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


METHODS
Ovarian 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).


RESULTS
For Groups 1 and 2, the mean densities of follicles per 1 mm3were 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).


CONCLUSIONS
The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany. v.isachenko@yahoo.com.
Vladimir
V
Isachenko
Institute of Biology and Immunology of Reproduction, Tzarigradsko shosse 73, 1113, Sofia, Bulgaria.
Plamen
P
Todorov
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.
Evgenia
E
Isachenko
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.
Gohar
G
Rahimi
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.
Bettina
B
Hanstein
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.
Mahmoud
M
Salama
Research Group for Reproductive Medicine and IVF-Laboratory, Department of Obstetrics and Genecology, Cologne University, Kerpener Str. 34, 50931, Cologne, Germany.
Peter
P
Mallmann
Laboratory of Experimental Immunology, Institute of Microbiology, Acad. G. Bonchev Street, Block 26, 1113, Sofia, Bulgaria.
Andrey
A
Tchorbanov
Institute of Women's Health, University College London, London, UK.
Paul
P
Hardiman
Institute of Women's Health, University College London, London, UK.
Natalie
N
Getreu
MedEvent Dr. Merzenich GmbH, Im Zollhafen 12, 50678, Cologne, Germany.
Markus
M
Merzenich
D000818:Animals; D015925:Cryopreservation / Q000379:methods; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016513:Mice, SCID; D010053:Ovary / Q000473:pathology / Q000637:transplantation*; D010718:Phosphatidylserines / Q000378:metabolism*; D014183:Transplantation, Heterologous / Q000379:methods
D010718:Phosphatidylserines
Cancer; Cryopreservation; Human ovarian tissue; Medulla; Phosphatidylserine translocation; Xenotransplantation