GNRH agonist leuprolide acetate neither activates antiapoptotic genes nor protects human ovary and granulosa cells from dna damage and apoptosis induced by cyclophosphamide

Abstract

Objective: Inconsistent results of randomized controlled trials (RCTs) and lack of a proven molecular mechanism of action with ovarian protection with co-administration GnRH agonists (GnRHa) with chemotherapy places GnRHa under scrutiny as a fertility preservation strategy. We aimed in this study to provide molecular evidence for-or-against the role of GnRHa in the prevention of cyclophosphamide induced damage in human ovarian tissue samples and granulosa cells. Design: A translational research study. Materials and Methods: Ovarian cortical pieces (n=15, age 14-37) and human mitotic non-luteinized (COV434, HGrC1) and non-mitotic luteinized (HLGC) granulosa cells were treated with 4-hydroperoxy cyclophosphamide (in vitro active metabolite of cyclophosphamide used at 50 and 100 μM) with and without GnRHa leuprolide acetate (50 ng/mL: peak intraovarian concentration of the drug) for 24 hrs. Cell proliferation (real-time quantitative assessment by xcelligence system),DNA damage (p-histone H2AX), apoptosis (cleaved caspase-3, YO-PRO-1), follicle counts, hormonal markers of ovarian function and reserve (estradiol, progesterone and AMH), and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy+GnRHa groups. Results: GnRH receptor expression and its activation by GnRHa were validated with qRT-PCR and measuring intracellular cAMP level, respectively. Exposure to cyclophosphamide resulted in massive follicle loss, arrested cell growth, increased DNA damage/apoptosis and decreased hormone productions in the tissue samples and granulosa cells. The co-administration of GnRHa with cyclophosphamide did not prevent or attenuate any of these cytotoxic effects. Furthermore, GnRHa did not up-regulate the anti-apoptotic genes compared to control and cyclophosphamide treated samples.Mcl-1 and BIRC2 expressions were further decreased after cyclophosphamide+GnRHa (Table). Conclusions: GnRH agonist leuprolide acetate does not offer any protection against cyclophosphamide induced damage in human ovary and granulosa cells via its cognate receptors.