Long Acting GnRHa-Treatment Increases Apoptosis in Cumulus Granulosa Cells (cGCs) of Patients with Endometriosis Undergoing IVF Program

Abstract

Objectives: Apoptosis is important during embryonic development, metamorphosis, tissue renewal, hormone-induced issue atrophy, and many pathological conditions. GnRHa had been reported to induce apoptosis in the granulosa cells of the rat ovary whereas hMG and FSH block apoptosis. There have been few reports concerning apoptosis in human IVF-ET programs until recently. In the present study, through measuring apoptosis and apoptotic-related proteins (Bcl-2, Bax, and Mcl-1) in cGCs following long acting GnRHa treatment in the patients with endometriosis in IVF program, the incidence of apoptosis of cGCs might be a marker to predict the outcome of oocyte and embryo quality. Design: A retrospective case control study in a university hospital. Materials and Methods: A total of 8 women enrolled with long acting GnRHa-treated endometriosis and 4 control undergoing IVF. The cGCs were collected after follicular aspiration, oocyte isolation and, cumulus separating process. Detection of apoptotic nuclei was accomplished by in situ nuclear labelling with terminal deoxynucleotidy transferase-mediated dUIP nick end-labelling (TUNEL) reaction. The expression of Bcl-2, Bax, and Mcl-1 protein products in cGCs were examined by immunohistochemistry with specific antibodies. Results: A typical apoptotic bodies of cGCs, which included fragmented and shrunken nuclei stained with the TUNEL technique, was observed under a fluorescence microscope. The GnRHa-treated endometriosis showed more TUNEL positive stained cells, than the control group (P<0.05). The results indicated that GnRHa-treated cases had higher incidence (mean ± SE) of apoptotic cells (13.6 ± 2.4% vs. 4.1 ± 0.9%, P<0.05) and apoptotic bodies (1.6 ± 0.3% vs. 0.7 ± 0.1%, P<0.05) in cGCs than the control group. We also found that the patients with higher incidence of apoptotic cGCs had smaller number of harvested oocyte and matured oocytes (P<0.05), but no significant influence on the embryo development (P>0.05). The expression of Bax and Mcl-1 were higher than the Bcl-2 in the cytoplasm and nuclei of cGCs indicated that the balance of these proteins might be involved in the mechanism of apoptosis in cGCs. Conclusion: The TUNEL technique provides more specific, sensitive, and objective way to examine the apoptotic cells and apoptotic bodies formation in cGCs than the Hoechst 33258 staining. Results indicated that GnRHa-treated endometriosis might increase the rates of apoptosis in cGCs and showed no significant change in embryo development compared with the control group. The results also suggested that the treatment of endometriosis with long acting GnRHa before the induction of ovulation would modify the apoptosis in cGCs, decrease oocyte number, but still retain the same cleavage rates. In conclusion, we have shown for the first time the distribution of Bcl-2, Bax, and Mcl-1 in cGCs from patients with endometriosis undergoing IVF-ET program. The results demonstrated that Bax and Mcl-1 were highly expressed in the cGCs, and suggested that the regulation of these apoptotic-related protein might be involved in the apoptosis of cGCs.