| ObjectiveEarly diagnosis and multiple treatments improve the long-term survival rates of young women with malignant ovarian cancer. However, cancer therapies such as radial and chemical therapy cause severe injuries to the ovarian reserve, which may, consequently, lead to ovarian failure and fertility. Preservation of an ovarian cortical fragment is most commonly used in clinical applications, but limited by ischemic injury and follicle loss occurs during the transplantation process. Whole ovary cryopreservation along with vascular reanastomosis could overcome this problem, theoretically. Similar to cryopreservation of an ovarian cortical piece, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS) and glycerine are widely used as cryoprotectant. However, some of which has been proven toxic to the human body. And it is more difficult for cryoprotectant to infiltrate and remove in freezing whole ovary than ovarian cortex. Our study aim to research the effects of freezing sheep intact ovary perfused with different cryoprotectants by concentration gradient cryo-perfusion apparatus, and to obtain the best cryoprotectants combination for cryopreservation of the whole sheep ovary.Methods28 ovaries collected from 6-8 months non-pregnant female sheep were randomly distributed into fresh control group (Group A), trehalose group (Group B), glycerin group (Group C) and DMSO group (Group D). Each experimental group contained 7 ovaries. Ovaries in group B, C, D were cannulated and ligated, and then connected to our newly designed apparatus--concentration gradient cryo-perfusion apparatus and perfused with appropriate cryoprotectant. We applied Conventional slow freezing for cooling ovaries and then plunge them in liquid nitrogen. Half size of Fresh and freezed-warmed samples were sectioned serially and stained either with HE for histological assessment or TUNEL assay. The other half were used for RNA isolation and then detected the mRNA level of BAX(Bcl-2 Associated X Protein, BAX)and CIRP (Cold inducible RNA-binding Protein, CIRP) by Real-time PCR.Results1.The rate of intact follicles in the fresh control group (93.46±2.30%) was significantly higher (P<0.05) than that in the Glycerin group (88.03±4.80%) and DMSO group (80.85±228%), and it was not different (P=0.07) from that in the trehalose group (90.29±2.30%).2.Quantitative assessment of stromal cell density indicated that ovaries in fresh control group had values(1114.68±184.24 nuclei/400×field) higher than group B, C, D, and variance is significant compared with DMSO group (906.14± 304.78 nuclei/400×field) (P<.005). The values in trehalose group(1020.83± 278.39 nuclei/400×field) and glycerin group(1089.38±573.13 nuclei/400×field) were comparable to those of the fresh group.3. The number of positive cells in the fresh group (13.86±13.22/400×field) is the lowest among all the groups (P<0.05), and the value in the trehalose group (73.14±32.01/400×field) was significantly lower than that of the glycerin group(166.43±60.88/400×field) and the DMSO group (250.29±85.90/400× field) (P<0.05).4.The level of BAX transcripts significantly increased in group C (0.081±0.075) and group D (0.099±0.125), which were higher than group A (0.016±0.019) and group B (0.026±0.024) (P<0.05); The level of CIRP transcript in all freezed-thawed groups were significant higher than that in the fresh control group (0.164±0.121) (P<0.05), and the value in trehalose group (0.512±0.226) was higher than that in the glycerin group (0.252±0.129) and the DMSO group (0.224±0.093) (P<0.05).Conclusions1. The frozen/thawed whole ovary of sheep still reserved normal morphology of follicles and stromal cells density, and apoptosis is not distinct after cryopreservation. It demonstrates that cryopreservation of whole sheep ovary is feasible.2. Cryoprotectant combination of group B appears to be better suited for cryopreservation of whole sheep ovary in aspect of histology and anti-apoptosis. |
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