This prospective study compares the effect of a GnRH agonist on the number of follicles in different developmental stages in cryopreserved human ovarian grafts transplanted into gonadotropin-stimulated or not stimulated severe combined immunodeficient mice (SCID mice). Human ovarian tissue from seven patients was cryopreserved with an open-freezing system and xenotransplanted in SCID mice. The SCID mice were then treated according to different stimulation protocols. The survival of the tissue after cryopreservation was examined by LIVE/DEAD viability staining or transplanted in the neck muscle of 41 SCID mice. Development of follicles, estradiol production, vaginal cytology, and uterus weight were assessed after 15 weeks with or without gonadotropin stimulation. Viable follicles were detected in all frozen/thawed specimens using the LIVE/DEAD assay. Triptorelin, a GnRH agonist, caused a significant reduction of follicles in all developmental stages in the non-gonadotropin-stimulated animals (P<0.001). In gonadotropin-stimulated animals, GnRH agonist treatment has no significant effect on primordial, primary and preantral follicle count, whereas the antral follicles were significantly fewer (P = 0.03). The GnRH agonist treatment is not able to prevent the primordial follicle depletion after the xenografting of ovarian tissue in SCID mice with or without gonadotropin stimulation. Furthermore, it causes an additional loss of follicles if administered during the critical neovascularization period after the transplantation.