| Currently,ovarian tissue(OT)cryopreservation and transplantation is most ideal option for fertility preservation for cancer survivors.About 60%of survivors can recover their endocrine function within three months after transplantation.It has resulted in more than 140 live births.Unfortunately,OT without surgical connections of vascular anastomosis,more than 70%of follicles are lost on account of the early ischemia injury between transplantation and revascularization.To date,several attempts have been applied to improve follicle survival in grafted ovarian tissue.Various angiogenesis and antioxidants,have been shown to yield some improvement.However,no effective drugs and dose were selected,no long-term follow-up were investigated in grafted ovarian tissue,further optimization studies are required to improve tissue grafting.The aim of this study was to investigate the protective substances of ovarian tissue cryopreservation and transplantation,to provide technical support for improving the effect of ovarian tissue cryopreservation and transplantation,and to contribute to the female fertility preservation.Part1 Evaluation of the effect of vitrified-warmed mouse ovarian tissueObjectiveTo assess the effect of vitrified-warmed mouse ovarian tissue.Methods1.6-8 week-old female ICR mice were randomly distributed into fresh control and vitrified-warmed groups.2.The morphology of ovarian follicles after thawing was analyzed by Hematoxylin-eosin(HE),antigen Ki67(Ki67)was used to detect the proliferation of oocytes and stromal cell,Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay,(TUNEL)was used to detect apoptosis.Results1.The rate of intact antral follicles decreased after vitrification.In morphology,antral follicles were atresia and the surrounding granular cells were loose.Most of follicles were primordial,primary and secondary,stromal cells showed an intact shape.2.Concerning ki67 protein,a positive nuclear staining was found in the granulosa cells and oocytes in fresh and frozen-thawed follicles,there were no obvious differences.3.TUNEL analysis showed positive cells were found in follicles at antral stage,the differences were significant between two groups.Conclusion1.Vitrification can preserve the morphology and function of ovarian tissue better.2.There was a remarkable reduction in the percentage of morphologically normal antral follicles.Part2 Effect of simvastatin on mouse ovarian vitrification and transplantationObjectiveTo study the effect of simvastatin on the quality of vitrified-warmed ovarian tissue after auto-transplantation in mice.Methods1.6-8 week-old female ICR mice were removed and vitrified.After thawing,ovarian tissues were auto-transplanted to the back muscles.Survival mice were randomly divided into two groups.The mice in simvastatin group were administered with simvastatin(5mg/kg,orally),whereas the mice in saline group received normal saline as control.Two treatments lasted for 7 days after auto-transplantation.2.Mice were sacrificed on the day 3,7 and 21.Ovarian tissues were collected for HE,CD34 antigen(CD34)and TUNEL.Serum was collected for detecting Estradiol(E2)and Follicle-stimulating hormone(FSH).Moreover mice were sacrificed on the day 3 and 7,malondialdehyde(MDA)and superoxide dismutase(SOD)were analyzed.Results1.On day 3 and 7 after transplantation,the rate of intact primordial follicles in the simvastatin group was higher,and the rate of apoptotic follicles was less,than those in the saline group.On day 21,the ovarian tissue grew well and antral follicles were found in both groups.The rate of intact antral follicles of simvastatin group was higher than saline group.2.In the simvastatin group,the number of CD34-positive in the simvastatin group was increased than that in the saline group on day 3 and 7.3.In the simvastatin group,the levels of serum E2 was significantly increased after transplantation,and the level of FSH was decreased on the day 21.4.On day 7,The SOD was higher than day 3 in the saline group,SOD was higher than day 3 and MDA was lower than day 3 after transplantation in the simvastatin group.The level of SOD was increased,and MDA decreased than saline group on day 3 and 7.Conclusion1.All stages of ovarian follicles deteriorated after auto-transplantation.2.Treatment with simvastatin after auto-transplantation of ovarian tissue could prevent the ovarian damage and restore ovarian function through the mechanisms of anti-oxidation and reconstruction of blood vessels.Part3 Effect and mechanism of resveratrol on mouse ovarian vitrification and transplantationObjectiveTo study the effect and mechanism of resveratrol on quality of vitrified-warmed ovarian tissue after auto-transplantation in miceMethods1.6-8 week-old female ICR mice were removed and vitrified.After thawing,ovarian tissues were auto-transplanted to the bilateral kidney capsules.Survival mice were randomly divided into three groups.The mice were administered resveratrol(15mg/kg,orally),carboxymenthyl cellulose nitrate(CMC)(5%,orally)and saline.Three treatments lasted for 7 days after auto-transplantation.2.Mice were sacrificed on the day 3,7 and 21.Ovarian tissues were collected for HE and TUNEL.Serum was collected for detecting E2 and FSH.Related genes'(MDA,SOD,Nuclear factor-k-gene binding(NF-?B),Interleukin-6(IL-6)and sirtuinl(SIRT1))expression was evaluated by Reverse transcription-Polymerase Chain Reaction(qRT-PCR).Embryonic development was evaluated after IVF of oocytes obtained from transplanted ovaries on day 21.Results1.On day3 and day7 after transplantation,the rate of intact primordial follicles significantly increased in resveratrol groups comparing with that in the saline and CMC groups,respectively.Moreover,resveratrol-treated ovarian grafts had an increased rate of intact follicles(primarily and antral follicles)compared with the other two groups three weeks after transplantation2.On day 21 after transplantation,the serum FSH level was significantly decreased in the resveratrol group comparing with the other two groups,serum E2 level was significantly increased in the resveratrol group3.qRT-PCR demonstrated that the level of MDA and NF-?B were significantly lower in the resveratrol group in comparison with saline group on day 3.IL-6 was lower than saline groups on day 7.Furthermore,a significant increase of SOD level was observed even on day 3 and day 7 compared with saline group.However,there was no difference of SIRT1 in auto-transplantation ovarian tissue treated with resveratrol comparing with saline group4.On day 21 after transplantation,cleavage rate in resveratrol group was higher than that in saline group,no significant difference was noted between the two groups,but blastocysts were not cultured in both groupsConclusion1.Resveratrol could reduce the loss of follicles on vitrified-warmed ovarian tissue after auto-transplantation in mice2.Resveratrol could inhibit the activation of NF-?B and resulted in reduced release of pro-inflammatory cytokines IL-6 after transplantation3.Resveratrol could decrease the level of MDA via increasing SOD to reduce oxidative stress in the early age of auto-transplantation4.We could not draw a conclusion that resveratrol can improver embryonic outcomes,and it needs further study. |
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