Marcadores de apoptose em células da granulosa humanas e sua correlação com parametros clínicos de pacientes submetidas a hiperestimulação ovariana controlada

Abstract

The quality of oocytes depends on interactions with surrounding granulosa cells. Granulosa cells are essential in normal follicular maturation process since they produce steroidal hormones and growth factors, and they play a crucial role in follicular atresia. An increase of granulosa cells apoptosis appears to have a negative effect on conception and pregnancy rates in IFV programs. Thus, this study evaluated the clinical correlation of pro caspase-3, cleaved caspase- 3 and other apoptosis related genes expression in human granulosa cells of patients undergoing controlled ovarian stimulation. We enrolled prospectively 35 IVF patients referred to a private clinic for couple infertility treatment and women fertility preservation. Luteinized granulosa cells were isolated from follicular fluid, their expression of pro caspase-3 and cleaved caspase- 3 was assessed by immunostaining of cellblock sections and the gene expression of apoptosis inducers (CASPASE 3, CASPASE 8 and BAX) and inhibitor (BCL2) was quantified by real- time polymerase chain reaction. Cleaved caspase-3 correlated positively with the length of COS (r = 0.445, p < 0.05) and the length of infertility (r = 0.476, p < 0.05). Gene expression of CASPASE 3 and CASPASE 8 also correlated directly with the length of COS (r = 0.462, p < 0.05; r = 0.420, p < 0.05; respectively). There was a major difference in the expression of pro caspase 3 and cleaved caspase 3 in granulosa cells (p < 0.0001), associated with a positive correlation of pro caspase 3 expression and the proportion of mature oocytes collected (r = 0.427, p < 0.05). Therefore, the present study demonstrated that patients submitted to control ovarian stimulation (COS) protocol for a longer period present a higher proportion of apoptosis in granulosa cells, and this event may be related to the time period needed to COS that triggers apoptosis in granulosa cells; or the higher proportion of apoptosis in granulosa cells is the cause for a longer time of COS in these patients. These previous findings suggest that pro caspase 3 is constitutively expressed in human granulosa cells, underling it’s role not as a cell death marker, but of physiological granulosa cell function. Moreover, the activation of caspase 3 in granulosa cells is associated with a longer COS in IVF protocols.