Cristina Fernández Molina

Endometriosis affects ~10% of reproductive-age women, yet targeted non-hormonal therapies remain unavailable, and treatment response is highly variable. Here, we apply a single-cell framework to resolve therapeutic heterogeneity at a resolution previously unattained in drug development efforts. Using scRNA-seq profiles from eutopic and ectopic tissues, combined with a machine learning-based drug response model, we identified compounds predicted to revert disease-associated transcriptional states and map cell-type-specific vulnerabilities across patients and tissues. Our analysis revealed pronounced tissue-specific and inter-patient heterogeneity in predicted responses. Stromal, endothelial, and stem cell populations emerged as the dominant therapeutic targets, collectively revealing selective sensitivity to two recurrent drug classes, histone deacetylase and tubulin polymerisation inhibitors. Transcriptomic comparison of predicted responders and non-responders to these drugs pointed to conserved molecular programmes involving extracellular matrix remodelling, angiogenesis, and proliferative activation. These signatures were shared between eutopic and ectopic stromal compartments, supporting the feasibility of assessing therapeutic response using readily accessible eutopic tissue. Our findings show that this single-cell framework can dissect therapeutic heterogeneity in endometriosis, support the development of precision non-hormonal therapies and identify responder subgroups relevant for patient stratification. Together, these results highlight that underlying molecular diversity in endometriosis necessitates therapeutic approaches beyond a one-size-fits-all model.

Endometriosis, despite its high prevalence, is underdiagnosed and poorly managed due to lack of clinically validated biomarkers and pathophysiological insight. Menstrual blood-derived stem cells (MenSCs) have been implicated in disease pathogenesis, but their diagnostic potential remains unexplored. We conducted a clinical study (n=42; 19 endometriosis, 23 controls) to assess whether DNA methylation profiles of freshly isolated MenSCs can identify disease-specific biomarkers. Whole-genome methylation sequencing revealed differentially methylated regions (DMRs) enriched in genes linked to hallmarks of endometriosis (e.g., inflammation, tissue remodelling, development). These DMRs robustly distinguished cases from controls, independent of technical and clinical variables. Machine learning models trained and validated on these DMRs achieved high diagnostic performance (specificity 83%, sensitivity 79%). Integration with an independent single-cell RNA sequencing dataset showed that the DMRs may modulate gene expression, further supporting their biological relevance. These findings position MenSC DNA methylation profiling as a promising, non-invasive approach for early endometriosis diagnosis and personalised care.

The maintenance of mitochondrial proteins homeostasis, which is essential for proper cell function, is affected in pathophysiological ageing, yet several underlying mechanisms remain unexplored. We show that in normal and accelerated ageing cells, POLG1, the enzyme responsible for mitochondrial DNA replication, is degraded by the protease cathepsin B, which is overexpressed, escapes from lysosomes, and is stabilized by the chaperone activity of another protease, HTRA3. This degradation is in part counteracted by RAC1, a small GTPase also stabilized by HTRA3. POLG1 depletion, that occurs in progeroid Cockayne syndrome and senescent cells, is linked respectively to impairment or downregulation of the CSB protein, which promote cellular senescence. Our experiments in engineered cells, demonstrate that senescence itself, and not the absence of CSB, triggers the accumulation of cathepsin B and HTRA3, leading to POLG1 degradation. In summary, we uncover a complex, multi-step process that controls the degradation of POLG1 in mitochondria, a process that is activated by cell senescence and becomes more pronounced in Cockayne syndrome cells, providing new insight in the regulation of mitochondrial proteostasis in ageing and progeroid disorders.