{"paper_id":"3d0c8a68-ddeb-4db7-8369-b9398eef1d43","body_text":"Dear Editor,\nEndometriosis (EM) is a common gynecological disease affecting ~190 million women worldwide, characterized by the growth of endometrial-like tissue outside the uterus that causes pelvic pain and infertility. Despite its high prevalence, diagnosis is often delayed for years, due to the lack of non-invasive biomarkers. Although various biomolecules have been proposed as diagnostic biomarkers, none are currently used in clinical practice [1]. Therefore, novel biomarkers for early EM diagnosis are urgently needed. Transfer RNA-derived fragments (tRFs) are small non-coding RNAs generated by a specific tRNA cleavage that regulate translation, epigenetic modification, and gene silencing [2]. Although aberrant expression of tRFs has been identified in endometriotic tissues [3], the presence and potential diagnostic value of circulating tRFs have not yet been reported. This is the first pilot study to report tRFs in the blood of women with endometriosis. We identified a distinct circulating tRF signature that differentiates women with endometriosis from those without the disease, highlighting the potential of tRFs as novel noninvasive biomarkers for the diagnosis of endometriosis. Women undergoing laparoscopy for suspected benign gynecologic conditions or chronic pelvic pain were enrolled. Blood was collected on the day of surgery after consent and processed for serum RNA extraction. Controls had no surgical evidence of endometriosis; endometriosis severity was classified using the revised American Society for Reproductive Medicine (rASRM) classification system.\nSerum-derived total RNA was used to generate small RNA sequencing libraries. tRF abundance was quantified using a customized analysis pipeline for tRFs, and differential expression was assessed using DESeq2 with FDR correction (adjusted p < 0.05). Results were internally validated and compared with edgeR analyses to assess the robustness of identified tRF profiles. We performed an internal validation of differential expression stability and results were validated by comparison with the edgeR quasi-likelihood F-test (RRID:SCR_012802), and concordance of log2 fold-changes was evaluated.\nThe demographic and clinical characteristics of participants with and without endometriosis are presented in Fig. 1a. A total of 11 participants were included in the study, comprising the endometriosis group (n = 4) and the control group (n = 7). No significant differences were observed between the endometriosis and control groups in age, BMI, race, hormonal treatment, or menstrual cycle phase (Fig. 1a). Controls had various benign gynecologic conditions other than endometriosis. DESeq2 analysis identified several tRFs that were significantly expressed between the two groups. Following Benjamini–Hochberg correction for multiple testing, 82 tRFs were identified as differentially expressed between women with endometriosis and controls (adjusted p < 0.05), including 19 upregulated and 63 downregulated tRFs (Fig. 1b). Significant tRFs were ranked by absolute log2 fold change. Differential expression results were highly concordant between DESeq2 and edgeR (Fig. 1b), with near-identical log2 fold-change estimates (Pearson r ≈ 0.99; Spearman ρ ≈ 1). While edgeR identified fewer FDR-significant tRFs, the direction and magnitude of effects were consistently preserved across methods. Leave-one-out (LOO) analysis demonstrated that the direction of effect for most significantly altered tRFs remained stable across sample perturbations, indicating that the findings were not driven by individual influential samples. To identify the most significantly altered tRFs, the 10 tRFs with the largest absolute log2 fold-changes (ranging from −3.81 to +4.49) were selected and visualized in a volcano plot (Fig. 1c). These included five upregulated tRFs (tRF-19-WB8US5IU, tRF-21-WR381H93E, tRF-21-WRN81H93E tRF-17-73VL4YK, and tRF-20-9PMNH5IS) and five downregulated tRFs (tRF-27-Q1QU9P9L842, tRF-28-Q1QU9P9L84DF, tRF-27-E1Q89P9L842, tRF-30-PW5SVP9N15WV, and tRF-27-Q1Q89P9L8N2) listed in a table as shown in Fig. 1c. The adjusted p-values for these candidates ranged from 0.0014 to 0.0471. Among the 82 significantly differentially expressed tRFs identified in this study, seven were also significantly altered in our previously reported baboon model of endometriosis [4]. Of these, three tRFs were upregulated (tRF-22-WB8US5652, tRF-16-W9RKM80, and tRF-18-3290Q4DZ) and four were downregulated (tRF-27-E1Q89P9L842, tRF-27-Q1Q89P9L842, tRF-27-Q1Q8XP9L842, and tRF-30-E1Q89P9L8422) (Fig. 1d). The concordant expression patterns observed in both human and baboon endometriosis support their potential biological relevance. Receiver operating characteristic (ROC) analyses were performed for the top 10 differentially expressed tRFs identified by DESeq2, with area under the curve (AUC) values calculated from normalized counts using the pROC package in R. ROC analyses demonstrated discriminatory performance, with area under the curve (AUC) values ranging from 0.68 to 0.96. The highest performance was observed for tRF-19-WB8US5IU (AUC = 0.96), followed by tRF-27-E1Q89P9L842 and tRF-21-WRN81H93E (AUC = 0.93) (Fig. 1b). These findings suggest potential diagnostic utility of circulating tRFs for endometriosis, although validation in larger independent cohorts is required.\nThese findings extend previous observations of altered circulating miRNAs in endometriosis [5] and identify tRFs as a previously unrecognized circulating small RNAs in endometriosis. Aberrant tRF expression has been linked to numerous pathological conditions, including multiple cancers, metabolic and neurodegenerative diseases, viral infections, and cellular stress responses [2], suggesting broad regulatory functions in health and disease. In the context of endometriosis, a chronic inflammatory disorder characterized by immune dysfunction, the observed changes in circulating tRFs may reflect systemic molecular responses that contribute to disease pathophysiology and offer novel opportunities for biomarker development. Among the top ten differentially expressed tRFs, only tRF-30-PW5SVP9N15WV has been previously implicated in disease, with elevated levels reported to correlate with poor prognosis in breast cancer. Interestingly, we observed reduced expression of this tRF in endometriosis, indicating that its function may be disease- and tissue-context dependent. The remaining nine top-ranked tRFs have not been previously associated with endometriosis or any other disease, highlighting the largely unexplored nature of the tRF landscape. These findings emphasize the need for mechanistic studies to define the biological functions of tRFs and to determine their utility as biomarkers and potential therapeutic targets in endometriosis. Unlike previous studies focused on endometriotic lesions, this work represents the first comprehensive analysis of circulating serum tRFs in endometriosis, revealing a distinct systemic small RNA signature associated with the disease. The absence of overlap between the specific tRFs identified here and those reported in lesion tissues suggests that local and systemic manifestations of endometriosis may be governed by different tRF populations. Tissue-derived tRFs are likely reflective of processes intrinsic to ectopic lesions, whereas circulating tRFs may represent broader host responses involving inflammatory, immune, and metabolic pathways. Importantly, the accessibility of blood sampling makes circulating tRFs more amenable to the development of noninvasive diagnostic assays than tissue-derived RNA biomarkers, which require surgical specimen collection. The limitations of this study include the small sample size, early-stage endometriosis cases, and the use of controls with other benign gynecologic conditions rather than pathology-free individuals, all of which may have influenced circulating tRF profiles. Another limitation emanates from lack of correction for menstrual cycle stage as this represents an important potential source of variation. In addition, the lack of functional validation precludes conclusions regarding the biological roles of the identified tRFs. Finally, circulating RNA signatures may be affected by systemic physiological and environmental factors. Larger validation studies and mechanistic investigations are therefore warranted.\nIn conclusion, this pilot study provides the first evidence that circulating tRFs are dysregulated in endometriosis. We identified a distinct serum tRF signature that was robust across analytical approaches and included candidates with promising diagnostic performance. These findings establish circulating tRFs as a previously unrecognized class of biomarkers for endometriosis and warrant validation in larger independent cohorts.\nData availability\nRNA-Seq data was deposited into the Gene Expression Omnibus database at NCBI under accession number GSE343024 and are available at the following URL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE343024\nReferences\nTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397:839–52. https://doi.org/10.1016/S0140-6736(21)00389-5.\nHu Y, Cai A, Xu J, Feng W, Wu A, Liu R, et al. An emerging role of the 5’ termini of mature tRNAs in human diseases: current situation and prospects. Biochim Biophys Acta Mol Basis Dis. 2022;1868:166314. https://doi.org/10.1016/j.bbadis.2021.166314.\nLi WS, Li YL, Cao R, Ha CF, Sun S, Yu L, et al. Differential expression and bioinformatics analysis of tRF/tiRNA in endometriosis patients. BioMed Res Int. 2022;2022:9911472. https://doi.org/10.1155/2022/9911472.\nSurmann H, Mamillapalli R, Joshi N, Burns G, Fazleabas AT, Taylor HS. Circulating transfer RNA-derived fragment abundance after experimental endometriosis induction in baboons. Mol Hum Reprod. 2026:32. https://doi.org/10.1093/molehr/gaag032.\nMoustafa S, Burn M, Mamillapalli R, Nematian S, Flores V, Taylor HS. Accurate diagnosis of endometriosis using serum microRNAs. Am J Obstet Gynecol. 2020;223:557.e551-557.e511. https://doi.org/10.1016/j.ajog.2020.02.050.\nAcknowledgements\nWe thank Monish Kumar for depositing the data in the NCBI database.\nFunding\nThis work was supported by grants from National Institutes of Health (NIH R01 HD100336) and the Endometriosis Foundation of America (AWD0003567).\nAuthor information\nAuthors and Affiliations\nContributions\nAVM recruitment of patients, blood collection, acquiring demographic data and read the manuscript. RM sample storage, performed experiments, acquired the data and supervision of the project. HS analyzed the data and wrote the initial draft of the manuscript. RM and HS prepared the figures. RM and HT conceptualization designed the study, methodology, reviewed and edited the manuscript. All authors read and approved the manuscript.\nCorresponding authors\nEthics declarations\nEthics approval and consent to participate\nThis study was conducted in accordance with the Declaration of Helsinki, and approved by Institutional Review Board of the Yale University, protocol # 1608018237.\nInformed consent was obtained from all individual participants included in the study, or from legal guardians as appropriate.\nConsent for publication\nPatients or their guardians, as appropriate, signed informed consent regarding publishing their data.\nCompeting interests\nAll authors declare that none of authors have any financial or non‐financial competing interests.\nAdditional information\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nSupplementary Information\nRights and permissions\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\nAbout this article\nCite this article\nVash-Margita, A., Surmann, H., Mamillapalli, R. et al. Circulating tRNA-derived fragments in women with endometriosis: a pilot study. Mol Biomed 7, 238 (2026). https://doi.org/10.1186/s43556-026-00629-8\nReceived:\nRevised:\nAccepted:\nPublished:\nVersion of record:\nDOI: https://doi.org/10.1186/s43556-026-00629-8","source_license":"CC0","license_restricted":false}