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.

Adjustment of progesterone administration after endometrial transcriptomic analysis does not improve reproductive outcomes in women with adenomyosis.

RESEARCH QUESTION: Do patients with adenomyosis present a dysregulated endometrial receptivity that can be reversed with personalized embryo transfer (PET) by transcriptomic-based progesterone adjustment, improving IVF outcomes? DESIGN: A multicentre, retrospective, cohort study that transcriptomically analysed the endometrial receptivity of the endometrium in patients with adenomyosis (n = 81) and healthy women (n = 231). Subsequently, implantation, biochemical and clinical miscarriage, and live birth rates between adenomyosis patients with one previous implantation failure using donor oocytes who received (n = 59) or did not receive (n = 66) PET based on endometrial receptivity, were observed to evaluate if adjusted progesterone improves reproductive outcomes of adenomyosis patients. RESULTS: Patients with adenomyosis significantly presented an altered endometrial receptivity (non-receptive) compared with healthy patients (53.1% versus 37.2%, P = 0.0179), elevating the risk of adenomyosis patients having a non-receptive endometrium 42.59% higher (95% CI 41.50 to 44.45). No significant differences were found in implantation (62.7% versus 78.8%, P = 0.0514), biochemical (13.5% versus 3.9%, P = 0.1223) and clinical (10.8% versus 15.4%, P = 0.7543) miscarriage, or live birth rates (75.7% versus 80.8%, P = 0.6066), in patients with PET compared with those without PET. CONCLUSIONS: Women with adenomyosis presented an altered expression of genes involved in decidualization, and a higher rate of non-receptive endometrial statuses than controls. Although progesterone is indispensable for implantation, adjusting progesterone before PET, using endometrial transcriptomic signatures, does not improve IVF outcomes in patients with adenomyosis. Other molecular mechanisms beyond progesterone regulation may be involved in implantation failure.

Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is unknown. Class I histone deacetylases (including HDAC1, 2, 3, and 8) are one of the major classes of the HDAC family and catalyze the removal of acetyl groups from lysine residues in histones and cellular proteins. Class I HDACs exhibit distinct cellular and subcellular expression patterns and are involved in many biological processes and diseases through diverse signaling pathways. However, the link between class I HDACs and uLMS is still being determined. In this study, we assessed the expression panel of Class I HDACs in uLMS and characterized the role and mechanism of class I HDACs in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that HDAC1, 2, and 3 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium. Immunoblot analysis demonstrated that the expression levels of HDAC 1, 2, and 3 exhibited a graded increase from normal and benign to malignant uterine tumor cells. Furthermore, inhibition of HDACs with Class I HDACs inhibitor (Tucidinostat) decreased the uLMS proliferation in a dose-dependent manner. Notably, gene set enrichment analysis of differentially expressed genes (DEGs) revealed that inhibition of HDACs with Tucidinostat altered several critical pathways. Moreover, multiple epigenetic analyses suggested that Tucidinostat may alter the transcriptome via reprogramming the oncogenic epigenome and inducing the changes in microRNA-target interaction in uLMS cells. In the parallel study, we also determined the effect of DL-sulforaphane on the uLMS. Our study demonstrated the relevance of class I HDACs proteins in the pathogenesis of malignant uLMS. Further understanding the role and mechanism of HDACs in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles.

Uterine leiomyomas (ULs) are the most common benign tumors in women of reproductive age. Despite the high prevalence, tumor pathology remains unclear, which hampers the development of safe and effective treatments. Epigenetic mechanisms appear to be involved in UL development, particularly via DNA methylation that regulates gene expression. We aimed to determine the relationship between DNA methylation and gene expression in UL compared with adjacent myometrium (MM) to identify molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, and a different global transcriptome profile. Integration of DNA methylation and whole-transcriptome RNA-sequencing data identified 93 genes regulated by methylation, with 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes and molecular functions involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation, as well as pathways related to uterine biology and cancer. Cellular components such as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1, RIMS2, TFAP2C) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2), which are related to proliferation, invasion, altered metabolism, deposition of extracellular matrix, and Wnt/ß-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, inhibition of DNA methyltransferases by 5-aza-2'-deoxycitidine increased the expression of hypermethylated/downregulated genes in UL cells in vitro. In conclusion, gene regulation by DNA methylation is implicated in UL pathogenesis, and reversion of this methylation could offer a therapeutic option for UL. © 2022 The Pathological Society of Great Britain and Ireland.

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.

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.

Novel therapeutic targets to improve IVF outcomes in endometriosis patients: a review and future prospects.

BACKGROUND: Patients with endometriosis often experience infertility and have poor IVF outcomes, with low fertilization and pregnancy rates. Although many theories have tried to explain the mechanisms underlying infertility in these patients, none of them is conclusive. OBJECTIVE AND RATIONALE: In this review, we discuss the pathologic mechanisms through which endometriosis likely leads to infertility along with the therapeutic options used to date to treat endometriosis-related infertility and, thereby, to improve IVF outcomes in patients with endometriosis. SEARCH METHODS: We performed a comprehensive literature search of clinical outcomes in endometriosis and the molecular mechanisms contributing to oocyte quality using the PubMed database to identify human and animal studies published from 1992 until September 2020. In total, 123 manuscripts were included. OUTCOMES: While some theories propose that endometriosis patients may have fertility problems as a result of decreased endometrial receptivity, others reinforce the idea that infertility could be associated with oocyte alterations and lower implantation rates. Single-cell RNA sequencing of oocytes from patients with endometriosis has identified dysregulated mechanisms involved in steroid metabolism and biosynthesis, response to oxidative stress and cell cycle regulation. Dysregulation of these mechanisms could result in the poor IVF outcomes observed in patients with endometriosis. Further, impaired steroidogenesis may directly affect oocyte and embryo quality. Increased oxidative stress in patients with endometriosis also has a detrimental effect on the follicular microenvironment, inducing cell cycle dysregulation in oocytes, poor oocyte quality, and infertility. Moreover, granulosa cells in the context of endometriosis undergo increased apoptosis and have an altered cell cycle that could adversely affect folliculogenesis, oocyte and embryo quality, and IVF outcomes. Endometriosis is also associated with inflammatory damage and impaired angiogenesis, which could be directly correlated with poor IVF outcomes. While therapeutic options using GnRH analogues, progestins and aromatase inhibitors do not improve endometriosis-related infertility, anti-inflammatory agents and antioxidant supplementation could improve oocyte quality as well as implantation and clinical pregnancy rates in patients with endometriosis. WIDER IMPLICATIONS: Endometriosis is a heterogeneous disease whose pathogenesis is complex and could affect fertility by altering a collection of molecular mechanisms in oocytes. Thus, a single model is not sufficient to describe endometriosis-related infertility. Dysregulation of steroidogenesis, oxidative stress, cell cycle progression, inflammation and angiogenesis in the follicular environment and oocytes in individuals with endometriosis are all possible contributors to endometriosis-related infertility. Therefore, treatments targeting these mechanisms could be therapeutic alternatives to improve IVF outcomes for these patients.

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.

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.

Single-cell RNA sequencing of oocytes from ovarian endometriosis patients reveals a differential transcriptomic profile associated with lower quality.

STUDY QUESTION: Do oocytes from women with ovarian endometriosis (OE) have a different transcriptomic profile than those from healthy women? SUMMARY ANSWER: Oocytes from endometriosis patients, independently of whether they came from the affected ovary, exhibited a differential transcriptomic profile compared to oocytes from healthy egg donors. WHAT IS KNOWN ALREADY: Studies of endometriosis have sought to determine whether OE affects oocyte quality. While many reports indicate that oocytes recovered from endometriotic ovaries may be affected by the disease, other studies have found no significant differences among oocyte/embryo quality and fertilization, implantation and pregnancy rates in women with endometriosis. STUDY DESIGN, SIZE, DURATION: This prospective study compared metaphase II (MII) oocytes (n = 16) from endometriosis patients (n = 7) to oocytes (n = 16) from healthy egg donors (n = 5) by single-cell RNA sequencing (scRNA-seq). Participants were recruited between December 2016 and February 2018 at IVI-RMA Valencia and Vigo clinics. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human MII oocytes were collected from healthy egg donors and OE patients aged 18-34 years, with a body mass index of <30 and >6 pre-antral follicles. RNA was extracted, cDNA was generated and libraries were constructed and sequenced. scRNA-seq data libraries were processed and statistically analysed. Selected genes were validated by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Our scRNA-seq results revealed an effect of endometriosis on global transcriptome behaviour in oocytes from endometriotic ovaries. The highest number of differentially expressed genes (DEGs) was found when oocytes from women with OE were compared to oocytes from healthy donors [520 DEGs (394 upregulated and 126 downregulated)], independently of whether oocytes came from an affected or unaffected ovary. Among the top 20 significant DEGs in this comparison, most were upregulated, including APOE, DUSP1, G0S2, H2AFZ, ID4, MGST1 and WEE1. PXK was the only downregulated gene. Subsequently, functional analysis showed 31 enriched functions deregulated in endometriosis patients (Benjamini P < 0.1), being 16 significant enriched functions considering Benjamini P < 0.05, which involved in biological processes and molecular functions, such as steroid metabolism, response to oxidative stress and cell growth regulation. In addition, our functional analysis showed enrichment for mitochondria, which are an important cellular component in oocyte development. Other functions important in embryo development, such as angiogenesis and methylation, were also significantly enriched. LARGE SCALE DATA: All raw sequencing data are submitted in Gene Expression Omnibus (GEO) under accession number (PRJNA514416). LIMITATIONS, REASONS FOR CAUTION: This study was restricted only to OE and thereby other anatomical entities, such as peritoneal and deep infiltrating endometriosis, were not considered. This is a descriptive study with a limited number of samples reflecting the difficulty to recruit human oocytes, especially from women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that OE exhibits a global transcriptomic effect on oocytes of patients in OE, independently if they come from an affected or unaffected ovary and alters key biological processes and molecular functions related to steroid metabolism, response to oxidative stress and cell growth regulation, which reduce oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by IVI Foundation, the Spanish Ministry of Economy and Competitiveness through the Miguel Servet programme (CPII018/00002 to F.D.), the Sara Borrell Program (CD15/00057 to H.F.) and the VALi+d Programe (Generalitat Valenciana); ACIF/2016/444 to A.C.). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: None.

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

OBJECTIVE: To assess the effect of vitamin D (VitD) on human uterine leiomyomas through Wnt/ß-catenin pathway inhibition, apoptosis induction, and cell growth arrest. DESIGN: A prospective study comparing leiomyoma vs. myometrium tissues. Paired design study comparing human uterine leiomyoma primary (HULP) cells treated with or without VitD. SETTING: University hospital. PATIENT(S): Human uterine leiomyoma and myometrium were collected from women (aged 35-52 years) without hormonal treatment. INTERVENTION(S): Samples were collected from women undergoing surgery due to symptomatic uterine leiomyoma pathology. MAIN OUTCOME MEASURE(S): Uterine leiomyoma and myometrium tissues were analyzed by western blot (WB) to determine proliferation, Wnt/ß-catenin, and apoptosis pathways. HULP cells were used to study VitD effect in cell proliferation (WB), cell cycle (flow cytometry), Wnt/ß-catenin and apoptosis genes (polymerase chain reaction arrays), Wnt-related proteins (protein array), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] assay). RESULTS: Human leiomyoma tissues compared with matched myometrium showed higher proliferation (fold change = 8.16; P=.0006) and altered Wnt/ß-catenin pathway (fold change = 5.5; P<.0001), whereas no differences in apoptosis were observed. VitD induced cell growth arrest and decreased proliferation in HULP cells (fold change = 0.74; P=.007). Moreover, VitD decreased Wnt-pathway expression in HULP cells at gene (activity score = -0.775; P<.001) and protein levels. However, VitD did not induce apoptosis expression. CONCLUSION: Increased proliferation and Wnt/ß-catenin pathway deregulation play a role in the development and growth of leiomyomas, whereas apoptosis appears not to contribute. VitD exerts an antiproliferative action on HULP cells through cell growth arrest and Wnt/ß-catenin pathway inhibition, but not through apoptosis regulation, suggesting VitD as an effective therapy to stabilize leiomyoma size and prevent its growth.