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.

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.

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.

Deciphering the Role of Histone Modifications in Uterine Leiomyoma: Acetylation of H3K27 Regulates the Expression of Genes Involved in Proliferation, Cell Signaling, Cell Transport, Angiogenesis and Extracellular Matrix Formation.

Uterine leiomyoma (UL) is a benign tumor arising from myometrium (MM) with a high prevalence and unclear pathology. Histone modifications are altered in tumors, particularly via histone acetylation which is correlated with gene activation. To identify if the acetylation of H3K27 is involved in UL pathogenesis and if its reversion may be a therapeutic option, we performed a prospective study integrating RNA-seq (n = 48) and CHIP-seq for H3K27ac (n = 19) in UL vs MM tissue, together with qRT-PCR of SAHA-treated UL cells (n = 10). CHIP-seq showed lower levels of H3K27ac in UL versus MM (p-value < 2.2 × 10−16). From 922 DEGs found in UL vs. MM (FDR < 0.01), 482 presented H3K27ac. A differential acetylation (FDR < 0.05) was discovered in 82 of these genes (29 hyperacetylated/upregulated, 53 hypoacetylated/downregulated). Hyperacetylation/upregulation of oncogenes (NDP,HOXA13,COL24A1,IGFL3) and hypoacetylation/downregulation of tumor suppressor genes (CD40,GIMAP8,IL15,GPX3,DPT) altered the immune system, the metabolism, TGFß3 and the Wnt/ß-catenin pathway. Functional enrichment analysis revealed deregulation of proliferation, cell signaling, transport, angiogenesis and extracellular matrix. Inhibition of histone deacetylases by SAHA increased expression of hypoacetylated/downregulated genes in UL cells (p < 0.05). Conclusively, H3K27ac regulates genes involved in UL onset and maintenance. Histone deacetylation reversion upregulates the expression of tumor suppressor genes in UL cells, suggesting targeting histone modifications as a therapeutic approach for UL.

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.

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.

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.

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.