**2.6 Cell invasion assays**

The influence of peritoneal fluid and culture media of 24-h cultures of peritoneal macrophages of women with endometriosis upon invasiveness of eutopic endometrial cells of women with endometriosis was estimated in the 2D Matrigel system using BD BioCoat Matrigel Invasion Chamber (BD Biosciences, USA). 50 μl of prepared endometrial stromal cells isolated from eutopic endometrium of women with endometriosis by enzymatic method were placed on top of porous membrane coated with Matrigel. Autologous peritoneal fluid or pooled culture media of 24-h cultures of peritoneal macrophages of women with endometriosis were added to the lower chamber and then incubated overnight. At the end of the incubation period the upper side of the membrane was cleaned with a cotton wool bud. For assessment of the number of invaded cells, the filters were stained with hematoxylin and eosin and mounted on glass microscope slides. Total number of cells that had invaded onto the underside of the filter was counted manually at 200x magnification.

#### **2.7 Statistics**

Results were presented as the mean ± standard error. All variables were checked for normal distribution with the Kolmogorov-Smirnov test. All the parameters studied showed a normal distribution. Student's t-test was used to compare results from different groups. Statistical significance was defined as p < 0.05.
