P-207: Viability testing of cryopreserved ovarian tissue

Abstract

ObjectiveOvarian tissue cryopreservation is now offered to women at risk of losing ovarian function, but few programs test for viability, normal morphology and developmental potential of this tissue post thaw and prior to grafting. In the current literature, Proliferating Cell Nuclear Antigen (PCNA) has been used as the marker of choice for cellular proliferation (Wandji et al., 1996; Oktay et al., 1998). This study provides a better assay to determine the impact of cryopreservation on follicle density and viability. The effect of cryopreservation on ovarian tissue viability was evaluated using conventional histology and immunohistochemical staining with PCNA, Ki-67 and CPP32 (Caspase-3 Antibody).DesignProspective, nonrandomized study using immunofluoresence staining and histological markers.Materials and methodsCryopreserved ovarian tissue from seven patients was thawed and cultured in vitro over three weeks in Transwell Plates (3.0 μm pore size, 24.5 mm diameter; Costar, Cambridge, MA, U.S.A) filled with 0.01ml Blastocyst Medium supplemented with 5% human serum albumin (Irvine Scientific, Santa Ana, CA), 100 mIU/mL follicle stimulating hormone (Serono, Norwell MA), and 1.0 ng/mL epidermal growth factor (Molecular Probes). Tissue was retrieved weekly for histological evaluation (of follicle size, density, and developmental stage)using phase contrast and confocal microscopy and for immunohistochemical staining with PCNA, Ki-67, and CPP32.ResultsNo histological differences were identified microscopically in either follicular or stromal cells in ovarian tissue samples before and after cryopreservation. The percentage of primordial follicles was greater than the percentage of primary follicles at all time points. The percentage of atretic follicles increased after the first week in culture. No difference in the intensity of staining with PCNA was observed between oocytes, granulosa cells, and stromal cells. Conversely, immunohistochemical staining for Ki-67 demonstrated minimal cell proliferation, while many follicles stained positive for CPP32, a marker for apoptosis.ConclusionThis study was undertaken to demonstrate follicular viability and normal morphology of cryopreserved ovarian tissue after thawing. Initially, PCNA was used to assess cell proliferation and metabolic activity in oocytes. However, the widespread staining of PCNA in the oocytes and stromal cells in all sections did not make it a useful marker for cell proliferation. PCNA is an auxiliary protein to DNA polymerase delta, which is involved in DNA synthesis and repair (Liu et al., 1989). Thus, it might be possible that the majority of cells in the ovarian tissue fragments were undergoing DNA repair rather than proliferation. PCNA did not appear to be a reliable quantitative marker of cell proliferation by the procedures followed in this study. Immunohistochemical staining with Ki-67 and CPP32, on the other hand, were good indicators of cell viability. Thus, we believe Ki67 is a superior indicator of true post thaw viability since it is utilized only if nuclear DNA is functioning at the time the stain is incorporated, unlike PCNA. This is the first study to demonstrate the utility of Ki67 staining for use in demonstrating viability of cryopreserved ovarian tissue. ObjectiveOvarian tissue cryopreservation is now offered to women at risk of losing ovarian function, but few programs test for viability, normal morphology and developmental potential of this tissue post thaw and prior to grafting. In the current literature, Proliferating Cell Nuclear Antigen (PCNA) has been used as the marker of choice for cellular proliferation (Wandji et al., 1996; Oktay et al., 1998). This study provides a better assay to determine the impact of cryopreservation on follicle density and viability. The effect of cryopreservation on ovarian tissue viability was evaluated using conventional histology and immunohistochemical staining with PCNA, Ki-67 and CPP32 (Caspase-3 Antibody). Ovarian tissue cryopreservation is now offered to women at risk of losing ovarian function, but few programs test for viability, normal morphology and developmental potential of this tissue post thaw and prior to grafting. In the current literature, Proliferating Cell Nuclear Antigen (PCNA) has been used as the marker of choice for cellular proliferation (Wandji et al., 1996; Oktay et al., 1998). This study provides a better assay to determine the impact of cryopreservation on follicle density and viability. The effect of cryopreservation on ovarian tissue viability was evaluated using conventional histology and immunohistochemical staining with PCNA, Ki-67 and CPP32 (Caspase-3 Antibody). DesignProspective, nonrandomized study using immunofluoresence staining and histological markers. Prospective, nonrandomized study using immunofluoresence staining and histological markers. Materials and methodsCryopreserved ovarian tissue from seven patients was thawed and cultured in vitro over three weeks in Transwell Plates (3.0 μm pore size, 24.5 mm diameter; Costar, Cambridge, MA, U.S.A) filled with 0.01ml Blastocyst Medium supplemented with 5% human serum albumin (Irvine Scientific, Santa Ana, CA), 100 mIU/mL follicle stimulating hormone (Serono, Norwell MA), and 1.0 ng/mL epidermal growth factor (Molecular Probes). Tissue was retrieved weekly for histological evaluation (of follicle size, density, and developmental stage)using phase contrast and confocal microscopy and for immunohistochemical staining with PCNA, Ki-67, and CPP32. Cryopreserved ovarian tissue from seven patients was thawed and cultured in vitro over three weeks in Transwell Plates (3.0 μm pore size, 24.5 mm diameter; Costar, Cambridge, MA, U.S.A) filled with 0.01ml Blastocyst Medium supplemented with 5% human serum albumin (Irvine Scientific, Santa Ana, CA), 100 mIU/mL follicle stimulating hormone (Serono, Norwell MA), and 1.0 ng/mL epidermal growth factor (Molecular Probes). Tissue was retrieved weekly for histological evaluation (of follicle size, density, and developmental stage)using phase contrast and confocal microscopy and for immunohistochemical staining with PCNA, Ki-67, and CPP32. ResultsNo histological differences were identified microscopically in either follicular or stromal cells in ovarian tissue samples before and after cryopreservation. The percentage of primordial follicles was greater than the percentage of primary follicles at all time points. The percentage of atretic follicles increased after the first week in culture. No difference in the intensity of staining with PCNA was observed between oocytes, granulosa cells, and stromal cells. Conversely, immunohistochemical staining for Ki-67 demonstrated minimal cell proliferation, while many follicles stained positive for CPP32, a marker for apoptosis. No histological differences were identified microscopically in either follicular or stromal cells in ovarian tissue samples before and after cryopreservation. The percentage of primordial follicles was greater than the percentage of primary follicles at all time points. The percentage of atretic follicles increased after the first week in culture. No difference in the intensity of staining with PCNA was observed between oocytes, granulosa cells, and stromal cells. Conversely, immunohistochemical staining for Ki-67 demonstrated minimal cell proliferation, while many follicles stained positive for CPP32, a marker for apoptosis. ConclusionThis study was undertaken to demonstrate follicular viability and normal morphology of cryopreserved ovarian tissue after thawing. Initially, PCNA was used to assess cell proliferation and metabolic activity in oocytes. However, the widespread staining of PCNA in the oocytes and stromal cells in all sections did not make it a useful marker for cell proliferation. PCNA is an auxiliary protein to DNA polymerase delta, which is involved in DNA synthesis and repair (Liu et al., 1989). Thus, it might be possible that the majority of cells in the ovarian tissue fragments were undergoing DNA repair rather than proliferation. PCNA did not appear to be a reliable quantitative marker of cell proliferation by the procedures followed in this study. Immunohistochemical staining with Ki-67 and CPP32, on the other hand, were good indicators of cell viability. Thus, we believe Ki67 is a superior indicator of true post thaw viability since it is utilized only if nuclear DNA is functioning at the time the stain is incorporated, unlike PCNA. This is the first study to demonstrate the utility of Ki67 staining for use in demonstrating viability of cryopreserved ovarian tissue. This study was undertaken to demonstrate follicular viability and normal morphology of cryopreserved ovarian tissue after thawing. Initially, PCNA was used to assess cell proliferation and metabolic activity in oocytes. However, the widespread staining of PCNA in the oocytes and stromal cells in all sections did not make it a useful marker for cell proliferation. PCNA is an auxiliary protein to DNA polymerase delta, which is involved in DNA synthesis and repair (Liu et al., 1989). Thus, it might be possible that the majority of cells in the ovarian tissue fragments were undergoing DNA repair rather than proliferation. PCNA did not appear to be a reliable quantitative marker of cell proliferation by the procedures followed in this study. Immunohistochemical staining with Ki-67 and CPP32, on the other hand, were good indicators of cell viability. Thus, we believe Ki67 is a superior indicator of true post thaw viability since it is utilized only if nuclear DNA is functioning at the time the stain is incorporated, unlike PCNA. This is the first study to demonstrate the utility of Ki67 staining for use in demonstrating viability of cryopreserved ovarian tissue.