Morphological and ultrastructural evaluation of cultured frozen–thawed human fetal ovarian tissue

ObjectiveTo investigate a defined culture condition for the culture of frozen-thawed human ovarian tissue.DesignProspective laboratory study.SettingReproductive biology laboratories in university hospitals.Patient(s)Fetal ovarian tissue from elective termination of pregnancy.Intervention(s)Culture of frozen–thawed fetal ovarian tissue for up to 63 days.Main Outcome Measure(s)Morphology, morphometry, and survival of follicles in relation to culture times.Result(s)The proportion of primordial, early primary, and primary follicles in frozen–thawed (day 0) ovarian tissue was 77.5%, 21.7%, and 0.8%, respectively. Pronounced degeneration was found in all cell types, and ≤36% of the follicles had signs of atresia at days 7–14, but this figure improved with culture time to <20% of the total follicular population. After 7–14 and 21–35 days of culture, the relative proportion of the follicles in the different classes remained nearly stable. Morphometric examination of healthy follicles showed a significant increase in both follicle and oocyte diameter compared with control. A few follicles had developed to the early secondary stage. Ultrastructural analysis demonstrated well-preserved morphological integrity of healthy primordial and early primary follicles. Immunohistochemical localization of proliferating cell nuclear antigen was positive in proliferating follicular cells at days 7–14 and 21–35 of culture.Conclusion(s)The present culture condition leads to good survival and progressive follicular growth and differentiation that is comparable to the physiological pattern of early folliculogenesis.