| The continued progress and enhanced effectiveness of chemotherapy andradiotherapy are leading to improved survival rate of young female cancersUnfortunately, the side effects accompany with these cytotoxic treatmentincluding premature ovarian failure, loss of endocrine and fertility function.Thus, quality-of-life issues have become a high priority on the patients'agenda.The different cryopreservation options available for fertility preservationare embryo, oocyte, and ovarian tissue cryopreservation. Embryoscryopreservation only refers to women who married. Oocyte cryopreservationis available for single women, but the pregnancy rate is still low. These twooptions are more complicated, and require long time hormone simulation toacquire oocyte. Moreover, hormone stimulation may be contraindicated inpatients with hormone-sensitive malignancies. On the contrary, ovarian tissuecryopreservation is the only technique for prepubertal girls and single women.Furthermore, ovarian tissue can be collectedat any stage of the menstrual cycle,avoiding delays of their anticancer therapy.Cryopreservation of ovarian tissue can be performed using slow-ratefreezing, conventional vitrification (CV) or direct cover vitrification (DCV).Despite successes using slow-cooled cryopreserved ovarian tissue, which ismuch more complicated, costly and time-consuming than vitrification, thelatter has gained popularity for better freezing efficiency. Recently,Chenreported a new method--direct cover vitrification, the ovarian tissue is directlycontacted with liquid nitrogen.This novel method is much more efficient thanthe above two ways. However, to the best of our knowledge, there is littlereport on this DCV method for animal and human ovarian tissue.At present, ovarian tissue grafting is the only option for potentially restoring ovarian function and fertility after cryopreservation and storage of ovarian tissue. Unfortunately, ischemic injury result in loss of follicles before neoangiogenesis occurs. The risks of ovary tissue harvesting appear to be small. Based on these observations, various strategies havebeen suggested to reduced graft ischemia and optimize the function of the transplant, such as graft of thin ovarian fragments, or pretransplantation treatment with FSH, antioxidant vitamin E, and growth factors etc. Improved ovarian tissue cryopreservation and transplantation protocols areneeded to ensure the best outcomes.In this study, we assessed the viability and function of ovarian tissue from adult mice that was cryopreserved by CV or DCV. Moreover,we investigated the viability and function of graft ovarian tissue that was cryopreserved by DCV with or without FSH and VEGF-treatment.Part I The comparison study of conventional vitrification(CV)and direct cover vitrification(DCV) of mice ovarian tissue.Objective: This study was conducted to investigate the effects of cryopreserve ovarian tissue using DCV and CV.Method: Thirty mature female mice were randomly subjected to3groups(10mice in each group): A) fresh control; B) conventionalvitrification(CV) group; C) direct cover vitrification(DCV) group; All thefresh and thawed ovarian tissues were autotransplanted back into the thighmuscle. Both fresh and warmed-vitrified tissue was compared by lightmicroscopic morphology of the primordial, primary and secondary follicleswithin the tissues. The percentages of each stage normal follicles=(the numberof normal follicles/total follicle number)×100%. The apoptosis positivefollicles in the fresh and thrawing tissue was detected by TUNEL. Once theoocyte nuclei or more than50%granulocyte show brown, it means the ovarianfollicle apoptosis. The TUNEL-positive follicles rate=(TUNEL-positivefollicles number/total follicle number)×100%. Results:(1) The percentages of normal primordial follicles were93.61±1.66%,46.86±4.56%and90.93±1.84%in fresh, CV and DCV group respectively.That of primary follicles is89.14±2.62%,33.39±3.06%,73.17±4.53%;secondary follicles were84.03±4.07%,25.31±4.57%and62.09±3.59%.Thenormal of all stage of follicles rates in CV group were lower than those of thefresh group(P<0.05);However, no significant differences of normal primordialfollicles were observed between fresh and DCV group(P>0.05). The normalform of the proportion of the follicles, from primordial follicles, primaryfollicles to secondary follicles presented a decrease trend.(2) The TUNEL-positive follicles rate were8.54±1.17%,40.31±3.87%,18.28±2.48%in fresh group, CV group and DCV group, respectively. TheTUNEL-positive follicles rate in the warmed tissues was higher than in thefresh group. A sharp (P<0.05) decrease in follicle viability was detected in CVgroup.Conclusions: Primordial follicles in the ovarian tissue can be wellpreserved by DCV. Part II The study of autotransplantation of mice ovarian tissue.Objective: This study was conducted to determine the viability andfunction of adult mice graft ovarian tissue that was cryopreserved byDCV with or without FSH and VEGF-treatment.Method:(1) Forty mature female mice were randomly subjected to5groups(10mice inA,B,C group,5mice in D and E group): A) fresh transplant control; B)direct cover vitrification(DCV) group; C) direct cover vitrification(DCV),mice received once daily injections of FSH and VEGF two days beforeautotransplant for7days; D) mice were castrated; E)sham operated group; All the fresh and thawed ovarian tissues were autotransplanted back intothe thigh muscle.(2) Approximately six weeks after autotransplantation, estrus was synchronized, the blood was collected through eye sockets for serum E2.(3) Mice were sacrificed, and ovarian tissues were collected for histological analyze and follicular density of ovarian tissue was counted(4) The activity of follicles in the grafts was detected by PCNA.(5) Weigh the uterus.Results:(1) The serum level of E2in group A, B, C, D, and E is59.61±6.85ng/L,28.38±8.47ng/L,58.99±8.63ng/L,2.59±1.19ng/L,79.56±12.07ng/L,respectively. The E2level in group A, B, C, E was much higher than groupD(P<0.05); The E2level in group A, B, C, D was lower than groupE(P<0.05), but we did not see any differences between group A and groupC(P>0.05). The E2level in group C was higher than group B.(2) The follicular density of ovarian tissue in A, B, C and E group is5.17±0.47n/mm3,2.07±0.28n/mm3,5.05±0.31n/mm3,7.75±0.38n/mm3,respectively. The follicular density of ovarian tissue in A, B, C group waslower than group E(P<0.05). The follicular density of ovarian tissue ingroup B was much lower in group C(P<0.05), but we did not see anydifferences between group A and group C(P>0.05).(3) When the ovarian tissues were transplanted back to mice, significant(P<0.05) decrease in follicle viability was detected in A,B,C group. A highpercentage of PCNA-positive follicles were presented in group A and Cthan group B. Graft recovery and follicle survival in group A were similarto group C.(P>0.05)(4) The weight of uterus in group A, B, C, D, and E is1.39±0.09g,1.10±0.12g,1.36±0.10g,1.02±0.06g,1.45±0.05g. The uterus weight in group B(without FSH and VEGF treatment) was much lower than group E, but wedid not see any differences between group A,C and group D(P>0.05).Conclusions: Fertility preservation using transplant warming-vitrified ovarian tissue is effective, especially the ones treated by VEGF and FSH. |
PDF Full Text Request