Cryopreservation of oocytes and embryos is commonly used to preserve fertility. However, women undergoing cancer treatment may not have the time or may not be good candidates for these options. Ovarian cortical tissue cryopreservation and subsequent tissue transplant has been proven successful yet inefficient in preserving larger secondary follicles, and is not recommended as a fertility preservation option for women with certain cancers. We evaluated cryopreservation of individual follicles as an alternative option in rodents, nonhuman primates, and human primates. Under optimal conditions, cryopreserved mouse secondary follicles were able to reestablish granulosa cell-oocyte interactions, which are essential for subsequent follicle growth. Individual secondary follicles survived cryopreservation, were able to be cultured in a three-dimensional alginate hydrogel matrix to the antral stage, and the enclosed oocytes were competent for fertilization. Using a vital imaging technique (pol-scope) employed in many fertility centers, we were able to bioassay the thawed, cultured follicles for the presence of transzonal connections between the somatic and germ cells. Perturbations in these linkages were shown to be reversed when follicles were cryopreserved under optimal freezing conditions. We applied the optimized cryopreservation protocol to isolated rhesus monkey and human secondary follicles, and using the birefringent bioassay, we were able to show good correlation between early follicle growth and healthy somatic cell-oocyte connections. Our results suggest that ovarian follicles can be cryopreserved, thawed, and analyzed noninvasively, making follicle preservation an additional option for young cancer patients.