Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue

Abstract

ObjectivesTo translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen.Materials and MethodsOvarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).ResultsFor groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05).ConclusionsLong time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.

The effect of delayed processing on ovarian tissue stored for fertility preservation

“…By extension, this data also suggests that a processing delay of 24 or 48 h did not cause loss of function of the vasculature and the supporting stromal cells. This finding is consistent with insight from other studies that also identified follicles in ovarian tissue xenotransplanted after a 24 h delay in cryopreservation (12,13).…”

“…Remarkably, a processing delay of 48 h resulted in a 44% decrease in the probability of follicles being graded as affected, degenerating or atretic after cryopreservation, despite showing the opposite effect in fresh tissue. This is consistent with findings from Isachenko et al who explored a 24h delay at 4°C suggesting a role for pre-cooling of human ovarian tissue before cryopreservation (13). It is possible that acclimatisation of the ovarian tissue at 4°C for 48 h provides a more gradual temperature transition, which primes the tissue to better respond to the challenges of cryopreservation and, thus, experience a reduced insult on the health of the primordial follicle pool.…”

“…This finding is consistent with the presence of morphologically normal primordial follicles after a 20 h processing delay at 4°C of human ovarian cortical tissue as reported by Rosendahl et al (12). Isachenko et al also provided evidence that a 24 h cryopreservation delay at 4°C does not affect the proportion of morphologically normal follicles or follicle density in frozen-thawed ovarian cortical tissue (13). Conversely, another study described a decrease in the proportion of morphologically normal follicles induced by a 24 h freeze-delay prior to thawing and in vitro culture of human ovarian tissue (14), however, this study used vitrification, which relies on very high concentrations of cryoprotectants, while the present study employed slow freezing which is the currently accepted gold-standard method of ovarian tissue cryopreservation (25).…”

“…Furthermore, there is some evidence to suggest that ischaemic preconditioning can improve the outcomes of organ transplantation (31) and in this context, our findings might suggest that the benefits of ischaemic preconditioning are not lost after cryopreservation and thawing of the tissue. This, taken together with studies using human tissue, which demonstrated that follicles can survive a processing delay of delay of up to 20 h at 4°C and lead to a live birth (8,12,13), indicates that ovarian tissue responds to delayed cryopreservation favourably without a negative impact on the primordial pool.…”

Isothiocyanate from Moringa oleifera seeds mitigates hydrogen peroxide-induced cytotoxicity and preserved morphological features of human neuronal cells

“…Thus, preventing translocation of phosphatidylserine (PS) to cytoplasm. Although, study have shown that when cells are exposed to oxidative stress conditions, the internally generated ROS promote the disruption of membrane asymmetrical status, causing translocation of PS [ 48 ]. This effect may translate through receptor activating signals to break mitochondrial membrane potentials and trigger the release of cytochrome C with consequent cell death via apoptosis [ 49 ].…”