Integrative Analysis of Alternative Splicing Regulation Reveals Genetic and RBP-Mediated Mechanisms in Endometriosis Pathogenesis

Alternative splicing (AS) plays a crucial role in various diseases, yet its functional significance in endometriosis remains poorly understood. In this study, we analyzed RNA-seq data from 146 endometriosis patients and 114 control endometrial samples (normal endometrium) to investigate aberrant AS events (AASEs) in endometriosis. Transcriptome profiling identified 1,005,616 transcripts, including 379,829 reference, 418,431 novel, and 207,356 intergenic transcripts, highlighting extensive transcriptomic diversity. A total of 91,767 alternative splicing events (ASEs) were detected, of which 4,850 were classified as AASEs. GO enrichment analysis showed that AASE-associated genes were significantly enriched in biological processes such as “regulation of small GTPase-mediated signal transduction” and “regulation of protein localization to plasma membrane,” which may contribute to disease progression by modulating cell proliferation, invasion, and adhesion. By correlating the expression of differentially expressed RNA-binding proteins (RBPs) with the percent spliced in (PSI) values of ASEs, we constructed a regulatory network comprising 20 RBPs and 644 AASEs, revealing potential post-transcriptional regulatory mechanisms. Additionally, splicing quantitative trait locus (sQTL) analysis identified 26,430 sQTLs associated with 29,654 ASEs. Notably, several high-confidence sQTLs were located within known endometriosis-associated genes, including androgen receptor (AR) and grainyhead like transcription factor 2 (GRHL2). Together, these findings provide new insights into the molecular mechanisms of endometriosis from the perspective of AS regulation. Similar content being viewed by others Data Availability The datasets analysed during the current study are available in the NCBI (National Center for Biotechnology Information), https://www.ncbi.nlm.nih.gov/ (under accession number PRJNA643835, PRJNA643840, PRJNA714537, PRJNA798414, PRJNA877250, PRJNA973632, PRJNA1122200, PRJNA1137416, PRJNA1174758, PRJNA347653, PRJNA415301, PRJNA553574, PRJNA559080 and PRJNA347653).

Vercellini P, Viganò P, Somigliana E, Fedele L. Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol. 2014;10:261–75. https://doi.org/10.1038/nrendo.2013.255. Fung JN, Rogers PAW, Montgomery GW. Identifying the biological basis of GWAS hits for endometriosis1. Biol Reprod. 2015;92:1-12,87. https://doi.org/10.1095/biolreprod.114.126458. Zhao L, Gu C, Ye M, Zhang Z, Han W, Fan W, et al. Identification of global transcriptome abnormalities and potential biomarkers in eutopic endometria of women with endometriosis: a preliminary study. Biomed Rep. 2017;6:654–62. https://doi.org/10.3892/br.2017.902. Faustino NA, Cooper TA. Pre-mRNA splicing and human disease. Genes Dev. 2003;17:419–37. https://doi.org/10.1101/gad.1048803. Garcia-Blanco MA, Baraniak AP, Lasda EL. Alternative splicing in disease and therapy. Nat Biotechnol. 2004;22:535–46. https://doi.org/10.1038/nbt964. Fu R-H, Liu S-P, Huang S-J, Chen H-J, Chen P-R, Lin Y-H, et al. Aberrant alternative splicing events in Parkinson’s disease. Cell Transplant. 2013;22:653–61. https://doi.org/10.3727/096368912X655154. Sun Y, Hu X. Aberrant alternative splicing in cancer: splicing events and their regulatory mechanisms (review). Int J Oncol. 2024;65:90. https://doi.org/10.3892/ijo.2024.5678. Yang Q, Zhao J, Zhang W, Chen D, Wang Y. Aberrant alternative splicing in breast cancer. J Mol Cell Biol. 2019;11:920–9. https://doi.org/10.1093/jmcb/mjz033. Fu X-D, Ares M. Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet. 2014;15:689–701. https://doi.org/10.1038/nrg3778. Shilo A, Ben Hur V, Denichenko P, Stein I, Pikarsky E, Rauch J, et al. Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development. RNA. 2014;20:505–15. https://doi.org/10.1261/rna.042259.113. Kono M, Kurita T, Yasuda S, Kono M, Fujieda Y, Bohgaki T, et al. Decreased expression of serine/arginine-rich splicing factor 1 in T cells from patients with active systemic lupus erythematosus accounts for reduced expression of RasGRP1 and DNA methyltransferase 1. Arthritis Rheumatol. 2018;70:2046–56. https://doi.org/10.1002/art.40585. Su Y, Tian W, Cheng L, Yin L, He X, Wei X. Inhibition of IGF2BP1 attenuates the progression of endometriosis through PTBP1. Folia Histochem Cytobiol. 2024;62:25–36. Abood A, Mesner LD, Jeffery ED, Murali M, Lehe MD, Saquing J, et al. Long-read proteogenomics to connect disease-associated sQTLs to the protein isoform effectors of disease. Am J Hum Genet. 2024;111:1914–31. https://doi.org/10.1016/j.ajhg.2024.07.003. Tian C, Zhang Y, Tong Y, Kock KH, Sim DY, Liu F, et al. Single-cell RNA sequencing of peripheral blood links cell-type-specific regulation of splicing to autoimmune and inflammatory diseases. Nat Genet. 2024;56:2739–52. https://doi.org/10.1038/s41588-024-02019-8. Bane K, Desouza J, Shetty D, Choudhary P, Kadam S, Katkam RR, et al. Endometrial DNA damage response is modulated in endometriosis. Hum Reprod. 2021;36:160–74. https://doi.org/10.1093/humrep/deaa255. Sarsenova M, Boggavarapu NR, Kask K, Modhukur V, Samuel K, Karro H, et al. Hypoxic conditions affect transcriptome of endometrial stromal cells in endometriosis and promote TGFBI axis. Front Endocrinol (Lausanne). 2024;15. https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2024.1465393. Frisendahl C, Tang Y, Boggavarapu NR, Peters M, Lalitkumar PG, Piltonen TT, et al. miR-193b-5p and miR-374b-5p are aberrantly expressed in endometriosis and suppress endometrial cell migration in vitro. Biomolecules. 2024;14:1400. Zhang L, Feng Y, Zhang Y, Sun X, Ma Q, Ma F. The sweet relationship between the endometrium and protein glycosylation. Biomolecules. 2024;14:770. Heldenbrand J, Ren Y, Asmann Y, Mainzer LS. Step-by-Step guide for downloading very large datasets to a supercomputer using the SRA Toolkit. 2017. https://doi.org/10.17504/protocols.io.kb6csre Chen S. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta. 2023;2:e107. https://doi.org/10.1002/imt2.107. Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–15. https://doi.org/10.1038/s41587-019-0201-4. Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10:giab008. https://doi.org/10.1093/gigascience/giab008. Liao Y, Smyth GK, Shi W. Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30. https://doi.org/10.1093/bioinformatics/btt656. Love M, Anders S, Huber M. Differential gene expression analysis based on the negative binomial distribution. Genome Biol. 2014;15:10–1186. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol. 2015;33:290–5. Pertea G, Pertea M. GFF utilities: GffRead and GffCompare. F1000Res. 2020;9:304. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. https://doi.org/10.1093/bioinformatics/bts635. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. https://doi.org/10.1186/1471-2105-12-323. Trincado JL, Entizne JC, Hysenaj G, Singh B, Skalic M, Elliott DJ, et al. SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions. Genome Biol. 2018;19:40. https://doi.org/10.1186/s13059-018-1417-1. Schafer S, Miao K, Benson CC, Heinig M, Cook SA, Hubner N. Alternative splicing signatures in RNA-seq data: percent spliced in (PSI). Curr Protoc Hum Genet. 2015;87:11.16.1-11.16.14. https://doi.org/10.1002/0471142905.hg1116s87. Wu Q, Zhang Y, An H, Sun W, Wang R, Liu M, et al. The landscape and biological relevance of aberrant alternative splicing events in esophageal squamous cell carcinoma. Oncogene. 2021;40:4184–97. https://doi.org/10.1038/s41388-021-01849-8. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27:1017–8. https://doi.org/10.1093/bioinformatics/btr064. Sebestyén E, Singh B, Miñana B, Pagès A, Mateo F, Pujana MA, et al. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. Genome Res. 2016;26:732–44. https://doi.org/10.1101/gr.199935.115. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–504. Poplin R, Ruano-Rubio V, DePristo MA, Fennell TJ, Carneiro MO, Van der Auwera GA, et al. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2018;201178. https://doi.org/10.1101/201178. Ongen H, Buil A, Brown AA, Dermitzakis ET, Delaneau O. Fast and efficient QTL mapper for thousands of molecular phenotypes. Bioinformatics. 2016;32:1479–85. https://doi.org/10.1093/bioinformatics/btv722. Yin L, Zhang H, Tang Z, Xu J, Yin D, Zhang Z, et al. rMVP: a memory-efficient, visualization-enhanced, and parallel-accelerated tool for genome-wide association study. Genomics Proteomics Bioinform. 2021;19:619–28. https://doi.org/10.1016/j.gpb.2020.10.007. Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innov. 2021;2. https://doi.org/10.1016/j.xinn.2021.100141. Villanueva RAM, Chen ZJ. ggplot2: Elegant graphics for data analysis (2nd ed.). Meas Interdiscip Res Perspect. 2019;17:160–7. https://doi.org/10.1080/15366367.2019.1565254. Conway JR, Lex A, Gehlenborg N. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics. 2017;33:2938–40. https://doi.org/10.1093/bioinformatics/btx364. Liu J, Lin C-X, Zhang X, Li Z, Huang W, Liu J, et al. Computational approaches for detecting disease-associated alternative splicing events. Brief Bioinform. 2023;24:bbad106. https://doi.org/10.1093/bib/bbad106. Xiong Y, Yang G, Wang K, Riaz M, Xu J, Lv Z, et al. Genome-wide transcriptional analysis reveals alternative splicing event profiles in hepatocellular carcinoma and their prognostic significance. Front Genet. 2020;11:879. https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2020.00879. Singh RK, Cooper TA. Pre-mRNA splicing in disease and therapeutics. Trends Mol Med. 2012;18:472–82. https://doi.org/10.1016/j.molmed.2012.06.006. Takata A, Matsumoto N, Kato T. Genome-wide identification of splicing QTLs in the human brain and their enrichment among schizophrenia-associated loci. Nat Commun. 2017;8:14519. https://doi.org/10.1038/ncomms14519. Park E, Pan Z, Zhang Z, Lin L, Xing Y. The expanding landscape of alternative splicing variation in human populations. Am J Hum Genet. 2018;102:11–26. https://doi.org/10.1016/j.ajhg.2017.11.002. Nishida M, Nasu K, Ueda T, Fukuda J, Takai N, Miyakawa I. Endometriotic cells are resistant to interferon-γ-induced cell growth inhibition and apoptosis: a possible mechanism involved in the pathogenesis of endometriosis. Mol Hum Reprod. 2005;11:29–34. https://doi.org/10.1093/molehr/gah133. Sharpe-Timms KL. Endometrial anomalies in women with endometriosis. Ann NY Acad Sci. 2001;943:131–47. https://doi.org/10.1111/j.1749-6632.2001.tb03797.x. Nguyen-Ngoc K-V, Cheung KJ, Brenot A, Shamir ER, Gray RS, Hines WC, et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc Natl Acad Sci U S A. 2012;109:E2595–604. https://doi.org/10.1073/pnas.1212834109. Schlie-Wolter S, Ngezahayo A, Chichkov BN. The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro. Exp Cell Res. 2013;319:1553–61. https://doi.org/10.1016/j.yexcr.2013.03.016. Daftary GS, Taylor HS. EMX2 gene expression in the female reproductive tract and aberrant expression in the endometrium of patients with endometriosis. J Clin Endocrinol Metab. 2004;89:2390–6. https://doi.org/10.1210/jc.2003-031389. Zhang Y, Zhang Y, Zhao C, Yu T, Liu Y, Shi W, et al. Reduced alternative splicing of estrogen receptor alpha in the endometrium of women with endometriosis. Oncotarget. 2017;8:110676. https://www.oncotarget.com/article/22701/text/. Hudson QJ, Proestling K, Perricos A, Kuessel L, Husslein H, Wenzl R, et al. The role of long non-coding RNAs in endometriosis. Int J Mol Sci. 2021;22:11425. Zhang Z, Lin R, Mingxuan L, Yao X. Analysis of key candidate genes and pathways of endometriosis pathophysiology by a genomics-bioinformatics approach. Gynecol Endocrinol. 2019;35:576–81. https://doi.org/10.1080/09513590.2019.1576609. Adamyan L, Aznaurova Y, Stepanian A, Nikitin D, Garazha A, Suntsova M, et al. Gene expression signature of endometrial samples from women with and without endometriosis. J Minim Invasive Gynecol. 2021;28:1774–85. https://doi.org/10.1016/j.jmig.2021.03.011. García-Pérez R, Ramirez JM, Ripoll-Cladellas A, Chazarra-Gil R, Oliveros W, Soldatkina O, et al. The landscape of expression and alternative splicing variation across human traits. Cell Genom. 2023;3:100244. https://doi.org/10.1016/j.xgen.2022.100244. Mathew DJ, Sánchez JM, Passaro C, Charpigny G, Behura SK, Spencer TE, et al. Interferon tau-dependent and independent effects of the bovine conceptus on the endometrial transcriptome. Biol Reprod. 2019;100:365–80. https://doi.org/10.1093/biolre/ioy199. Börschel CS, Stejskalova A, Schäfer SD, Kiesel L, Götte M. miR-142-3p reduces the size, migration, and contractility of endometrial and endometriotic stromal cells by targeting integrin- and Rho GTPase-related pathways that regulate cytoskeletal function. Biomedicines. 2020;8:291. Bondy-Chorney E, Baldwin RM, Didillon A, Chabot B, Jasmin BJ, Côté J. RNA binding protein RALY promotes protein arginine methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells. Int J Biochem Cell Biol. 2017;91:124–35. https://doi.org/10.1016/j.biocel.2017.07.008. Belanger KK. CELF1, PTBP1, and RBFOX2-mediated alternative splicing regulation in cardiovascular diseases. 2019. https://hdl.handle.net/2152.3/11588 Horn T, Gosliga A, Li C, Enculescu M, Legewie S. Position-dependent effects of RNA-binding proteins in the context of co-transcriptional splicing. NPJ Syst Biol Appl. 2023;9:1. https://doi.org/10.1038/s41540-022-00264-3. Van Nostrand EL, Pratt GA, Yee BA, Wheeler EC, Blue SM, Mueller J, et al. Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins. Genome Biol. 2020;21:90. https://doi.org/10.1186/s13059-020-01982-9. Yang F, Qi T, McRae AF, Rogers PAW, Montgomery GW, Mortlock S. Regulation of RNA splicing in endometrial tissue and its association with endometriosis. iScience. 2025;28. https://doi.org/10.1016/j.isci.2025.113207. Balasubramanian V, Saravanan R, Balamurugan SSS, Rajendran S, Joseph LD, Dev B, et al. Genetic alteration of mRNA editing enzyme APOBEC3B in the pathogenesis of ovarian endometriosis. Reprod Biomed Online. 2024;49:104111. https://doi.org/10.1016/j.rbmo.2024.104111. Lee N-K, Lee J-W, Woo J-H, Choi YS, Choi J-H. Upregulation of SPI1 in ectopic endometrium contributes to an invasive phenotype. Arch Med Res. 2023;54:86–94. https://doi.org/10.1016/j.arcmed.2022.12.011. Zhao X, Kong W, Zhou C, Deng B, Zhang H, Guo H, et al. Bioinformatics-based analysis of the roles of sex hormone receptors in endometriosis development. Int J Med Sci. 2023;20:415–28. https://doi.org/10.7150/ijms.79516. Hao Y, Li Y, Wu J, Hao N, Qin Y, zhang H, et al. Hypermethylation of the GRHL2 promoter region is associated with ovarian endometriosis. Reproduction. 2022;163:379–86. https://doi.org/10.1530/REP-21-0383. Laudanski P, Gorodkiewicz E, Ramotowska B, Charkiewicz R, Kuzmicki M, Szamatowicz J. Determination of cathepsins B, D and G concentration in eutopic proliferative endometrium of women with endometriosis by the surface plasmon resonance imaging (SPRI) technique. Eur J Obstet Gynecol Reprod Biol. 2013;169:80–3. https://doi.org/10.1016/j.ejogrb.2013.01.024.

The authors would like to thank all members of the Department of Gynaecology at the Women’s Hospital of Zhejiang University School of Medicine for their valuable scientific advice and encouragement. We also sincerely thank Haining Maternity and Child Health Care Hospital for its support and assistance in sample collection and experimental procedures. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author information Authors and Affiliations Contributions ZW and HY conceived and designed the experiments. ZW, LZ, SN, LQ, MY and JU contributed to paper writing. ZW, LZ and MY contributed to the data analysis. All authors reviewed the final manuscript. Corresponding author Ethics declarations Ethics Approval and Consent to Participate This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was approved by the Ethics Committee of Haining Maternity and Child Health Care Hospital (approval number: 2026–002). Written informed consent was obtained from all participants. Consent for Publication Not applicable. Competing 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. 43032_2026_2064_MOESM1_ESM.pdf (download PDF ) Supplementary file1 (PDF 201 KB) The circle diagram shows the enrichment analysis of 2057 parent genes associated with AASE 43032_2026_2064_MOESM2_ESM.pdf (download PDF ) Supplementary file2 (PDF 408 KB) The relative expression of TNRC6B, XRN1, PTBP2, MACF1, ANKHD1 and SYNE1 in Control (n=3) and Endometriosis (n=3) samples was detected by qRT-PCR. **p < 0.01; t-test was used 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 Wang, Z., Yan, H., Zhu, L. et al. Integrative Analysis of Alternative Splicing Regulation Reveals Genetic and RBP-Mediated Mechanisms in Endometriosis Pathogenesis. Reprod. Sci. 33, 550–563 (2026). https://doi.org/10.1007/s43032-026-02064-0 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-026-02064-0