A novel protocol for the establishment of in vitro intrauterine adhesions model

Intrauterine adhesions(IUAs), also known as Asherman'ssyndrome, is a prevalent gynecological condition that presents with various clinical manifestations, including atypical menstrual patterns, pelvic discomfort, reduced endometrial thickness, repeated pregnancy loss, and potential infertility. Despite advancements in therapy, the successful pregnancy rate remains low, especially in severe cases, posing significant therapeutic challenges and poor prognoses. Therefore, extensive research is essential to understand the underlying mechanisms of IUAs and develop effective therapeutic interventions. The objective was to examine the co-culture endometrial stem cells(EnSCs) of the human uterus and intraperitoneal macrophages as an in vitromodel for IUAs. The aim was to establish a physiologically relevant model that replicates the complex cellular interactions and microenvironment observed in the endometrium during IUAs development.

This study introduces a novel method of incubating co-culture of stem cells and intraperitonealmacrophages with hydrogen peroxide (H2O2) to mimic impaired endometrial stromal cells, considering the regenerative properties of macrophages, and prominent leukocytes in the endometrium. We investigate the influence of macrophages on the response of stemcells to H2O2 by examining the amount of cell migration after scratching and the cell viability by MTT test and evaluating the amount of secreted NO in the presence of H2O2and without it. Statistical analysis utilized Student's t-testor one-way ANOVA, with GraphPad Prism version 6 software, considering results with p< 0.05 as statistically significant.

The study involved culturing and preparing EnSCsin a 24-well plate, while peritoneal macrophages were isolated through lavage in mice. Four experimental groups were established: EnSCs (group A), EnSCs treated with H2O2 (group B), EnSCs co-cultured with macrophages(group C), and EnSCs co-cultured with macrophages treated with H2O2(group D).Cell viability was assessed using the MTT test, measuring the reduction of tetrazolium salt to formazan crystals. Nitric oxide(NO) production was measured using the Griess reaction, and cell migration was evaluated through a scratch wound healing assay.

The findings indicated that H2O2treatment had a negative impact on EnSCs viability, but co-culturing with macrophages provided a protective effect. Moreover, H2O2treatment led to increaseNO levels, suggesting macrophage activation against oxidative stress. Interestingly, H2O2treatment promoted cell migration in both EnSCs alone and the co-culture, while macrophages inhibited migration. These results underscore the significance of the interaction between EnSCs and macrophages in understanding IUAs and developing effective treatments.