Menstrual Blood-Derived Stem Cell Applications in Endometriosis Diagnosis: A Review of Molecular Approaches

Abstract:
Recent research highlights the potential of menstrual blood-derived stem cells (MenSCs) as a powerful, non-invasive tool for improving the diagnosis and understanding of endometriosis. These studies demonstrate that MenSCs carry a distinct molecular "memory" of the disease, reflecting the biological changes that occur within the uterine environment. By analyzing various DNA methylation patterns, gene activity, and protein levels, scientists have identified specific signatures that can distinguish patients with endometriosis from healthy individuals with high accuracy. Key findings include significant shifts in how genes are regulated and a decrease in the activity of enzymes responsible for processing cellular messages. These molecular changes are linked to critical disease processes such as increased cell migration, tissue remodeling, and chronic inflammation, which allow the disease to establish itself and spread. Collectively, this work suggests that the unique profile of MenSCs could serve as the foundation for novel, non-invasive diagnostic tests, potentially reducing the long delays many patients face before receiving a diagnosis.

Introduction:
Endometriosis is a chronic gynecological disease that affects 5-10% of women around the world, representing about 190 million individuals, and 50% of women experiencing gynecological symptoms. This condition is caused by the growth of tissue similar to the endometrium—the lining of the uterus—on the ovaries, fallopian tubes, and pelvic tissues. Women with endometriosis can experience debilitating pain during periods and infertility, but these symptoms are often overlooked because the they overlap with other gynecological conditions or are dismissed as “normal.”

Even though it is such a common condition, endometriosis is remarkably difficult to diagnose. On average, it takes 7 to 10 years from the onset of symptoms to receive a diagnosis. These diagnostic delays have serious health consequences, as women with longer delays experience more severe symptoms, higher healthcare costs, and worse fertility outcomes. The current gold standard for diagnosis is an expensive and invasive laparoscopic surgery that typically requires the biopsy of a lesion, which can only detect visible lesions and accounts for only a fraction of endometriosis cases. Clinical imaging is also used to detect specific types of endometriosis but fails to detect superficial peritoneal lesions, the most common form of the disease.

Menstrual blood contains stem cells that may hold the key to diagnosing endometriosis without surgery. Scientists are now studying the molecular characteristics of these cells to find disease-specific biomarkers. The following review highlights three important studies that use different molecular techniques—gene and protein analysis, microRNA processing pathways, and DNA methylation profiling to identify patterns that distinguish women with endometriosis from healthy controls. These approaches could transform endometriosis diagnosis from an invasive surgical procedure to a simple menstrual blood test.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Paper 1: Transcriptome and Proteome Analysis of Menstrual-Blood Derived Stem Cells
Penariol and colleagues investigated whether Menstrual-Blood Derived Stem Cells (MenSCs) from women with endometriosis showed different characteristics compared to healthy women. They collected menstrual blood from 10 women with endometriosis and 10 healthy controls and isolated the stem cells in order to analyze the gene expression and protein levels.

The researchers used RNA sequencing to identify which genes were active in the cells, as well as mass spectrometry to measure protein levels. By using both methods, they were able to identify 41 differentially expressed genes and 15 differentially expressed proteins in endometriosis MenSCs. Many of these genes are involved in inflammation and tissue remodeling processes. For example, genes like ATF3, ID1, and SNAI1 were more active in endometriosis cells, and several had already been linked to endometriosis in previous studies. At the protein level, they found elevated levels of collagen proteins that are known to be involved in scarring, aligning with the tissue scarring characteristic of endometriosis lesions.

The study also revealed that multiple biological pathways were dysregulated in the endometriosis MenSCs, including ones that were related to cell migration, inflammation, and response to low oxygen conditions. The researchers suggested that MenSCs may be able to produce ectopic lesions due to the chronic inflammatory environment in the endometrium.

This study was significant because it was the first to combine transcriptomics and proteomics in a multi-omics approach to analyze menstrual blood stem cells from endometriosis patients. Their findings suggest that the MenSCs carry molecular signatures that could be used as biomarkers, although the differences were subtle, indicating that endometriosis involves complex molecular alterations.

Paper 2: DROSHA Downregulation and miRNA Processing
While the first paper focused on the overall gene and protein expression of MenSCs, this paper focuses on the specific cellular machinery involved with the production of microRNAs (miRNAs), which are small molecules that control gene expression. Making miRNA properly is crucial for stem cell function, including self-renewal and differentiation.

Using RT-qPCR, a sensitive technique for measuring gene expression, Cressoni and colleagues examined eight genes in miRNA production in MenSCs in 10 women with endometriosis and compared them to healthy controls. They found that DROSHA, an enzyme critical for the first steps of miRNA processing, was reduced to half its amount in endometriosis MenSCs (p = 0.008). DROSHA acts like molecular scissors, which cut long pre-miRNA transcripts into shorter segments in the nucleus, which is a fundamental processing step. Interestingly, the other genes involved in the miRNA biogenesis pathway did not show significant changes, but the study acknowledged that this could be attributed to the small sample size.

To understand what was causing the reduction in DROSHA expression, the researchers used computational analysis. They identified nine miRNAs that are predicted to negatively regulate DROSHA expression, meaning that they could be reducing the amount of DROSHA through a feedback mechanism. These nine are predicted to target DROSHA and have been previously associated with endometriosis in other studies.

This finding is relevant, because as the first enzyme in the miRNA synthesis pathway, DROSHA’s reduction could lead to changes in miRNA levels throughout the cell. Previous studies have shown that DROSHA dysregulation affects stem cell proliferation, differentiation, and cell cycle progression. The authors propose that impaired miRNA biogenesis might contribute to the altered functional characteristics of MenSCs in endometriosis, though they noted that future studies on the global expression of miRNA would be needed to confirm this hypothesis.

Paper 3: Whole-Genome Methylation Analysis and Machine Learning
The authors of this paper took a different molecular approach by looking at DNA methylation in MenSCs. DNA methylation is a chemical modification that controls when certain genes are turned on and off without altering the DNA sequence itself. Methylation patterns make good biomarkers because they are relatively stable and don’t change frequently like gene expression does.

This study was the most comprehensive of the three, analyzing 19 women with endometriosis and 23 controls. By using enzymatic methylation, Tiniakou and colleagues analyzed methylation patterns across the entire genome. They also used freshly isolated MenSCs instead of cultured cells in order to preserve the natural state of the cells and avoid any changes that could occur during the culture.

This analysis identified 466 sections of DNA that had different methylation in endometriosis patients compared to the controls. 458 of these had an increased rate of methylation, meaning that those genes were more likely to be inhibited. The deviated regions have genes involved in processes that are involved in the progression of endometriosis, like WNT signaling, angiogenisis, and tissue organization and development. Changes in these genes could explain the mechanisms that allow endometrial cells to grow outside of the endometrium and cause pain and damage.

Tiniakou and colleagues then used machine learning to build a potential diagnostic model based on the methylation data. The most successful version used four methylation markers and was 81% accurate. Previous biomarker studies have struggled to produce reliable results, so although the model is not the most accurate, it represents significant progress towards a biomarker-based diagnostic test.

They validated their findings by comparing the methylation data to the Human Endometrial Cell Atlas (HECA) and found that 90% of the cells that had increased methylation from their research also had reduced expression in the reference database. The correlation showed that the changes in methylation actually affected the expression of genes in the endometrium.

By using multiple techniques, this research provides strong evidence that DNA methylation patterns in MenSCs have the potential to act as reliable biomarkers that could be used to create a noninvasive and accessible diagnostic test. However, the researchers did acknowledge that further testing with larger and more diverse patient samples would be necessary before this biomarker approach could be implemented in a clinical setting.

Conclusion:
Research across methylomic, transcriptomic, and proteomic layers identifies menstrual blood-derived stem cells (MenSCs) as a high-resolution, non-invasive "window" into the uterine microenvironment of endometriosis. These studies reveal that MenSCs in affected women carry a distinct molecular "memory" of the disease, including 466 differentially methylated regions (DMRs)—predominantly hypermethylated—that enable machine learning models to achieve an 81% diagnostic accuracy. Beyond DNA methylation, researchers identified a significant two-fold downregulation of DROSHA, a critical enzyme for miRNA biogenesis, and dysregulation in key genes and proteins. These molecular alterations consistently point to the activation of pathways driving Epithelial-Mesenchymal Transition (EMT), WNT signaling, angiogenesis, and chronic inflammatory responses via TNFA/NFkB and hypoxia-related targets, reflecting how the hostile uterine environment "programs" these cells with enhanced invasive and migratory properties. Collectively, this integrated evidence positions MenSC-based profiling as a robust framework for developing novel, non-invasive biomarkers and personalized therapeutic strategies for this complex disorder.

References:
Penariol, L. B., Thomé, C. H., Tozetti, P. A., Paier, C. R., Buono, F. O., Peronni, K. C., ... & Meola, J. (2022). What do the transcriptome and proteome of menstrual blood-derived mesenchymal stem cells tell us about endometriosis?. International Journal of Molecular Sciences, 23(19), 11515.
Cressoni, A. C. L., Penariol, L. B., Padovan, C. C., Orellana, M. D., Rosa-e-Silva, J. C., Poli-Neto, O. B., ... & Meola, J. (2023). Downregulation of DROSHA: could it affect miRNA Biogenesis in Endometriotic Menstrual Blood mesenchymal stem cells?. International Journal of Molecular Sciences, 24(6), 5963.
Tiniakou, I., Bafligil, C., Pérez-Moraga, R., Harden, S., Ribeiro-Volturo, S., Rodríguez, A. S., ... & Fernández-Molina, C. (2025). Whole-genome methylation profiling of menstrual stem cells identifies novel biomarkers for endometriosis. bioRxiv, 2025-07.
Wang, M. H., Chen, J. H., Qi, X. Y., Li, Z. X., & Huang, Y. (2025). Global prevalence of adenomyosis and endometriosis: a systematic review and meta-analysis. Reproductive Biology and Endocrinology, 23(1), 148.
Cordeiro, M. R., Carvalhos, C. A., & Figueiredo-Dias, M. (2022). The emerging role of menstrual-blood-derived stem cells in endometriosis. Biomedicines, 11(1), 39.
Feng, Y., & He, Y. (2025). The secrets of menstrual blood: emerging frontiers from diagnostic tools to stem cell therapies. Frontiers in Cell and Developmental Biology, 13, 1623959.
Nisenblat, V., Bossuyt, P. M., Farquhar, C., Johnson, N., & Hull, M. L. (2016). Imaging modalities for the non‐invasive diagnosis of endometriosis. Cochrane Database of Systematic Reviews, (2).