Endometriosis is an estrogen-driven, inflammatory condition that affects roughly 10% of menstruators and commonly causes severe pain. Current therapies that reduce estrogen or inflammation often show inconsistent efficacy and have tolerability problems. The authors used a drug-repositioning rationale to nominate candidate therapeutics predicted to reverse endometriosis-associated gene expression signatures and sought proof-of-concept validation in a human in vitro model. The study focuses on two repurposed drugs identified by prior analyses—simvastatin and primaquine—and compares them with nonsteroidal anti-inflammatory drugs.
The investigators established human endometrial assembloids composed of immortalized epithelial (12Z) and stromal fibroblast (iEc) cell lines and supplemented experiments with primary tissue from patients. Assembloids were formed in 96-well agarose molds to permit co-culture of both cell types and to model epithelial–stromal interactions.
Four test compounds were evaluated: ibuprofen, fenoprofen, primaquine, and simvastatin. Each compound was applied for a 24-hour exposure. Parallel monolayer cultures were also treated to provide comparative data.
After treatment, samples (assembloids and monolayers) were harvested and processed for stranded messenger RNA sequencing. Libraries were sequenced on an Illumina NovaSeqX Plus System. The authors used gene set enrichment analysis to evaluate pathway-level effects and to identify reversals of endometriosis-associated pathways following drug treatment.
Gene set enrichment analysis revealed drug- and context-specific pathway reversals:
Primaquine reversed a chemical carcinogenesis — reactive oxygen species (ROS) pathway specifically in assembloids.
Simvastatin produced the largest overall gene expression changes. Notably, simvastatin reversed the cytokine–cytokine receptor interaction pathway in both the epithelial (12Z) and stromal (iEc) cell lines. In stromal cell lines, simvastatin also reversed a cytoskeleton-in-muscle-related pathway.
Fenoprofen reversed a calcium signaling pathway in stromal cell lines.
Ibuprofen was included in the screen but the abstract does not report a prominent pathway reversal attributed to ibuprofen in these experiments.
The authors emphasize that simvastatin’s reversal of cytokine-related pathways in both cell types mirrors findings from their animal behavioral testing, where simvastatin and primaquine reduced endometriosis-associated pain in a rat model referenced in the paper.
The data support the potential of simvastatin and primaquine to reverse disease-associated transcriptional programs in human-derived assembloids. Simvastatin, in particular, elicited the most substantial transcriptomic responses and specifically targeted inflammatory cytokine signaling pathways that are plausibly relevant to endometriosis pathophysiology and pain.
Primaquine’s activity in reversing a ROS-related pathway in assembloids suggests an effect on oxidative stress–linked biology in the co-culture context, which was not equally evident in monolayers according to the reported summary.
These in vitro assembloid results are presented as mechanistic and translational evidence complementing prior in vivo behavioral findings in a rat model; together, these observations motivated the authors to propose simvastatin and primaquine as candidate therapeutics for further evaluation.
The authors explicitly note a small sample size as a limitation of the reported experiments. The article is a preprint and has not undergone peer review. Specific experimental details beyond those in the abstract (for example, exact sample numbers, concentrations, and statistical metrics) were not reported in the provided source text and would need to be consulted in the full manuscript or supplementary materials.
Competing interests reported: L.C. Giudice disclosed recent advisory and consulting relationships with multiple companies. Tomiko T. Oskotsky, Marina Sirota, and Linda C. Giudice have a patent related to this work. The remaining authors declared no competing interests.
Funder information included support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and fellowships/awards noted in the manuscript metadata.
Using a human epithelial–stromal assembloid screening platform, the investigators observed that simvastatin and primaquine reversed multiple endometriosis-associated gene expression pathways, with simvastatin showing the largest transcriptional impact and reversal of inflammatory cytokine signaling in both epithelial and stromal lines. The authors present these findings as proof-of-concept that assembloid models can prioritize and provide mechanistic insight into drug-repositioning candidates for endometriosis. Further validation with larger sample sizes, detailed dose–response characterization, and formal peer review is required to advance these candidates toward clinical testing.