A non-invasive, patient-derived organoid platform from uterine lavage fluid for modeling endometrial implantation and disease.

Abstract

Background: Endometrial organoids, with their three-dimensional structure and robust hormonal responsiveness, have emerged as valuable models for investigating uterine physiology and embryo implantation. However, conventional derivation from endometrial biopsies is invasive, carries surgical risks, and limits repeated sampling. This study introduces uterine lavage fluid as a non-invasive source for generating endometrial organoids.

Methods: We established endometrial organoids from uterine lavage fluid (UF-EOs) and compared them with matched tissue-derived endometrial organoids (T-EOs) for morphology, proliferation, epithelial marker expression, transcriptomic profiles, and hormone responsiveness. Lavage fluid was collected from reproductive-age women with normal endometrium and from patients with intrauterine adhesions (IUA). Differential gene-expression analysis between IUA-derived and control organoids was performed to identify IUA-associated signatures. A microinjection platform was developed to deliver HTR8/SVneo trophoblast spheroids into the organoid lumen.

Results: UF-EOs were successfully derived from 90% of control individuals (9/10) and 60% of IUA patients (6/10). UF-EOs and T-EOs were morphologically indistinguishable, with comparable proliferation rates (104.6 ± 6.21 versus 106.4 ± 5.16; P > 0.05) and equivalent expression of epithelial markers (EpCAM, Cytokeratin 7, E-cadherin). Transcriptomic analysis showed tight paired clustering of UF-EOs with matched T-EOs across three independent patients. Both organoid types demonstrated equivalent hormone responsiveness, with progesterone-induced secretory differentiation evidenced by glandular folding, subnuclear vacuolization, glycogen accumulation, and upregulation of SPP1, PAEP, and 17HSDβ2. IUA-derived UF-EOs displayed 190 differentially expressed genes, with enrichment of cell cycle, TGF-β, and Hippo signaling pathways. TGF-β2 and Smad2 were significantly upregulated and SOX9, PCNA, and Ki67 were downregulated, consistent with compromised regenerative capacity. HTR8/SVneo spheroids were successfully microinjected into organoid lumens, with luminal localization observed at 24 h.

Conclusion: We have introduced uterine lavage fluid as a feasible source for generating endometrial organoids with equivalent fidelity to tissue-derived counterparts. UF-EOs provide a non-invasive alternative that enables organoid-based research in populations previously excluded from invasive sampling, including patients with intrauterine adhesions. Our findings demonstrate applications in disease mechanism investigation, endometrial receptivity assessment, and modeling of early embryo-epithelium interactions. Future studies employing larger cohorts and integrated multicellular systems will be essential to validate the clinical utility of this approach.