Comparison of chromatin accessibility remodeling of granulosa cells in patients with endometrioma or pelvic/tubal infertility

Purpose To elucidatethe epigenetic alteration associated with impaired oogenesis in endometrioma using multi-omic approaches.

ATAC-seq was performed on the granulosa cells (GCs) of 6 patients (3 with endometrioma and 3 without). Follicular samples from another 20 patients (10 with endometrioma and 10 without) were collected for mRNA-seq analysis of GCs and extracellular vesicles (EVs) of follicular fluid. qRT-PCR validated candidate genes in GCs from 44 newly enrolled patients (19 with endometrioma and 25 without). mRNA abundance was compared with the Mann–Whitney test. Pearson’s correlation analyzed relationships between candidate genes and oocyte parameters.

Chromatin accessibility and gene expression profiles of GCs from endometrioma patients differed significantly from the pelvic/tubal infertility group. RNA-seq revealed most differentially expressed genes were downregulated (6216/7325) and enriched in the cellular localization pathway. Multi-omics analyses identified 22 significantly downregulated genes in the GCs of endometrioma patients, including PPIF (P < 0.0001) and VEGFA (P = 0.0148). Both genes were further confirmed by qRT-PCR. PPIF (r = 0.46, p = 0.043) and VEGFA (r = 0.45, p = 0.048) correlated with the total number of retrieved oocytes.

GC chromatin remodeling may disrupt GC and EV transcriptomes, interfering with somatic cell-oocyte communication and leading to compromised oogenesis in endometrioma patients. Similar content being viewed by others Data availability All sequencing data presented in this study can be found in Sequence Read Archive (SRA) with accession no. PRJNA806789. Code availability All analysis codes used in this study can be obtained by application to the authors.

Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. 2020;382(13):1244–56. Nnoaham KE, et al. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril. 2011;96(2):366-373 e8. Yilmaz Hanege B, GulerCekic S, Ata B. Endometrioma and ovarian reserve: effects of endometriomata per se and its surgical treatment on the ovarian reserve. Facts Views Vis Obgyn. 2019;11(2):151–7. Macer ML, Taylor HS. Endometriosis and infertility: a review of the pathogenesis and treatment of endometriosis-associated infertility. Obstet Gynecol Clin North Am. 2012;39(4):535–49. Kaponis A, et al. Current treatment of endometrioma. Obstet Gynecol Surv. 2015;70(3):183–95. Galczynski K, et al. Ovarian endometrioma - a possible finding in adolescent girls and young women: a mini-review. J Ovarian Res. 2019;12(1):104. Giacomini E, et al. Characteristics of follicular fluid in ovaries with endometriomas. Eur J Obstet Gynecol Reprod Biol. 2017;209:34–8. Sanchez AM, et al. The distinguishing cellular and molecular features of the endometriotic ovarian cyst: from pathophysiology to the potential endometrioma-mediated damage to the ovary. Hum Reprod Update. 2014;20(2):217–30. Shah DK. Diminished ovarian reserve and endometriosis: insult upon injury. Semin Reprod Med. 2013;31(2):144–9. Seyhan A, Ata B, Uncu G. The impact of endometriosis and its treatment on ovarian reserve. Semin Reprod Med. 2015;33(6):422–8. Ferrero H, et al. Single-cell RNA sequencing of oocytes from ovarian endometriosis patients reveals a differential transcriptomic profile associated with lower quality. Hum Reprod. 2019;34(7):1302–12. Sanchez AM, et al. Is the oocyte quality affected by endometriosis? A review of the literature. J Ovarian Res. 2017;10(1):43. Xu B, et al. Oocyte quality is decreased in women with minimal or mild endometriosis. Sci Rep. 2015;5:10779. Ben Maamar M, Nilsson EE, Skinner MK. Epigenetic transgenerational inheritance, gametogenesis and germline development dagger. Biol Reprod. 2021;105(3):570–92. Li Y, et al. Abnormal PIWI-interacting RNA profile and its association with the deformed extracellular matrix of oocytes from recurrent oocyte maturation arrest patients. Fertil Steril. 2021;115(5):1318–26. Tomizawa S, Nowacka-Woszuk J, Kelsey G. DNA methylation establishment during oocyte growth: mechanisms and significance. Int J Dev Biol. 2012;56(10–12):867–75. Sendzikaite G, Kelsey G. The role and mechanisms of DNA methylation in the oocyte. Essays Biochem. 2019;63(6):691–705. Sha QQ, Zhang J, Fan HY. Function and regulation of histone H3 lysine-4 methylation during oocyte meiosis and maternal-to-zygotic transition. Front Cell Dev Biol. 2020;8: 597498. Yu C, et al. CFP1 regulates histone H3K4 trimethylation and developmental potential in mouse oocytes. Cell Rep. 2017;20(5):1161–72. Sha QQ, et al. CFP1 coordinates histone H3 lysine-4 trimethylation and meiotic cell cycle progression in mouse oocytes. Nat Commun. 2018;9(1):3477. Miyamoto K, et al. Chromatin accessibility impacts transcriptional reprogramming in oocytes. Cell Rep. 2018;24(2):304–11. Gu C, et al. Integrative single-cell analysis of transcriptome, DNA methylome and chromatin accessibility in mouse oocytes. Cell Res. 2019;29(2):110–23. Zhang C, et al. The chromatin remodeler Snf2h is essential for oocyte meiotic cell cycle progression. Genes Dev. 2020;34(3–4):166–78. Buenrostro JD, et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10(12):1213–8. Bajic M, Maher KA, Deal RB. Identification of open chromatin regions in plant genomes using ATAC-seq. Methods Mol Biol. 2018;1675:183–201. Buenrostro JD, et al. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr Protoc Mol Biol. 2015;109:21 29 1-21 29 9. Picelli S, et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 2014;24(12):2033–40. Li Z, et al. Identification of transcription factor binding sites using ATAC-seq. Genome Biol. 2019;20(1):45. Hendrickson DG, et al. Simultaneous profiling of DNA accessibility and gene expression dynamics with ATAC-seq and RNA-seq. Methods Mol Biol. 2018;1819:317–33. Doyle LM, Wang MZ. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 2019;8(7):727. Borges FT, Reis LA, Schor N. Extracellular vesicles: structure, function, and potential clinical uses in renal diseases. Braz J Med Biol Res. 2013;46(10):824–30. Bebelman MP, et al. Biogenesis and function of extracellular vesicles in cancer. Pharmacol Ther. 2018;188:1–11. Miranda KC, et al. Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease. Kidney Int. 2010;78(2):191–9. Wang J, et al. Plasma exosomes as novel biomarker for the early diagnosis of gastric cancer. Cancer Biomark. 2018;21(4):805–12. Chen IH, et al. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc Natl Acad Sci U S A. 2017;114(12):3175–80. Dumesic DA, et al. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril. 2015;103(2):303–16. Andronico F, et al. Extracellular vesicles in human oogenesis and implantation. Int J Mol Sci. 2019;20(9):2162. Quinn MC, et al. Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates. Reprod Fertil Dev. 2006;18(5):501–8. Nottola SA, et al. Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization. Microsc Res Tech. 2006;69(6):508–16. Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270–83. https://doi.org/10.1093/humrep/der037. Machtinger R, Racowsky C. Morphological systems of human embryo assessment and clinical evidence. Reprod Biomed Online. 2013;26(3):210–21. Yu G, Wang LG, He QY. ChIPseeker: an R/bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31(14):2382–3. Wu DY, et al. Identifying differential transcription factor binding in ChIP-seq. Front Genet. 2015;6:169. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. Han H, et al. TRRUST: a reference database of human transcriptional regulatory interactions. Sci Rep. 2015;5:11432. Hsueh AJ, et al. Intraovarian control of early folliculogenesis. Endocr Rev. 2015;36(1):1–24. Zhang Y, et al. Transcriptome landscape of human folliculogenesis reveals oocyte and granulosa cell interactions. Mol Cell. 2018;72(6):1021-1034 e4. Ahmadi SE, et al. MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies. J Hematol Oncol. 2021;14(1):121. Xianjun F, et al. Momordin Ic induces G0/1 phase arrest and apoptosis in colon cancer cells by suppressing SENP1/c-MYC signaling pathway. J Pharmacol Sci. 2021;146(4):249–58. Wen L, et al. Phenylethanol glycosides from herba cistanche improve the hypoxic tumor microenvironment and enhance the effects of oxaliplatin via the HIF1alpha signaling pathway. Mol Med Rep. 2021;24(1):1. Song H, et al. HIF-1alpha-mediated telomerase reverse transcriptase activation inducing autophagy through mammalian target of rapamycin promotes papillary thyroid carcinoma progression during hypoxia stress. Thyroid. 2021;31(2):233–46. Sanchez-Alvarez M, et al. Signaling networks converge on TORC1-SREBP activity to promote endoplasmic reticulum homeostasis. PLoS ONE. 2014;9(7): e101164. Xie XJ, et al. CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the ecdysone receptor in Drosophila. PLoS Biol. 2015;13(7): e1002207. Rodriguez AC, et al. ETV4 is necessary for estrogen signaling and growth in endometrial cancer cells. Cancer Res. 2020;80(6):1234–45. Fonseca AS, et al. ETV4 plays a role on the primary events during the adenoma-adenocarcinoma progression in colorectal cancer. BMC Cancer. 2021;21(1):207. Holtz G, et al. Luteinized unruptured follicle syndrome in mild endometriosis. Assessment with biochemical parameters. J Reprod Med. 1985;30(9):643–5. Mio Y, et al. Luteinized unruptured follicle in the early stages of endometriosis as a cause of unexplained infertility. Am J Obstet Gynecol. 1992;167(1):271–3. Xiao T, Li X, Felsenfeld G. The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization. Proc Natl Acad Sci U S A. 2021;118(7):e2023127118. Luo W, et al. MYC associated zinc finger protein promotes the invasion and metastasis of hepatocellular carcinoma by inducing epithelial mesenchymal transition. Oncotarget. 2016;7(52):86420–32. Javadov S, et al. Mitochondrial permeability transition in cardiac ischemia-reperfusion: whether cyclophilin D is a viable target for cardioprotection? Cell Mol Life Sci. 2017;74(15):2795–813. Xiao A, et al. The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells. Biochem Biophys Res Commun. 2017;483(1):765–71. Sargent KM, et al. Loss of vascular endothelial growth factor A (VEGFA) isoforms in granulosa cells using pDmrt-1-Cre or Amhr2-Cre reduces fertility by arresting follicular development and by reducing litter size in female mice. PLoS ONE. 2015;10(2): e0116332. Rico C, et al. HIF1 activity in granulosa cells is required for FSH-regulated Vegfa expression and follicle survival in mice. Biol Reprod. 2014;90(6):135. Rose M, et al. The ECM modulator ITIH5 affects cell adhesion, motility and chemotherapeutic response of basal/squamous-like (BASQ) bladder cancer cells. Cells. 2021;10(5):1038. Liu J, et al. ITIH5, a p53-responsive gene, inhibits the growth and metastasis of melanoma cells by downregulating the transcriptional activity of KLF4. Cell Death Dis. 2021;12(5):438. Yang Z, et al. MORC4 is a novel breast cancer oncogene regulated by miR-193b-3p. J Cell Biochem. 2019;120(3):4634–43. Tencer AH, et al. Molecular mechanism of the MORC4 ATPase activation. Nat Commun. 2020;11(1):5466.

We appreciate Epibiotek (Guangzhou, China) for performing the ATAC-seq and mRNA-seq. Funding This work was supported by the National Natural Science Foundation of China grant 82171642 (ZQX); National Natural Science Foundation of China grant 81671523 (YL); National Natural Science Foundation of China grant 32170612 (CWC); Natural Science Funding of Guangdong Province grant 4210015092 (YL); Natural Science Funding of Guangdong Province grant 2017A030313895 (YL); Funding of Yat-sen Scholarship for Young Scientist (YL); Guangdong Science and Technology Department grant 2020B121200600180. Author information Authors and Affiliations Corresponding authors Ethics declarations Ethics approval This study has been reviewed by the scientific research ethics committee of Sun Yat-sen Memorial Hospital and approved by the institutional review board of Sun Yat-sen Memorial Hospital (no. 201717). All experiments related to human tissue were performed in accordance with the Declaration of Helsinki. Consent to participate All the patients have signed an informed consent before donating their abandoned GCs and FF. Conflict of interest The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Ou, S., Jiao, X., Li, Y. et al. Comparison of chromatin accessibility remodeling of granulosa cells in patients with endometrioma or pelvic/tubal infertility. J Assist Reprod Genet 42, 599–609 (2025). https://doi.org/10.1007/s10815-024-03302-7 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s10815-024-03302-7