Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate

Reproductive Biology and Endocrinology

Research

Plamen Todorov¹  Paul Hardiman²  Natalie Getreu²  Andrey Tchorbanov³  Markus Merzenich⁴  Peter Mallmann⁵  Evgenia Isachenko⁵  Bettina Hanstein⁵  Gohar Rahimi⁵  Vladimir Isachenko⁵  Mahmoud Salama⁵ 

[1] Institute of Biology and Immunology of Reproduction, Tzarigradsko shosse 73, 1113, Sofia, Bulgaria;Institute of Women’s Health, University College London, London, UK;Laboratory of Experimental Immunology, Institute of Microbiology, Acad. G. Bonchev Street, Block 26, 1113, Sofia, Bulgaria;MedEvent Dr. Merzenich GmbH, Im Zollhafen 12, 50678, Cologne, Germany;Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany;

关键词: Cancer;    Cryopreservation;    Human ovarian tissue;    Phosphatidylserine translocation;    Medulla;    Xenotransplantation;   

DOI  :  10.1186/s12958-016-0213-6

 received in 2016-07-29, accepted in 2016-11-03,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundPhosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments.MethodsOvarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).ResultsFor Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative).ConclusionThe presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.

【 授权许可】

CC BY   
© The Author(s). 2016