Passive slow freezing is an efficacious and cost-effective alternative to controlled slow freezing for ovarian tissue cryopreservation

Abstract

We aimed to assess the feasibility of passive slow freezing (PSF using Mr. Frosty container, Nalgene) as an alternative to controlled slow rate freezing (CSF using (Freezal™, Air liquide)) for human ovarian tissue (OT) cryopreservation. Validation studies needed were determined after assessing the risk associated (EuroGTP-II ART tool) and were conducted in 66 OT samples from 10 transgender men aged 23.4 ± 5.1 y. Folliculogenesis was assessed in vitro (after 2 h and 2 days of culture) and in vivo (2, 4 and 6 weeks xenotransplantation in Balbc/nude mice) by haematoxilin-eosin staining. Fibrosis was assessed by Masson's trichrome staining. Immunohistochemistry was used to study cell proliferation (PCNA and Ki-67) and apoptosis (caspase-3 and TUNEL). Differences in percentages were estimated using a generalized estimated equations method. After 2 days of in vitro culture, higher odds of primordial follicles (PF) (OR 1.626; 95%CI (1.162-2.266); P = 0.004) and lower odds of growing follicles (GF) (OR 0.616; 95%CI (0.441-0.861); P = 0.004) were associated with the established CSF technique. No statistical differences were found in the mean estimated proportion of proliferating (Ki-67+ or PCNA+) or apoptotic (caspase-3+ or Tunel+) follicles. Two and 6 weeks after xenotransplantation, respectively lower odds of GF (OR 0.419; 95%CI (0.217-0.809); P = 0.010) and secondary follicles (OR 0.135; 95%CI (0.071-0.255); P < 0.001) were associated with CSF. Proportion of fibrosis was similar. This validation study shows a higher follicle activation after 2 days in vitro and after 2 weeks following xenotransplantation in mice using PSF. PSF may be an easy, cost-effective low-risk alternative to CSF for cryopreservation of human OT.

Keywords: Cell proliferation; Controlled slow freezing; Follicle; Follicle activation; Ovarian tissue; Passive slow freezing; Xenografting.