A Study of Fertility Preservation Options Using Mouse Ovarian Tissue

Abstract

Introduction: As the diagnosis and treatment of cancer have been dramatically improved, fertility preservation in female cancer patients has been up in light to improve their quality of life. Oocyte banking and embryo cryopreservation commonly used as fertility preservation options, however, they cannot be applied to pre-pubertal girls and unmarried women. Fertility preservation with ovarian tissue (OT) can provide alternative instead of oocyte and embryo while there are major hurdles to enhance the efficiency of the procedures. First, cryoinjury is usually occurred during cryopreservation process. Second, ischemic injury is also spontaneously happened until re-vascularization, such as 2 days and 5 days are needed to initiate the re-vascularization. Finally, OT has a risk to re-implant the malignant cells after transplantation. This study was aimed to 1) compare the deleterious effects of cryoinjury and ischemic injury on the quality of OT after cryopreservation and transplantation process 2) decrease the cryoinjury after vitrification-warming process using several types of antifreeze proteins 3) diminish the ischemic injury via enhancement of re-vascularization after auto-transplantation 4) optimize the two-dimensional follicle culture system using mouse model. Methods: For the comparison of cryoinjury and ischemic injury (Exp I), a total of 160 ovaries were harvested from 6-week-old female B6D2F1 mice. Ovaries were randomly divided into eight different groups consisting of two control groups (fresh and vitri-con) and six experimental groups according to the presence or absence of vitrification and transplantation. (fresh OT [FrOT]-day [D] 2, FrOT-D7, FrOT-D21, vitrified OT [VtOT]-D2, VtOT-D7, and VtOT-D21). In the fresh control group, OT was fixed immediately after ovariectomy, and in the vitri-con group, OT was fixed after the vitrification-warming procedure. All six experimental groups were auto-transplanted with fresh or vitrified-warmed OT, and then the mice were sacrificed by cervical dislocation at 2, 7, or 21 days after grafting. To investigate the detrimental impacts of these injuries, histology, cell-death, blood vessel distribution in OT and ELISA for FSH level For decrease cryoinjury during vitrification-warming process (Exp II.), a total of 140 mice were sacrificed to collect sexually mature ovaries from 6-week-old aged female B6D2F1 mice. In Exp II-I, a total of 240 whole ovaries were randomly distributed to one of three groups: the fresh control group, the vitrification control group, or the AFP-treated group. The AFP-treated group was further divided into nine subgroups according to AFP type (e.g., FfIBP, LeIBP, and type III AFP) and dose (0.1, 1.0, and 10 mg/mL). After two-step vitrification and four-step warming process, the quality of ovary was assessed. Then, auto-transplantation of ovary was carried out to determine whether the cryoprotective effects of LeIBP could also be seen in OT after transplantation (Exp II-II). A total of 20 B6D2F1 mice were randomly divided into two groups: one group that received 10mg/ml of LeIBP-treated ovaries and another that received the vitrification control ovaries. We used only 10 mg/ml of LeIBP-treated group for this experiment because this group showed the best results in Experiment II-I. The quality was evaluated by histology, TUNEL, immunohistochemistry and serum FSH level on each evaluation days (Day 2, 7 and 21 days after transplantation). Next, a combination of simvastatin and/or methylprednisolone was treated to diminish of ischemic injury during avascular period after transplantation (Exp III). Following comparison study, the mice were treated with 5 mg/kg of simvastatin and/or methylprednisolone 2 h before ovareictomy and then the ovaries were cryopreserved by two-step vitrification process as previously described in Exp I. One week later, vitrified OTs were warmed by four-step warming process and then auto-transplanted under bilateral kidney capsules. Similar to the Exp II-II, the mice were sacrificed by cervical dislocation on 2nd, 7th or 21st of the transplantation period to assess the quality of OT. Macroscopic and microscopic examination, immunohistochemistry for blood vessel, flow cytometry for CD45, serum AMH ELISA were carried out on each evaluation days. Moreover, oocyte retrieval from graft and further in vitro fertilization were also performed to evaluate the drug safety on gametogenesis and embryogenesis. Finally, Pre-antral follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned into two groups according to the culture methods (with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layer with or without oil layerwith or without oil layerwith or without oil layerwith or without oil layer with or without oil layer , Exp IV). In vivo matured oocytes were collected using superovulation to compare the growth, cytoplasmic normality, gene expression and embryonic development. Ovarian follicles were in vitro cultured for 10 days and cumulus-oocyte complexes were harvested at 16-18 hours after hCG and EGF treatment. Mature oocytes were assessed their maturational ability and developmental competence in vitro. Results: In Exp I, The vitrification-warming procedure decreased the intact (grade 1, G1) follicle ratio in the vitri-con and FrOT-D2 groups compared with that in the fresh control, and this ratio was reduced more by ischemic injury after transplantation (fresh: 64.2%, vitri-con: 50.3%, and FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovarian tissue than in the fresh control, and it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0%, and FrOT-D2: 26.8%). The mean number of follicles per section and CD31-positive area was significantly reduced after vitrification and transplantation. (the number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, and FrOT-D2: 17.9 ± 2.1

CD31-positive area, fresh: 10.6 ± 1.3%, vitri-con: 5.7 ± 0.9%, and FrOT-D2: 4.2 ± 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1%, and FrOT-D21: 70.7%

CD31-positive area, FrOT-D2: 4.2 ± 0.4%, FrOT-D7: 5.4 ± 0.6%, and FrOT-D21: 7.5 ± 0.8%). In Exp II-I, the percentage of grade 1 total follicles was significantly higher in only the 10 mg/mL LeIBP group than in the vitrification control while all of AFP-treated groups had significantly improved grade 1 primordial follicle ratio compared with the vitrification control. The apoptotic (TUNEL-positive) follicle ratio was significantly decreased in the 1 and 10 mg/ml of LeIBP treated groups. The proportion of τH2AX positive follicles was significantly reduced in all AFP-treated groups while the Rad51-positive follicle ratio was significantly decreased in only FfIBP and LeIBP treated groups. In ExpII-II, after auto-transplantation of OT vitrified with 10 mg/ml of LeIBP, the percentage of total Grade 1 follicles and primordial Grade 1 follicles, the extent of the CD31-positive area were significantly increased. Moreover, the level of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated group than in the control group. In Exp III, The group that received simvastatin and methylprednisolone showed a significantly improved intact (G1) follicle ratio (D2: p<0.001, D7: p<0.05 and D21: p<0.001), apoptotic follicle ratio (D21: p<0.05), CD31-positive area (D7: p<0.05 and D21: p<0.05), and serum AMH level (D7: p<0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rate, the number of total and apoptotic blastomere per blastocyst and ICN/TE ratio among the four transplantation groups. In Exp IV, With respect to the follicular growth, the diameter of follicles in oil layer culture was significantly higher than that of without oil layer. In addition, maturational criteria including survival in oil layer, pseudo-antral like cavity formation, ovulation and oocyte maturation, also significantly increased compared with the without oil layer. Late stage of culture, estradiol on D10 and progesterone on D11 in spent medium of oil layer was statistically significant different between without oil layer culture condition. When comparing the mRNA expression in matured oocytes, no significant difference was observed between in vivo and in vitro derived oocytes. On the other hand, in vitro grown and matured oocytes increased the level of reactive oxygen species and decreased the mitochondrial activity when compared with the in vivo mature oocytes with statistical significance. Moreover, cortical granules of both in vitro derived oocytes seemed to be more clumped and unevenly distributed than in vivo control. However, no significant difference was noted in actin filament configuration and spindle normality between in vitro and in vivo derived oocytes. Conclusions: Cryoinjury and ischemic injury are main cause of follicular depletion during fertility preservation process using ovarian tissue. Inevitable post-transplantation ischemia seems to be more deleterious than cryoinjury during cryopreservation process. Then, cryoinjury and ischemic injury could be decreased by use of AFPs and a combination of simvastatin and methylprednisolone. It can improve the quality of OT via promotion of vessel integrity in OT after cryopreservation and transplantation process. Therefore, minimizing cryoinjury and ischemic injury by enhancing vascularization is needed to improve the ovarian function after fertility preservation. Finally, optimization of two-dimensional in vitro follicle culture method was also attempted to avoid the re-implantation of residual malignancy cells in OT after transplantation and found the cause of reduction in embryonic development competence from in vitro derived oocytes.