P-430 Characterization of secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue

Abstract Study question Can we obtain secondary follicles from cultured cryopreserved-thawed human cortical tissue? Summary answer We obtained a comparable percentage of secondary follicles after culture of cryopreserved-thawed human cortical tissue to that reported after culture of fresh human cortical tissue. What is known already The complete in vitro maturation of oocytes starting from primary oocytes (present in unilaminar follicles) until mature MII oocytes has been achieved previously by two different multi-step culture protocols. These protocols were applied to culture fresh ovarian cortical tissue from adult cisgender donors. However, the efficiency of these two protocols for growing unilaminar follicles to secondary follicles starting from cryopreserved cortical tissue from cisgender patients has not been investigated. As the ovarian tissue available for fertility preservation is cryopreserved, it is important to understand the potential of cryopreserved unilaminar follicles to mature in vitro. Study design, size, duration Cryopreserved ovarian cortical tissue from 4 cisgender adult donors was used for in vitro culture. According to the existing culture protocols, cortical ovarian tissue was cultured either for 8 days using the first step medium (Telfer’s medium) reported by M.McLaughlin 2018 (doi: 10.1093/molehr/gay002) or for 7 and 21 days using the first step medium (Xu’s medium) reported by Xu 2021 (doi: 10.1093/humrep/deab003). Participants/materials, setting, methods Ovarian cortical tissue obtained from adult cisgender donors undergoing oophorectomy for fertility preservation purposes was cryopreserved before chemotherapy. The cryopreserved ovarian cortical tissue was thawed and cut into small pieces. Several pieces were fixed immediately (day 0), the others were either cultured in Telfer’s medium or Xu’s medium. After culture, follicle survival, growth and morphology were assessed by histology and immunofluorescence. Main results and the role of chance We performed quantification of the different follicular stages (primordial, primary, secondary and atretic) after culture and observed that the percentage of secondary follicles increased independently of the culture media used. However, the ovarian cortical tissue cultured using Telfer’s protocol resulted in a higher percentage of secondary follicles and lower percentage of atretic follicles compared to that using Xu’s protocol. After culture, the ovarian cortical tissue was further immuno