Antifreeze Protein Supplementation During the Warming of Vitrified Bovine Ovarian Tissue Can Improve the Ovarian Tissue Quality After Xenotransplantation.

The occurrence of ice crystallization during ovarian tissue (OT) cryopreservation causes unavoidable cryodamage, and ice recrystallization during the warming is more detrimental than ice crystallization. Here, we investigated that antifreeze protein (AFP) treatment during the warming procedure can improve the bovine OT quality after xenotransplantation (XT). Bovine OTs (n=120) were evenly assigned to four groups: fresh, vitrified-warmed, vitrified-warmed with 10 mg/mL Leucosporidium ice-binding protein (LeIBP, a type of AFP) (LeIBP-10), and vitrified-warmed with 20 mg/mL LeIBP (LeiBP-20). LeIBPs were added to the first warming solution. Twenty pieces of OTs were assigned to each category. The remaining 10 OTs from each category were assigned to the XT-Fresh control, XT-Vitrified-warmed control, XT-LeIBP-10, and XT-LeIBP-20 groups, respectively, and xenotransplanted to 9-week-old ovariectomized nude mice for one week. LeIBP treatment during the warming step increased morphological follicle normality and decreased apoptotic follicle ratios after vitrification-warming and XT. The XT-vitrified-warmed control group showed significantly reduced microvessel density and increased fibrosis when compared to that of the XT-fresh group. Microvessel density and fibrosis were recovered in both LeIBP treated groups. There was no significant difference between the LeIBP-10 and LeIBP-20 groups in all outcomes. AFP treatment during the warming procedure can prevent OT damage, and improve ovarian follicle morphology and apoptosis in both the vitrified-warmed bovine OT and its graft. After confirmation in a human study, AFPs can potentially be applied to human OT cryopreservation to reduce cryodamage and improve the OT quality.

Molecular basis of ice-binding and cryopreservation activities of type III antifreeze proteins.

Antifreeze proteins (AFPs) can inhibit the freezing of body fluid at subzero temperatures to promote the survival of various organisms living in polar regions. Type III AFPs are categorized into three subgroups, QAE1, QAE2, and SP isoforms, based on differences in their isoelectric points. We determined the thermal hysteresis (TH), ice recrystallization inhibition (IRI), and cryopreservation activity of three isoforms of the notched-fin eelpout AFP and their mutant constructs and characterized their structural and dynamic features using NMR. The QAE1 isoform is the most active among the three classes of III AFP isoforms, and the mutants of inactive QAE2 and SP isoforms, QAE2ACT and SPACT, displayed the full TH and IRI activities with resepect to QAE1 isoform. Cryopreservation studies using mouse ovarian tissue revealed that the QAE1 isoform and the active mutants, QAE2ACT and SPACT, more effectively preserved intact follicle morphology and prevented DNA double-strand break damage more efficiently than the inactive isoforms. It was also found that all active AFPs, QAE1, QAE2ACT, and SPACT, formed unique H-bonds with the first 310 helix, an interaction that plays an important role in the formation of anchored clathrate water networks for efficient binding to the primary prism and pyramidal planes of ice crystals, which was disrupted in the inactive isoforms. Our studies provide valuable insights into the molecular mechanism of the TH and IRI activity, as well as the cryopreservation efficiency, of type III AFPs.

Establishment of an improved vitrification protocol by combinations of vitrification medium for isolated mouse ovarian follicles.

In vitro follicle growth (IVFG) is an emerging alternative option for fertility preservation in women instead of ovarian tissue cryopreservation and transplantation. To widen the application of this technique, follicle cryopreservation should be established prior to clinical use. In the present study, we tried to determine the optimal vitrification protocol of mouse ovarian follicle for in vitro culture and oocyte maturation by comparing four different compositions of cryoprotective agents (CPA). Secondary follicles were mechanically isolated from 2-week-old BDF-1 mice and randomly assigned to fresh control and four different groups by the composition of CPAs (ES, EDS, EFS and EPS groups; E: ethylene glycol, D: dimethyl sulfoxide, S: sucrose, F: ficoll, P: 1,2-propanediol (PROH)). After vitrification and warming procedures, the follicles were cultured in vitro for 10 days and then treated with human chorionic gonadotropin and epidermal growth factor to induce oocyte maturation. Fourteen to 16 h later, oocyte maturation and quality were assessed. Follicle viability was evaluated by Calcein-AM/ethidium homodimer-1 staining immediately after warming, and their survival and diameters were measured during follicle culture periods. Antral cavity formation was observed at the end of the culture period (on the 10th day of culture). Following oocyte maturation, its maturational ability and meiotic spindle formation were assessed to evaluate their competence. There was no significant difference in viability after warming among the vitrification groups. From the 8th day of culture, the survival rate of ES and EDS were significantly higher than those of other vitrification groups (EPS and EFS). The follicle diameter was largest in the fresh-control group from the 6th day, while smallest in the EFS with statistical significance. On the 10th day of culture, the antral-cavity formation rate of EDS was comparable to that of the fresh control group. However, the oocyte maturation was significantly decreased in all four vitrification groups when compared with control group; especially, the EFS showed a more marked reduction in the oocyte maturation. There were no significant differences in meiotic spindle formation among all of those groups. Our results suggest that EDS combination for mouse follicle vitrification are the most effective vitrification protocols for mouse follicle and evaluated by an in vitro culture and oocyte maturation after warming.

Comparison of the Oocyte Quality Derived from Two-Dimensional Follicle Culture Methods and Developmental Competence of In Vitro Grown and Matured Oocytes.

In vitro follicle growth (IVFG) is an emerging fertility preservation technique, which can obtain fertilizable oocytes from an in vitro culture system in female. This study aimed to compare efficiency of the most widely used two-dimensional follicle culture methods [with or without oil layer (O+ or O- group)]. Preantral follicles were isolated from mice and randomly assigned. Follicles were cultured for 10 days and cumulus-oocyte complexes harvested 16-18 hours after hCG treatment. Follicle and oocyte growth, hormones in spent medium, meiotic spindle localization, expression of reactive oxygen species (ROS), mitochondrial activity, and gene expression were evaluated. In follicle growth, survival, pseudoantral cavity formation, ovulation, and oocyte maturation were also significantly higher in O+ group than O- group. Hormone production was significantly higher in follicles cultured in O+ than O-. There were no significant differences in mRNA expression related to development. On the other hand, the level of ROS was increased while the mitochondrial activity of in vitro grown matured oocyte was less than in vivo matured oocytes. In conclusion, follicle culture with O+ group appears to be superior to the culture in O- group in terms of follicle growth, development, oocyte growth, maturation, and microorganelles in oocyte.

Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue.

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Effect of treatment with angiopoietin-2 and vascular endothelial growth factor on the quality of xenografted bovine ovarian tissue in mice.

Cryopreservation and transplantation of ovarian tissue (OT) represents a method for fertility preservation. However, as the transplantation is performed without vessel anastomosis, unavoidable ischemic damage occurs. To reduce this ischemic damage and improve outcomes after transplantation, we used two kind of angiogenic factors, angiopoietin-2 (ang-2) and vascular endothelial growth factor (VEGF). Fresh or vitrified-warmed bovine OTs were prepared for xenotransplantation (XT). Fresh OTs were immediately xenografted into nude mice (XT-Fresh). Vitrified-warmed OTs were xenografted into four subgroups of mice, which were injected intraperitoneally before XT with saline (XT-Vitri), Ang-2 (XT-Ang-2), VEGF (XT-VEGF), and a combination of Ang-2 and VEGF (XT-Combined). Seven or 28 days post-grafting, grafted OTs and blood samples were collected for evaluation. Follicle normality was higher in the angiogenic factor-treated groups than in the XT-Vitri group. The XT-VEGF and the XT-Combined showed higher (P<0.05) follicular density than the XT-Vitri group. The highest apoptotic follicle ratio was observed in the XT-Vitri group on day 7; this was decreased (P<0.05) in the XT-Combined group. Microvessel densities were higher in the angiogenic factor-treated groups than in the XT-Vitri group. The largest fibrotic area was showed in the XT-Vitri group on day 28, and it was decreased (P<0.05) in the XT-combined group. Based on these results, administration of Ang-2 and VEGF to recipients prior to XT appeared to alleviate ischemic damage by enhancing angiogenesis, which resulted in the maintenance of follicle integrity and density, and reduced follicle apoptosis and OT fibrosis.

Effects of supplementation with antifreeze proteins on the follicular integrity of vitrified-warmed mouse ovaries: Comparison of two types of antifreeze proteins alone and in combination.

OBJECTIVE: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. METHODS: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. RESULTS: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. CONCLUSION: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

Effects of Angiopoietin-2 on Transplanted Mouse Ovarian Tissue.

Transplantation of ovarian tissue (OT) is currently the only clinical option to restore fertility with cryopreserved OT. However, follicle loss caused by ischemia and slow revascularization occurs in transplanted OT. To shorten the ischemic period and promote angiogenesis, some angiogenic factors have been used. Angiopoietin-2 (Ang2) is one of the major angiogenic factors and has been reported to promote blood vessels and increase vascular permeability in ischemic and/or hypoxic environment. This study was performed to investigate the effects of Ang2 on follicle integrity and revascularization of transplanted mouse OT. Five-week-old B6D2F1 female mice were divided into a control group and two Ang2 groups, followed by ovary collection and vitrification. After warming, the ovaries were autotransplanted into kidney capsules with/without Ang2 injection (50 or 500 ng/kg), and then the mice were sacrificed at days 2, 7, 21, and 42 after transplantation. A total 2,437 follicles in OT grafts were assessed for follicular density, integrity, and classification by using hematoxylin and eosin staining. Apoptosis and revascularization were evaluated by using TUNEL assay and CD31 immunohistochemistry, respectively. Serum follicle-stimulating hormone (FSH) levels were measured by using enzyme-linked immunosorbent assay. Both Ang2 groups showed remarkable increase in morphologically intact follicle ratio across all grafting durations except D21. The numbers of CD31(+) vessels were significantly increased in both Ang2 groups compared with the control group at all durations, except in the 50 ng Ang2 group at D42. However, the mean numbers of follicles of the grafts, apoptosis ratios, and serum FSH levels showed no significant differences among the groups. Our results show that Ang2 treatment significantly increased the intact follicle ratios and the number of blood vessels of the mouse OT grafts. However, further studies performed with large animal or human OT are necessary before clinical application for fertility preservation in cancer patients, and the reliability of the systemic effects of Ang2 should be verified.

Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury.

STUDY QUESTION: What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury? SUMMARY ANSWER: Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function. WHAT IS KNOWN ALREADY: During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve. STUDY DESIGN, SIZE, DURATION: A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days). PARTICIPANTS/MATERIALS, SETTING, METHODS: In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but 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 percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 ± 3.6, vitri-con: 20.6 ± 2.9, 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%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time. LIMITATIONS AND REASONS FOR CAUTION: It is unclear how far these results can be extrapolated from mice to the human ovary. WIDER IMPLICATIONS OF THE FINDINGS: Minimizing ischemic injury should be the first priority rather than preventing cryoinjury alone, and decreasing the combination of cryoinjury and ischemic injury is necessary to improve ovarian quality after cryopreservation and transplantation. STUDY FUNDING/COMPETING INTEREST: This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.

Effect of Exogenous Anti-Müllerian Hormone Treatment on Cryopreserved and Transplanted Mouse Ovaries.

Follicle loss occurs after ovary cryopreservation and transplantation. To preserve the follicle pool of cryopreserved or grafted ovaries, anti-Müllerian hormone (AMH), which inhibits ovarian follicle recruitment, was used in a mouse model. In experiment 1, ovaries were vitrified warmed with different doses of AMH (0, 5, 15, or 45 µg/mL) supplementation. In experiment 2, AMH (0, 50, 250, and 1250 µg/mL) was injected into mice before and/or after cryopreserved ovary autotransplantation, and the recipients remained for 7 or 28 days after grafting. Ovaries were evaluated by follicle morphology, density, and apoptosis ratio. Additionally, serum follicle-stimulating hormone was measured in experiment 2. Significantly decreased follicle apoptosis were detected in AMH-treated groups when compared to the control ovaries in experiment 1, meanwhile no positive effect of exogenous AMH was found in experiment 2. Thus, we suggest AMH supplementation during ovary vitrification warming has beneficial effect on reducing follicle apoptosis.

A combination of simvastatin and methylprednisolone improves the quality of vitrified-warmed ovarian tissue after auto-transplantation.

STUDY QUESTION: Does the preoperative administration of simvastatin and methylprednisolone enhance mouse ovarian quality after auto-transplantation of vitrified-warmed ovarian tissue (OT)? SUMMARY ANSWER: Treatment with combined simvastatin and methylprednisolone enhances the quality of transplanted mouse OTs. WHAT IS KNOWN ALREADY: The prevention of ischemic injury after transplantation of OT is critical for preserving the ovarian follicles. Preoperative administration of simvastatin (a cholesterol-lowering drug) has beneficial effects on various organ transplantations. Moreover, donor treatment with simvastatin and methylprednisolone (main effects are on immune response) prevents ischemia-reperfusion injury and has a beneficial effect on allograft survival in rat cardiac allografts. STUDY DESIGN, SIZE, DURATION: A total of 232 6-week-old B6D2F1 mice were randomly distributed into fresh control, vitrified-warmed control and experimental groups (n = 10-17 per group). The experimental groups were as follows: sham control, simvastatin, methylprednisolone and co-treatment groups. In the experimental groups, the mice were administered simvastatin (5 mg/kg, orally), methylprednisolone (15 mg/kg, i.v.) or a combination of simvastatin and methylprednisolone 2 h before ovariectomy, whereas the sham control mice received normal saline. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules. The ovaries and blood samples were collected 2, 7 and 21 days (D) after transplantation for histological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, immunohistochemistry for CD31 and serum anti-Mullerian hormone (AMH) level estimation. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovary. MAIN RESULTS AND THE ROLE OF CHANCE: The group that received simvastatin and methylprednisolone showed a significantly improved intact (Grade 1) 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 rates, number of total and apoptotic blastomeres per blastocyst and inner cell mass/trophectoderm ratio among the four transplantation groups. LIMITATIONS, REASONS FOR CAUTION: Although we evaluated the beneficial effects of simvastatin and methylprednisolone in the present study, we did not unravel the corresponding protective mechanisms. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that a combination of simvastatin and methylprednisolone has beneficial effects on the quality and functioning of transplanted OT. This combined treatment can potentially be applied clinically to humans and domestic animals subject to further studies.