Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders.

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.

Hybrid Endometrial-Derived Hydrogels: Human Organoid Culture Models and In Vivo Perspectives.

The endometrium plays a vital role in fertility, providing a receptive environment for embryo implantation and development. Understanding the endometrial physiology is essential for developing new strategies to improve reproductive healthcare. Human endometrial organoids (hEOs) are emerging as powerful models for translational research and personalized medicine. However, most hEOs are cultured in a 3D microenvironment that significantly differs from the human endometrium, limiting their applicability in bioengineering. This study presents a hybrid endometrial-derived hydrogel that combines the rigidity of PuraMatrix (PM) with the natural scaffold components and interactions of a porcine decellularized endometrial extracellular matrix (EndoECM) hydrogel. This hydrogel provides outstanding support for hEO culture, enhances hEO differentiation efficiency due to its biochemical similarity with the native tissue, exhibits superior in vivo stability, and demonstrates xenogeneic biocompatibility in mice over a 2-week period. Taken together, these attributes position this hybrid endometrial-derived hydrogel as a promising biomaterial for regenerative treatments in reproductive medicine.

Human blastocysts uptake extracellular vesicles secreted by endometrial cells containing miRNAs related to implantation.

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 futuro¼' (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.

Advances of xenogeneic ovarian extracellular matrix hydrogels for in vitro follicle development and oocyte maturation.

Research aimed at preserving female fertility is increasingly using bioengineering techniques to develop new platforms capable of supporting ovarian cell function in vitro and in vivo. Natural hydrogels (alginate, collagen, and fibrin) have been the most exploited approaches; however they are biologically inert and/or biochemically simple. Thus, establishing a suitable biomimetic hydrogel from decellularized ovarian cortex (OC) extracellular matrix (OvaECM) could provide a complex native biomaterial for follicle development and oocyte maturation. The objectives of this work were (i) to establish an optimal protocol to decellularize and solubilize bovine OC, (ii) to characterize the histological, molecular, ultrastructural, and proteomic properties of the resulting tissue and hydrogel, and (iii) to assess its biocompatibility and adequacy for murine in vitro follicle growth (IVFG). Sodium dodecyl sulfate was identified as the best detergent to develop bovine OvaECM hydrogels. Hydrogels added into standard media or used as plate coatings were employed for IVFG and oocyte maturation. Follicle growth, survival, hormone production, and oocyte maturation and developmental competence were evaluated. OvaECM hydrogel-supplemented media best supported follicle survival, expansion, and hormone production, while the coatings provided more mature and competent oocytes. Overall, the findings support the xenogeneic use of OvaECM hydrogels for future human female reproductive bioengineering.

H3K4me3 mediates uterine leiomyoma pathogenesis via neuronal processes, synapsis components, proliferation, and Wnt/ß-catenin and TGF-ß pathways.

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/ß-catenin, and TGF-ß 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/ß-catenin, and TGF-ß pathways, that ultimately promote uterine leiomyoma progression. The reversal of these histone modifications may be a promising new therapeutic alternative for uterine leiomyoma patients.

Extracellular vesicles secreted by adenomyosis endometrial organoids contain miRNAs involved in embryo implantation and pregnancy.

RESEARCH QUESTION: Do extracellular vesicles secreted by the endometrium of women with adenomyosis contain miRNAs involved in adenomyosis-related infertility? DESIGN: A descriptive study using organoids from eutopic endometrium of women with adenomyosis (n = 4) generated and differentiated to secretory and gestational phases, in which miRNA cargo from extracellular vesicles secreted by these differentiated organoids in each phase was analysed by next-generation sequencing. miRNAs in secretory-extracellular vesicles and gestational-extracellular vesicles were selected based on the counts per million. miRNAs target genes in each phase were obtained from miRNet and gene ontology was used for enrichment analysis. RESULTS: miRNA sequencing identified 80 miRNAs in secretory-phase extracellular vesicles, including hsa-miR-21-5p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-92a-3p, hsa-miR-92b-3p, hsa-miR-200c-3p and hsa-miR-423a-5p, related to adenomyosis pathogenesis and implantation failure. Further, 60 miRNAs were identified in gestational-phase extracellular vesicles, including hsa-miR-21-5p, hsa-miR-26a-5p, hsa-miR-30a-5p, hsa-miR-30c-5p, hsa-miR-222-3p and hsa-miR-423a-5p were associated with preeclampsia and miscarriage. Among the target genes of these miRNAs, PTEN, MDM4, PLAGL2 and CELF1, whose downregulation (P = 0.0003, P < 0.0001, P = 0.0002 and P = 0.0003, respectively) contributes to adenomyosis pathogenesis, and impaired early embryo development, leading to implantation failure and miscarriage, are highlihghted. Further, functional enrichment analyses of the target genes revealed their involvement in cell differentiation, proliferation, apoptosis, cell cycle regulation and response to extracellular stimuli. CONCLUSIONS: Eutopic endometrium in secretory and gestational phase from women with adenomyosis releases extracellular vesicles containing miRNAs involved in adenomyosis progression, impaired embryo implantation and pregnancy complications.

Human umbilical cord platelet-rich plasma to treat endometrial pathologies: methodology, composition and pre-clinical models.

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 INTERESTS: 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.

Endometrial gene expression differences in women with coronavirus disease 2019.

OBJECTIVE: To study the potential effect of coronavirus disease (COVID-19) on the endometrial transcriptome of affected, symptomatic women for the detection of altered gene expression. DESIGN: Pilot study of the endometrial transcriptomes of women manifesting COVID-19 compared with those of women without COVID-19 undergoing hysteroscopic procedures for benign gynecologic disorders using RNA sequencing. SETTING: Hospital and university laboratories. PATIENT(S): Women with (n = 14) and without a COVID-19 (n = 10) diagnosis based on a nasopharyngeal swab analysis using quantitative reverse-transcription polymerase chain reaction. The endometrium of the patients with COVID-19 had previously been tested for severe acute respiratory syndrome coronavirus 2 infection, revealing the absence of the virus in this tissue. INTERVENTION(S): Endometrial biopsy sample collection. MAIN OUTCOMES MEASURE(S): Endometrial gene expression and functional analysis of symptomatic patients with COVID-19 vs. individuals without the infection. RESULT(S): The systemic disease COVID-19 altered endometrial gene expression in 75% of the women, with the patients exhibiting a preponderance of 163 up-regulated (e.g., UTS2, IFI6, IFIH1, and BNIP3) and 72 down-regulated genes (e.g., CPZ, CDH3, and IRF4) (false discovery rate<0.05). A total of 161 dysregulated functions (36 up-regulated and 125 down-regulated) were typically enriched in the endometria of the patients with COVID-19, including up-regulation in pathways involved in the development of immune responses to viruses and cytokine inflammation, reflecting elicitation of a COVID-19 response pathway. CONCLUSION(S): Coronavirus disease 2019 affects endometrial gene expression despite the absence of severe acute respiratory syndrome coronavirus 2 RNA in endometrial tissues.

Establishment of Adenomyosis Organoids as a Preclinical Model to Study Infertility.

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 17ßHSD2 expression and SPP1 secretion). Adenomyosis organoids showed higher expression of TGF-ß2 and SMAD3 and increased gene expression of SPP1, PAEP, LIF, and 17ßHSD2 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.

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

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.

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 ß3 (TGF-ß3) 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-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter (Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. RESULT(S): The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 µM SAHA, 73.46% at 10 µM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 µM SAHA, FC = 0.556; 10 µM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 µM SAHA, FC = 0.828) and CCND1 (5 µM SAHA, FC = 0.583; 10 µM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 µM SAHA, FC = 0.815; 10 µM SAHA, FC = 0.673) and COLLAGEN I (5 µM SAHA, FC = 0.599; 10 µM SAHA, FC = 0.635), in HULP cells. TGFß3 and MMP9 gene expression was also significantly down-regulated by 10 µM SAHA (TGFß3, FC = 0.596; MMP9, FC = 0.677). CONCLUSION(S): SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-ß3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

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 uterine horns of female C57BL/6 mice were first injected with 70% ethanol, then four days later, they were treated with: saline (negative control); biotin-labeled EndoECM; or biotin-labeled EndoECM plus platelet-derived GF, basic fibroblast GF, and insulin-like GF 1 (EndoECM+GF). Endometrial regeneration and fertility restoration were evaluated by assessing the number of glands, endometrial area, cell proliferation, neaoangiogenesis, reduction of collagen deposition, and fertility restoration. Interestingly, regenerative effects such as an increased number of endometrial glands, increased area, high cell proliferative index, development of new blood vessels, reduction of collagen deposition, and higher pregnancy rate occurred in mice treated with EndoECM+GF. Thus, a bioengineered system based on EndoECM hydrogel supplemented with GFs may be promising for the clinical treatment of endometrial conditions such as Asherman's syndrome and endometrial atrophy. STATEMENT OF SIGNIFICANCE: In the last years, the bioengineering field has developed new and promising approaches to regenerate tissues or replace damaged and diseased tissues. Bioengineered hydrogels offer an ideal option because these materials can be used not only as treatments but also as carriers of drugs and other therapeutics. The present work demonstrates for the first time how hydrogels derived from pig endometrium loaded with growth factors could treat uterine pathologies in a mouse model of endometrial damage. These findings provide scientific evidence about bioengineered hydrogels based on tissue-specific extracellular matrix offering new options to treat human infertility from endometrial causes such as Asherman's syndrome or endometrial atrophy.

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 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.

5-aza-2'-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/ß-catenin pathway in human uterine leiomyomas.

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/ß-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/ß-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

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 two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture.

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.

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/ß-catenin and transforming growth factor-ß (TGFß) 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 OUTCOME MEASURE(S): Analysis of Wnt/ß-catenin and TGFß pathways and proliferation by quantitative real-time polymerase chain reaction in leiomyoma and myometrial tissue as well as in VitD-treated HULP cells analyzed by Sanger sequencing. RESULTS: Sequencing data showed that 46% of leiomyomas presented MED12 mutation, whereas no mutations were detected in adjacent myometrium. Expression of Wnt/ß-catenin and TGFß pathway genes was significantly increased in MED12-mutated leiomyomas compared to matched myometrium; no significant differences were found in wild-type (WT) leiomyomas. In HULP cells, VitD significantly decreased PCNA expression of both MED12-mutated and WT groups. VitD treatment decreased WNT4 and ß-catenin expression in both groups compared to controls, with significance for WNT4 expression in MED12-mutated samples. Similarly, VitD significantly inhibited TGFß3 expression in cells from both groups. MMP9 expression also decreased. CONCLUSION: Despite molecular differences between MED12-mutated and WT leiomyomas, VitD inhibited Wnt/ß-catenin and TGFß pathways in HULP cells, suggesting VitD as an effective treatment to reduce proliferation and extracellular matrix formation in different molecular subtypes of uterine leiomyomas.

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-damaged NOD/SCID mice were treated with plasma samples from several origins. MAIN OUTCOME MEASURE(S): To investigate the possible beneficial effects of PRP from Asherman syndrome/endometrial atrophy patients. To test if plasma from human umbilical cord blood had a stronger effect than adult PRP in endometrial regeneration. To demonstrate if PRP from Asherman syndrome/endometrial atrophy patients was as effective as PRP from a healthy woman and could therefore be used for autologous treatment. RESULT(S): All plasma samples contained molecules with a high potential for regeneration (stem cell factor, platelet-derived growth factor BB, thrombospondin-1, von Willebrand factor). Furthermore, the highest increase in in vitro proliferation and migration rate was found when endometrial stromal cells were treated with umbilical cord plasma; adult PRP also revealed a significant increment. In the mouse model, a higher expression of Ki67 and Hoxa10 in the endometrium was detected after applying adult PRP, and the proteomic analysis revealed a specific protein expression profile depending on the treatment. The damaged uterine tissue showed more proregenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) compared with the other treatments (nonactivated umbilical cord plasma, activated adult PRP, and no treatment). CONCLUSION(S): Human PRP possesses regeneration properties usable for endometrial pathologies. Besides that, these regenerative effects seem to be more apparent when the source of obtaining is umbilical cord blood.

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/computerized tomography and macroscopic examination), cell proliferation (immunohistochemistry and quantitative real-time polymerase chain reaction [qRT-PCR]), ECM (Western blot), transforming growth factor (TGF) ß3 (qRT-PCR), and apoptosis (Westrn blot and TUNEL). RESULT(S): Short-term treatment with vitamin D did not appear to alter leiomyoma size, based on in vivo monitoring and macroscopic examination. However, long-term high-dose treatment induced a significant reduction in leiomyoma size. Cell proliferation was not decreased in the short term, whereas 1 µg/kg/d vitamin D in the long term significantly reduced proliferation compared with control. Although collagen-I and plasminogen activator inhibitor 1 were not modified by short-term treatment, they were both significantly reduced by long-term high-dose vitamin D. Similarly, long-term high-dose vitamin D significantly reduced TGF-ß3 expression. Finally, apoptosis significantly increased with both short- and long-term high-dose vitamin D treatment. CONCLUSION(S): Long-term vitamin D acts as an antiproliferative, antifibrotic, and proapoptotic therapy that provides a safe, nonsurgical therapeutic option for reducing uterine leiomyoma size without side-effects.