Pelvic organ prolapse is one of the most common gynecological conditions worldwide, occurring when the anterior or posterior vaginal wall, the uterus, or the vaginal apex descends from its normal position. The consequences are far from trivial: women experience pelvic pressure, voiding dysfunction, and defecatory difficulties that can severely compromise daily life. Epidemiological projections cited in the study estimate that the global incidence of prolapse will reach 156 million by 2036, placing a substantial socioeconomic burden on healthcare systems. Despite this scale, the cellular and molecular mechanisms that drive the condition remain poorly understood, and treatment has largely plateaued at the surgical level. Up to 30 percent of patients require repeat surgery within five years, a recurrence rate that reflects how little is known about the biology of the tissues that hold pelvic organs in place.

The researchers argue that this knowledge gap stems in large part from a shortage of reliable experimental models. Animal models, while informative, fail to recapitulate the unique biomechanical and anatomical features of the human pelvic floor. Conventional two-dimensional cell cultures grown from patient tissue allow basic mechanistic experiments but rapidly lose tissue-specific phenotypes and cellular heterogeneity as they are passaged in flat dishes. Tissue biopsies, meanwhile, provide only static snapshots of disease, precluding longitudinal studies of how prolapse progresses or how tissues respond to potential therapies. Organoid technology, which leverages the self-renewal and differentiation capacities of tissue-resident adult stem cells to build miniature versions of organs in a three-dimensional matrix, has transformed research on the intestine, brain, and endometrium, and has already yielded insights into implantation, trophoblast invasion, and ovarian carcinogenesis in gynecology. Its application to pelvic floor disorders, however, had remained essentially unexplored until now.

To build the protocol, the team recruited women undergoing hysterectomy at Chengdu Women’s and Children’s Central Hospital between April 2024 and October 2024. The prolapse group comprised three patients with stage III or higher disease according to the Pelvic Organ Prolapse Quantification system, who underwent total hysterectomy with concurrent partial vaginectomy when clinically indicated. The control group included two patients undergoing hysterectomy for benign gynecological conditions, specifically uterine fibroids, none of which were known to significantly affect vaginal or ligamentous tissues. All participants had no history of pelvic radiation, connective tissue disorders, or hormonal therapy within six months before surgery, and the study was conducted with written informed consent under approval from the hospital’s ethics committee in accordance with the Declaration of Helsinki.

The technical core of the protocol lies in how the tissue is processed. Immediately after surgical excision, vaginal wall and uterosacral ligament samples were rinsed in sterile saline, cut into pieces of roughly two to three cubic centimeters, and transported on ice, with processing completed within two hours to maximize cell viability. In the laboratory, the tissue was minced into fragments of one to three cubic millimeters and subjected to enzymatic digestion with a combination of 2.5 milligrams per milliliter collagenase II and 0.5 milligrams per milliliter DNase I, agitated at 37 degrees Celsius for one hour. The resulting suspension was filtered through a 100-micrometer cell strainer, separating dissociated primary cells from undigested tissue fragments. Crucially, the team pursued two parallel culture strategies for every sample, each tested in three independent technical replicates: one starting from fully dissociated primary cells, and one starting from the retained tissue fragments, which preserve cell-cell and cell-matrix interactions that enzymatic dissociation destroys.

Both strategies relied on a three-dimensional Matrigel system, in which cells or fragments are embedded in droplets of the basement membrane extract and bathed in a specialized organoid culture medium supplemented with the growth factors Wnt3a, R-Spondin 1, and Noggin, a formulation optimized for epithelial stem cell expansion. For the first two days of culture, the medium included 10 micromolar Y-27632, a ROCK inhibitor added to prevent anoikis, the form of cell death that occurs when epithelial cells lose their attachment to a substrate. The researchers prospectively defined success before the experiment began: an organoid had to be a discrete, three-dimensional, spherical or cystic structure with a clearly visible border, sustain growth for at least seven days under bright-field microscopy, and reach a diameter of at least 100 micrometers. Loose cell aggregates, single cells, and undigested fragments did not count.

The results from the vaginal wall told a story of partial success. Primary cells from benign control tissue consistently formed organoids within the first week of culture, and by Day 11, well-defined three-dimensional structures ranging from approximately 100 to 500 micrometers in diameter were clearly visible under higher magnification. Organoids derived from the prolapse patients showed a similar morphology under the microscope but were conspicuously fewer and less dense at the corresponding time points. The authors suggest that pathological vaginal tissue may possess either a reduced pool of epithelial stem or progenitor cells or an impaired capacity for self-organization under the same culture conditions. They are careful, however, to flag the limits of this observation: with only three prolapse patients and two controls, and no quantitative counting or statistical analysis performed, these findings are qualitative and preliminary, though the reduced yield was consistently seen in all three patients.

The tissue-fragment approach produced a notable methodological insight. When undigested fragments retained on the filter were mixed with Matrigel and plated in low-adhesion wells, some tissue cells coalesced into compact cell aggregates after 48 hours of culture. When these aggregates were then dissociated and reembedded in fresh Matrigel, they gave rise to a greater number of organoids than parallel cultures initiated solely from dissociated primary cells. Organoid formation was observed by Day 5 after plating, most organoids increased in volume by Day 9, and after another round of dissociation and passaging, growth was still evident on Day 9 after replating. Growth was documented up to passage 2 in this study, but the authors emphasize that long-term self-renewal and sustained expandability beyond this level were not established and require further demonstration.

The uterosacral ligament experiments delivered the study’s most striking negative result. Primary cells and tissue fragments from benign control uterosacral ligaments both successfully gave rise to solid, three-dimensional organoids, confirming that this fibroblast- and smooth muscle-rich connective tissue contains organoid-competent cell populations. In marked contrast, despite identical culture conditions and parallel strategies, no organoid formation was observed from pathological uterosacral ligament tissue of the prolapse patients, whether cultures were started from dissociated primary cells or from undigested fragments. The authors urge caution in interpreting this failure. Because the culture medium was optimized primarily for epithelial stem cell expansion while the uterosacral ligament is dominated by stromal and smooth muscle cells, the absence of organoids may reflect a mismatch between the culture system and the target cell population, a depleted or dysfunctional progenitor compartment, or an altered stromal microenvironment. In prolapse, the ligament undergoes structural remodeling including smooth muscle cell loss, increased collagen degradation, and altered fibroblast activity, and the disorganized extracellular matrix may no longer support stem cell survival and self-organization. Lineage-specific media or coculture systems will be needed to assess mesenchymal progenitor function more precisely.

Immunofluorescence characterization added a molecular portrait of the vaginal organoids, performed only on the organoids successfully cultured from the prolapse patients. Hematoxylin and eosin staining first confirmed that the matched vaginal wall tissue displays a well-organized stratified squamous epithelium overlying dense stroma, while the organoids appeared as small spherical or cystic structures with a central eosinophilic, keratinized core surrounded by an epithelial cell layer, lacking the complex stromal architecture of native tissue. Immunostaining then revealed positive expression of the epithelial marker KRT14, forming epithelial rings or clusters, whereas the fibroblast-associated marker vimentin, the myofibroblast and smooth muscle marker alpha-SMA, the endothelial marker von Willebrand factor, and the smooth muscle marker SM22 were not detected within the organoid structures. These findings indicate that the organoids were predominantly composed of epithelial cells. Interestingly, vimentin-positive cells were observed surrounding, but not within, the KRT14-positive organoids, possibly representing residual stromal or accessory cells whose origin and relationship to the organoid niche remain to be investigated.

The authors are candid about the study’s limitations. The very small number of patient-derived samples means organoid formation was assessed qualitatively by microscopic observation rather than by quantitative efficiency assays, and no statistical comparison between groups was performed, making the data descriptive and preliminary. The culture system favored epithelial expansion, and stable coculture of epithelial and stromal cells within a single organoid was not achieved, although such coculture would more faithfully recapitulate native tissue. The study also lacks molecular comparisons between the generated organoids and their matched tissues of origin, so KRT14 expression and the absence of selected stromal, endothelial, and smooth muscle markers suggest, but do not establish, a tissue-specific identity. Future work, the team writes, should focus on optimizing coculture systems containing both epithelial and stromal components, generating organoids from larger and more clinically diverse cohorts, quantifying formation efficiency with statistical comparisons, and applying qPCR, RNA sequencing, or single-cell profiling to determine whether disease-associated characteristics are retained in culture. If those steps succeed, this preliminary protocol could become a standardized platform for dissecting the cellular mechanisms of pelvic organ prolapse and, ultimately, for developing the nonsurgical therapies that current treatment options so clearly lack.

Subject of Research: A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues

Article Title: A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues

Article References: Yu, X., Wu, S., Tao, Q., Luo, X., Huang, Y., Yang, X., Yang, X., & Lin, Y. (2026). A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues. Frontiers in Cell and Developmental Biology, 14, Article 1977193. https://doi.org/10.3389/fcell.2026.1977193

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1977193

Keywords: protocol, vaginal, wall-derived, organoids, women, pelvic, organ, prolapse, benign, controls, limited, applicability