Many therapies indicated for the treatment of malignancy in female cancer patients unfortunately carry a severe risk of subsequent infertility. Preservation of the follicular reserve before gonadotoxic treatment can be studied on three different levels: isolated follicles, follicles embedded in ovarian cortical strips or in an intact ovary. For this purpose, enzymatic isolation, intact ovary cryopreservation and xenografting are different treatments currently under investigation in our laboratories. Our aim is to evaluate by transmission electron microscopy (TEM) the impact of enzymatic isolation, cryopreservation or xenografting on the tissue and cell integrity of human ovarian tissue. Prospective experimental study. Ovarian tissue was analyzed by TEM after the following treatments: Treatment 1- Enzymatic isolation: eight human ovarian biopsies were processed for follicle isolation by collagenase or Liberase enzymatic digestion. One hundred and nine isolated human primordial/primary follicles were fixed and processed for TEM. Treatment 2- Intact ovary cryopreservation : three human ovaries were frozen with their vascular pedicle using a passive cooling device. At three different steps (freshly removed ovary, after perfusion with cryoprotectant and after thawing), samples were taken and processed for TEM. Treatment 3- Xenografting of ovarian cortical strips: sixteen human ovarian cortical biopsies were frozen-thawed (FT). Eight FT biopsies were grafted in the peritoneum of nude mice and removed after 3 weeks. At three different steps (fresh, FT and grafted tissue), samples were taken and processed for TEM. In treatment 1, the majority of isolated follicles were well preserved, but surrounded by a discontinous basal lamina. Numerous connective tissue fibres were found on the outer aspect of the residual basal lamina. Some follicles showed ultrastructural signs of atresia, such as patches of heterochromatin, focal discontinuities at the oolemma-follicular cell interface, irregularities in the nuclear profile, clusters of lipid droplets or small vacuoles and myelin-like structures. In treatment 2, we found a well preserved follicular ultrastructure, stromal and vascular compartments after freezing and thawing. Occasionally, ultrastructural signs of atresia were observed in some follicles (as described above). In treatment 3, healthy-looking and altered primordial and primary follicles were found in the 3 groups. Altered follicles showed oocyte vacuolization and/or fragmentation, degeneration of follicular cells and increased thickness of the basement membrane. A few growing preantral follicles observed after xenografting contained intact oocytes, in which organelles showed a perinuclear distribution typical of earlier developmental stages. We evaluated the degree of maturity of neo-vessels in xenografts. TEM allows enhanced evaluation of actual cell structure, providing some accurate information, hardly obtained by histology or other analysis. We could evidence early signs of atresia in all cellular compartments, such as swelling of the mitochondria; alterations in follicular basal membrane and follicular cell-oocyte interactions, crucial for follicular maturation; or integrity and maturity of vessels. Morpho-functional analysis by TEM allows a marked improvement in the evaluation of integrity of human ovarian tissue.