A comparative analysis of the cell surface markers and immunological properties of cell cultures originating from normal endometrium and endometrioid heterotopias of women with extragenital endometriosis was carried out. Both types of cell cultures expressed surface molecules typical of mesenchymal stromal cells and did not express hematopoietic and epithelial markers. Despite similar phenotype, the mesenchymal stromal cells derived from the two sources had different immunomodulation capacities: the cells of endometrioid heterotopias but not eutopic endometrium could suppress dendritic cell differentiation from monocytes as well as lymphocyte proliferation in allogeneic co-cultures. A comparative multiplex analysis of the secretomes revealed a significant increase in the secretion of pro-inflammatory mediators, including IL6, IFN-γ, and several chemokines associated with inflammation by the stromal cells of ectopic lesions. The results demonstrate that the stromal cells of endometrioid heterotopias display enhanced pro-inflammatory and immunosuppressive activities, which most likely impact the pathogenesis and progression of the disease.

Data compatibility remains a major challenge in metabolomics, as commonly used measures of biological material—such as sample weight or cell count—are often poorly reproducible. Here, we systematically evaluated practical normalization strategies for GC × GC-MS-based metabolomic profiling of two widely used model cell lines: human hepatoblastoma (HepG2) and mesenchymal stromal cells (MSCs). We compared orthogonal biomass estimates, including total protein and double-stranded DNA quantified either directly in aliquots of the cell suspension lysate aliquots or in the post-extraction cell precipitate, alongside normalization based on extracted ion current (XIC). We also assessed three widely used extraction mixtures—methanol/chloroform/water (7:2:1); methanol/water (8:2); acetonitrile/isopropanol/water (3:3:2)—for metabolome coverage and normalization robustness. Under realistic biological variability, signal-to-biomass dependencies were moderate. In contrast, under strictly controlled conditions, DNA- and protein-based normalization yielded near-linear relationships with metabolite abundances (R² > 0.90), demonstrating that biological variability is the dominant source of dispersion rather than technical factors. Methanol/chloroform/water system provided the broadest metabolome coverage and strongest correlation with injected biomass. Based on these findings, we recommend normalization to total precipitate protein or DNA using the methanol/chloroform/water extraction protocol, with XIC as a complementary quality control metric.

Endometriosis is a gynecological disease affecting more than 190 million women of reproductive age worldwide, characterized by a complex molecular pathophysiology encompassing genetic, epigenetic, inflammatory, and immunological components. A fundamental methodological challenge in the study of endometriosis is the cellular heterogeneity of ectopic lesions: conventional analysis of bulk tissue specimens reflects averaged molecular profiles of mixed cell populations comprising glandular epithelium, stroma, fibrous and muscular tissue, and immune microenvironmental cells, substantially impeding the identification of cell-specific molecular alterations. This article presents an analysis of the capabilities of laser capture microdissection as a technology for the selective isolation of cell populations from heterogeneous tissues, enabling researchers to overcome this limitation. The methodological principles of the technology are described in detail, including critical parameters of biological material preparation, integration with downstream molecular analysis platforms, and the specificities of protocol standardization and validation across different tissue types. The broad applicability of laser capture microdissection in contemporary biomedical research is reviewed. Particular emphasis is placed on its application in endometriosis research specifically: data on transcriptomic profiling and differential gene expression analysis in the epithelial and stromal components of ectopic lesions are described; studies employing next-generation sequencing aimed at investigating somatic mutations, clonality, and mutational profiles of endometriotic cell populations are reviewed; and work in the field of multi-omics profiling across different disease forms is examined. The review also addresses existing methodological limitations of the technology and prospects for its further development, including integration with spatial transcriptomics, single-cell sequencing, and artificial intelligence-based approaches. Laser capture microdissection constitutes an instrument that opens fundamentally new avenues for investigating the molecular heterogeneity of endometriosis and establishing the foundation for the development of targeted, pathogenetically justified approaches to its diagnosis and treatment. This article also presents the authors’ own experience with the application of laser capture microdissection in endometriosis research. At the time of manuscript preparation, laser capture microdissection had been performed on 15 endometriotic lesion specimens obtained from patients with various forms of endometriosis, with separate isolation of epithelial and stromal components intended for subsequent molecular-genetic analysis.