterogeneous population possibly harboring endometrial stem cells, has the optimum capacity to regenerate endometrial-like tissue. In contrast, cells positive for single stem cell markers SUSD2 and ICAM1 have minimally functional regenerative capacities in the absence of niche-like cells. [ABSTRACT FROM AUTHOR]

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 female recipients with spontaneous or induced menstruations were selected and their endometrium was examined for the contribution of XY donor-derived cells using fluorescent in situ hybridization (FISH), telomapping and SP method investigation. We confirm the presence of XY donor-derived cells in the recipient endometrium ranging from 1.7% to 2.62%. We also identify 0.45-0.85% of the donor-derived cells in the epithelial compartment displaying CD9 marker, and 1.0-1.83% of the Vimentin-positive XY donor-derived cells in the stromal compartment. Although the percentage of endometrial SP cells decreased, possibly being due to chemotherapy applied to these patients, they were not formed by XY donor-derived cells, donor BM cells were not associated with the stem cell (SC) niches assessed by telomapping technique, and engraftment percentages were very low with no correlation between time from transplant and engraftment efficiency, suggesting random terminal differentiation. In conclusion, XY donor-derived cells of a BM origin may be considered a limited exogenous source of transdifferentiated endometrial cells rather than a cyclic source of BM donor-derived stem cells. [ABSTRACT FROM AUTHOR]

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. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45-) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERa) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis. [ABSTRACT FROM AUTHOR]

Adenomyosis is related to infertility and miscarriages, but so far there are no robust in vitro models that reproduce its pathological features to study the molecular mechanisms involved in this disease. Endometrial organoids are in vitro 3D models that recapitulate the native microenvironment and reproduce tissue characteristics that would allow the study of adenomyosis pathogenesis and related infertility disorders. In our study, human endometrial biopsies from adenomyosis (n = 6) and healthy women (n = 6) were recruited. Organoids were established and hormonally differentiated to recapitulate midsecretory and gestational endometrial phases. Physiological and pathological characteristics were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, and ELISA. Secretory and gestational organoids recapitulated in vivo glandular epithelial phenotype (pan-cytokeratin, Muc-1, PAS, Laminin, and Ki67) and secretory and gestational features (α-tubulin, SOX9, SPP1, PAEP, LIF, and 17βHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-β2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17βHSD2 compared with control organoids. Our results demonstrate that organoids derived from endometria of adenomyosis patients and differentiated to secretory and gestational phases recapitulate native endometrial-tissue-specific features and disease-specific traits. Adenomyosis-derived organoids are a promising in vitro preclinical model to study impaired implantation and pregnancy disorders in adenomyosis and enable personalized drug screening. [ABSTRACT FROM AUTHOR]

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 EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids. [ABSTRACT FROM AUTHOR]