Complete protection against cryodamage of cryopreserved whole bovine and human ovaries using DMSO as a cryoprotectant

Abstract

PurposeThis study aims to determine the optimal cryopreservation protocol for whole ovaries intended for preservation of fertility in women.MethodsWe investigated the optimal cryopreservation procedure for whole ovaries in a bovine model. The following parameters were investigated to determine their effect on ovarian tissue viability: type of cryoprotectant, administration route of the cryoprotectant (perfusion and/or submersion), and the maximum tolerable interval between death of the animal and start of the cryopreservation process. The resulting optimal cryopreservation procedure for bovine ovaries was subsequently tested on human ovaries. In vitro glucose uptake, histology, and immunohistochemistry were used to assess the integrity of the ovarian tissue.ResultsStarting the cryopreservation procedure (including perfusion with and submersion in DMSO) within 10–15 min after death of the animal proved critical, resulting in a 90–100% protection level against cryodamage. When cryopreserving human ovaries using the same protocol, over 95% protection against cryodamage was observed on all tissue levels. In addition, no apparent morphological damage to either the follicles or the vascular endothelium was observed.ConclusionOur findings suggest that using the optimized protocol presented in this paper allows good cryopreservation of whole human ovaries and represents an important step in considering whole ovary autotransplantation for clinically applied fertility preservation.

Whole Ovary Cryopreservation and Transplantation: A Systematic Review of Challenges and Research Developments in Animal Experiments and Humans

“…Indeed, the ovarian pedicle must be long enough (≥5 cm in humans) to allow processing of the graft and ensure perfect suture of donor vessels to recipient vessels of similar diameter [ 42 , 43 ]. Another crucial aspect is shortening the ischemic interval between ovary removal and cryopreservation as much as possible to avoid injury to ovarian cells [ 42 , 44 , 45 , 46 ]. For this reason, in the majority of studies, whole ovary removal is performed by laparotomy.…”

“…Some teams have demonstrated that perfusion for as long as 60 min is required to ensure optimal preservation of the different layers of intact ruminant ovaries [ 38 , 46 ]. Westphal et al investigated the optimal cryopreservation protocol for whole ovaries in a bovine model [ 46 ].…”

Gene banking and transplantation of (mammalian) ovarian tissue

“…A number of groups applied vitrification to cryopreserve bovine ovarian tissue (Kagawa et al, 2009, Campos et al, 2016. Cryopreservation after whole ovary perfusion, to load the tissue with cryoprotectants, was reported by three groups (Isachenko et al, 2013, Gao et al, 2016, Westphal et al, 2017. The study by Kagawa and colleagues (2009) reported grafting of the cryopreserved bovine ovarian tissue, showing no loss of oocyte viability after autotransplantation of vitrified-warmed tissue.…”

“…Whole sheep ovaries were also slow-frozen using directional solidification (Maffei et al, 2013). Perfusion was also applied in freezing whole ovaries of pig, as mentioned above (Imhof et al, 2004), and in human (Bedaiwy et al, 2006), guinea pig (Xu et al, 2012), and cattle (Isachenko et al, 2013, Westphal et al, 2017. In sheep, live offspring was obtained by Imhof and co-workers (2006) after whole frozen-thawed sheep ovary reanastomosis.…”

Cryopreservation of Preantral Follicles

“…It involved special CPA perfusion techniques because the structure is much larger. Such techniques can include immersion of the ovary in a CPA solution and also perfusion of CPAs with needles and clamps in order to inject the solution through the ovarian artery [41].…”