| Background: In recent years,women suffering from malignant tumors have shown an increasing incidence and a younger age.Radiotherapy and chemotherapy have caused great damage to ovarian endocrine and reproductive functions.With the cure of cancer increasing,women with cured cancer concern about their fertility and quality of life much more.Ovarian tissue cryopreservation and transplantation is the only way for pre-adolescent female’s fertility preservation,and without delaying cancer treatment,which has an important clinical significance and far-reaching application prospects.However,the main reasons that limit the survival of ovarian tissue post-transplantation are the freezing injury of ovarian tissue and the ischemic damage.Therefore,it is important to find new methods to reduce the freezing damage of ovarian tissue and the ischemic damage post ovarian tissue transplantation.Platelet-rich plasma(PRP)is composed by platelet-rich fibrin(PRF)and various cytokines which promote tissue growth,regeneration,and healing.It is convenient,safe,and could induce the regeneration of primordial follicles.It shows great potential in promoting ovarian tissue transplantation.Sufficient experiments and clinical study in sports medicine and aesthetic medicine showed befits of PRP.In reproductive medicine,PRP has got satisfied results in the study of thin endometrium and primary ovarian insufficiency.There is no relevant research on cryo-thawed and transplantation of ovarian tissue by using PRP,however,the special characteristics of PRP made it possible for a successful ovarian tissue transplantation.Purpose: This research aims to clarify the effect of PRP on ovarian tissue vitrification,and to find the best vitrification solution incubation time for PRPassisted ovarian tissue cryopreservation,and to study the effect of PRP on ovarian tissue freeze-thawed process,reproductive endocrine function and vascular remodeling,even possible mechanisms,provide some experimental basis and inspire for more clinical research and application of PRP in ovarian tissue transplantation.Methods: The rats were randomly divided into following groups,experimental group(Both vitrification solution and resuscitation solution containing 100μL/m L PRP),negative control group(Both vitrification solution and resuscitation solution without PRP)and fresh control group(fresh ovarian tissue without freeze-thawed).According to the incubation time of the ovarian tissue in vitrification solution,the PRP group was randomly divided into 5 subgroups.Respectively,they are 5 min group,10 min group,15 min group,20 min group and 30 min group,with 8 rats in each group.Through four chapters including the influence of PRP on ovarian tissue’s vitrification,the effect of PRP on the recovery of reproductive endocrine function after autologous ovarian tissue transplantation,the transcriptomics study of the influence of PRP on ovarian tissue transplantation,the influence of PRP on the vascular reconstruction after ovarian tissue transplantation and the possible mechanism,we explored the effects and possible mechanisms of the experimental group and the control group on the cryo-resuscitation effect of ovarian tissue,reproductive endocrine function,vascular reconstruction,as well as the best time for incubation in the vitrification solution with PRP.Indicators including the number of primitive follicles,the number of primary follicles,the number of secondary follicles,the number of blood vessels,the expression of VEGF,PDGF-B,HIF-1α m RNA and protein in ovarian tissue,and the concentration of serum AMH and E2 in each group were statistically analyzed.Results: 1.The number of primordial follicles per sight in PRP group was higher than control group post-vitrification,the difference was statistically significant(P <0.05).In comparison,there was no statistical difference in the number of primary and secondary follicles between the groups after cryopreservation of ovarian tissue(P>0.05).When vitrification solution incubation time was 5 min or 30 min,the m RNA and protein expressions of VEGF,PDGF-B and HIF-1α in ovarian tissue were lower than those in the control group post-vitrification and 3 weeks posttransplantation,the difference were statistically significant(P <0.05).The serum AMH and E2 concentrations in rats were lower than 10-20 min and the control group 3 weeks post-transplantation,the difference were statistically significant(P <0.05).The expression of VEGF,PDGF-B and HIF-1α m RNA and proteins were higher than those in the control group post-transplantation,the difference was statistically significant(P <0.05).However,1-or 2-weeks post-transplantation,the expression level of VEGF m RNA in PRP group was higher than which in the control group,and the difference was statistically significant(P<0.05).There was no statistical difference in PDGF and HIF-1α m RNA between the two groups.Conclusion: This study mainly showed that the vitrification solution with 100 μL/m L PRP could improve the freezing-thawing effect of ovarian tissue,promote the functional recovery and vascular reconstruction after ovarian tissue transplantation.The successful transplantation of ovarian tissue is related to the incubation time of the ovarian tissue in the vitrification solution.It’s not conducive for the functional recovery and vascular reconstruction after ovarian tissue transplantation when the vitrification solution incubation time is too short or too long during in vitrification.When the rat ovarian tissue was incubated in the vitrification solution was 5 minutes,the freezing-thawing effect of ovarian tissue was not satisfied as 10-20 minutes.When incubated for 10-20 min,it was beneficial to ovarian tissue’s vitrification and revascularization post-transplantation.When incubated for 30 min,PRP group could get an effect better than that of the negative control group,but not as satisfied as incubated for 10-20 min.Through sequencing analysis of rat ovarian tissue samples and KEGG enrichment analysis,it was suggested that the role of PRP in promoting the successful autologous transplantation of rat ovarian tissue be related to the HIF-α signaling pathway and the promotion of VEGF expression.PRP could promote the expression of VEGF,PDGF-B and HIF-1α factors in ovarian tissue.The mechanism may be that PRP induces the expression of VEGF gene through the HIF-1α signal pathway,however,the specific mechanism still needs to be further studied. |
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