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Purpose: Although adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition is yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most of the previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Methods: In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify, and evaluate the viability of isolated ectopic endometrial cells. Results: Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37 and 93.49%, respectively. Conclusion: Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests that excess iron accumulates in endometriotic and adenomyotic lesions. However, the role iron overload plays in the pathogenesis of endometriosis or adenomyosis remains unknown. Primary human eutopic endometrial stromal cells (EuESCs) from endometriosis or adenomyosis patients were used as the in vitro model of endometriosis or adenomyosis in this study. We found that iron, manifesting as ferric ammonium citrate (FAC; 0.05–4.8 mM), significantly inhibited cell growth, induced oxidative stress through the Fenton reaction, and functionally activated autophagy in EuESCs, as measured by 5-ethynyl-2′-deoxyuridine incorporation assay, MitoSOX™ Red staining, LC3 turnover assay, and tandem mCherry-eGFP-LC3B fluorescence microscopy. Immunohistochemistry analysis of Ki67 expression in proliferative-phase endometrial tissues revealed that cell proliferation in ectopic tissues was dramatically compromised, suggesting that iron overload may play a role in cell growth inhibition in vivo. We observed that autophagy may alleviate the FAC-induced inhibition of endometrial stromal cell proliferation. Furthermore, sequential FAC (0.8 mM, 24 h) and hydrogen peroxide (H 2 O 2 ; 300 μM, 2 h) treatment successfully induced the Fenton reaction in EuESCs and caused extensive apoptosis, whereas the disruption of autophagy by the knockdown of BECN1 further aggravated cell death. MitoSOX™ Red staining showed that autophagy may promote the survival of EuESCs by decreasing of the Fenton reaction-induced reactive oxygen species generation. In addition, we observed that the Fenton reaction-induced oxidative stress significantly suppressed iron overload-induced autophagy. Moreover, we found that FAC treatment impaired poly(ADP-ribose)-polymerase 1 (PARP1) expression while simultaneously upregulating SIRT1 expression in EuESCs. Our data further showed that PARP1 expression decreased in endometriotic lesions, which may partially result from iron overload. We also found that PARP1 inhibition aggravated iron overload-induced cell growth suppression, and was implicated in iron overload-induced autophagy. In addition, SIRT1 silencing alleviated iron overload-induced PARP1 downregulation and autophagy activation. Overall, our data suggest that iron overload in endometrial stromal cells of endometriotic or adenomyotic lesions may be involved in the inhibition of cell proliferation, simultaneously with the activation of protective autophagy via PARP1/SIRT1 signaling. [ABSTRACT FROM AUTHOR]