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

Abstract

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 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).

Conclusion: The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.

Keywords: Cancer; Cryopreservation; Human ovarian tissue; Medulla; Phosphatidylserine translocation; Xenotransplantation.

Fig. 1

Xenografting of cryopreserved human ovarian tissue. a Cryopreserved ovarian pieces just after thawing, b, c Transplantation of these pieces, d Ovarian piece after 45 d in vivo culture. Scale bar: 2 mm

Fig. 2

Effect of the containing of medula on the quality of follicles (expressed as quantity and normality of follicles). No statistical differences between respective rates (P > 0.1)

Fig. 3

Translocation of phosphatidylserine in ovarian tissue frozen in form of medulla-free and medulla-contained strips and then xenografted in SCID mice: representative example of one experiment. a, b, c, d, e tissue frozen in form of medulla-free cortex, f, g, h, i, j tissue frozen in form of medulla-contained cortex, a, f forward and scatter dot plot used to select the interest population, c, h histograms displaying fluorescence of PE channel, used to measure fluorescence intensity of Propidium Iodide (PI), (D, I) dot plot analysis of FITC-Annexin V and PE channels, (Q1) cells negative to Annexin V (FITC A) and positive to PI (could indicate necrotic cells), (Q2) cells positive to both Annexin V and PI (could indicate late apoptotic stage), (Q3) cells negative for both Annexin V and PI (could indicate viable cells), (Q4) cells positive to FITC-Annexin V and negative to PI (could indicate early apoptotic state)

Fig. 4

Translocation of phosphatidylserine in ovarian tissue frozen in form of medulla-free and medulla-contained strips and then xenografted in SCID mice. * Cells in early apoptotic state (FITC-Annexin V positive, PI negative). ** Cells in late apoptotic state or dead cells (FITC-Annexin V positive, PI positive). *** Viable cells (FITC-Annexin V negative, PI positive). **** Necrotic cells (FITC-Annexin V negative, PI positive). Different superscripts indicate statistical differences between respective rates (P < 0.05)