Effect of gonadotropin-releasing hormone analogs on the apoptosis and release of IL-1 and VEGF in endometrial cultures from patients with endometriosis

Abstract

Objective: To evaluate the effect of the treatment with GnRH analogs (agonist = ag and antagonist = ant) on the apoptosis and interleukin-1 (IL-1) and vascular endothelial growth factor (VEGF) release by cultures of epithelial cells from eutopic endometrial tissue of patients with endometriosis (EDT). Design: Prospective study. Materials/Methods: For cultures of epithelial endometrial cells (EEC), we have employed biopsies obtained in proliferative phase by laparoscopy from 16 untreated patients with EDT and 14 controls (without EDT). Purification and culture of EEC were done according to the technique described by Bongso et al. (Hum. Reprod. 1988;3:705–713). Cultures were treated with Leuprolide acetate (LA) 100 ng/ml as GnRHag, or Antide (A) 10 -7 M as GnRHant or both, LA+A. Percentages of apoptotic cells (ApC) were measured using the acridine orange - ethidium bromide technique and levels of IL-1 and VEGF in culture supernatants were measured using ELISA commercial kits. Comparisons among groups were performed by Kruskal-Wallis nonparametric ANOVA test, and Dunn’s multiple comparison test. Results: Treatment with LA enhanced values of ApC in cultures from EDT patients ( 23.9 +/− 8.0% to 41.7 +/− 9.6%, p <0.01) and from controls (18.2 +/− 6.3% to 42.5 +/− 14.7%, p <0.05). Simultaneously LA treated cultures showed significantly decreased levels of IL-1 and VEGF. Values of IL-1 obtained in EDT patients were 223.0 +/− 56.0 pg/ml in basal conditions vs 83.6 +/− 16.3 pg/ml in LA treated cultures, p <0.05, and values obtained in controls were 201.5 +/− 58.6 pg/ml vs. 98.1 +/− 54.8 pg/ml in basal and LA treated cultures respectively (p <0.03). VEGF levels were 283.64 +/− 83.9 pg/ml vs 134.4 +/− 37.3 pg/ml in basal and LA treated ECC cultures from EDT patients respectively (p <0.04), and in controls VEGF values were 201.5 +/− 58.6 pg/ml in basal conditions vs 96.1 +/− 54.8 pg/ml in LA treated cultures (p <0.03). Cultures of ECC from patients and controls treated with GnRHant did not show any significant differences compared to basal conditions. Also, the addition of A to cultures of ECC pre-treated with LA, reverted the increased apoptosis and the drop in IL-1 and VEGF levels. We found no difference in any of the parameters studied between cell cultures from EDT patients and controls. Conclusions: GnRHag has local effects, by enhancing the percentage of ApC and decreasing the release of pro-mitogenic cytokines as IL-1 and VEGF in endometrial cells. GnRHant alone did not produce any significant effect but reverted the changes caused by treatment with GnRHag. Supported by: Agencia de Promocion Cientifica y Tecnologica de la Republica Argentina.