Hye Won Youm

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival.

STUDY QUESTION What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? SUMMARY ANSWER The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. WHAT IS KNOWN ALREADY Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. STUDY DESIGN, SIZE, DURATION A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. PARTICIPANTS/MATERIALS, SETTING, METHODS The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. MAIN RESULTS AND THE ROLE OF CHANCE The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. LIMITATIONS, REASONS FOR CAUTION The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. WIDER IMPLICATIONS OF THE FINDINGS We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.

Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation.

Background Ovarian tissue (OT) cryopreservation is effective in preserving fertility in cancer patients who have concerns about fertility loss due to cancer treatment. However, the damage incurred at different steps during the cryopreservation procedure may cause follicular depletion; hence, preventing chilling injury would help maintain ovarian function. Objective This study was designed to investigate the beneficial effects of different antifreeze proteins (AFPs) on mouse ovarian tissue cryopreservation and transplantation. Methodology Ovaries were obtained from 5-week-old B6D2F1 mice, and each ovary was cryopreserved using two-step vitrification and four-step warming procedures. In Experiment I, ovaries were randomly allocated into fresh, vitrification control, and nine experimental groups according to the AFP type (FfIBP, LeIBP, type III) and concentration (0.1, 1, 10 mg/mL) used. After vitrification and warming, 5,790 ovarian follicles were evaluated using histology and TUNEL assays, and immunofluorescence for τH2AX and Rad51 was used to detect DNA double-strand breaks (DSBs) and repair (DDR), respectively. In Experiment II, 20 mice were randomly divided into two groups: one where the vitrification and warming media were supplemented with 10 mg/mL LeIBP, and the other where media alone were used (control). Ovaries were then autotransplanted under both kidney capsules 7 days after vitrification together with the addition of 10 mg/mL LeIBP in the vitrification-warming media. After transplantation, the ovarian follicles, the percentage of apoptotic follicles, the extent of the CD31-positive area, and the serum FSH levels of the transplanted groups were compared. Principal Findings In Experiment I, the percentage of total grade 1 follicles was significantly higher in the 10 mg/mL LeIBP group than in the vitrification control, while all AFP-treated groups had significantly improved grade 1 primordial follicle numbers compared with those of the vitrification control. The number of apoptotic (TUNEL-positive) follicles was significantly decreased in the groups treated with 1 and 10 mg/mL LeIBP. The proportion of τH2AX-positive follicles was significantly reduced in all AFP-treated groups, while the proportion of Rad51-positive follicles was significantly decreased in only the FfIBP- and LeIBP-treated groups. In Experiment II, after autotransplantation of OT vitrified with 10 mg/mL of LeIBP, the percentage of total grade 1 and primordial grade 1 follicles, and the extent of the CD31-positive area, were increased significantly. Moreover, the levels of serum FSH and the percentage of TUNEL-positive follicles were significantly lower in the LeIBP-treated than in the control group. Conclusion A supplementation with high concentrations of AFPs had protective effects on follicle preservation during OT vitrification-warming procedures. The group treated with LeIBP was protected most effectively. The beneficial effects of LeIBP were also observed after autotransplantation of vitrified-warmed OT. Further studies are necessary to determine the exact mechanism of these protective effects.

Effect of Antifreeze Protein on Mouse Ovarian Tissue Cryopreservation and Transplantation

Purpose To investigate the effect of antifreeze protein (AFP) supplementation on ovarian vitrification and transplantation. Materials and Methods In this experimental study, we researched a total of 182 ovaries from 4-week-old ICR mice. The equilibration solution included 20% ethylene glycol (EG), and the vitrification solution included 40% EG, 18% Ficoll, and 0.3 M sucrose. Intact ovaries were first suspended in 1 mL of equilibration solution for 10 min, and then mixed with 0.5 mL of vitrification solution for 5 min. Ovaries were randomly assigned to 3 groups and 0, 5, or 20 mg/mL of type III AFP was added into the vitrification solution (control, AFP5, and AFP20 groups, respectively). The vitrified ovaries were evaluated after warming and 2 weeks after autotransplantation. The main outcome measurements are follicular morphology and apoptosis assessed by histology and the TUNEL assay. Results A significantly higher intact follicle ratio was shown in the AFP treated groups (control, 28.9%; AFP5, 42.3%; and AFP20, 44.7%). The rate of apoptotic follicles was significantly lower in the AFP treated groups (control, 26.6%; AFP5, 18.7%; and AFP20, 12.6%). After transplantation of the vitrified-warmed ovaries, a significantly higher intact follicle ratio was shown in the AFP20 group. The rate of apoptotic follicles was similar among the groups. Conclusion The results of the present study suggest that supplementing AFP in the vitrification solution has beneficial effects on the survival of ovarian tissue during cryopreservation and transplantation.

Transplantation of mouse ovarian tissue: comparison of the transplantation sites.

Many studies have shown that ischemic injuries during the transplantation process were more detrimental than cryoinjuries for follicle survival and death, and it has been reported that transplantation sites can affect the outcomes of grafted ovarian tissue (OT). The purpose of this study was to assess the impact of different OT transplantation sites on follicular integrity and function of OT grafts. B6D2F1 mice were randomly assigned to control (sham) and four experimental groups according to transplantation sites (back muscle [BM], fat pad [FP], kidney capsule [KC], and subcutaneous [SC]). The ovaries from four groups were autotransplanted to each site. The OT recovery ratios on Days 2, 7, and 21 were significantly decreased in the FP group. The mean numbers of follicles were significantly lower in all the grafting groups compared with the sham group, except in the KC group on Days 7 and 21 and the BM group on Day 21. On Day 2, all the experimental groups showed low intact (G1) follicle ratio when compared with the sham group; however, the BM, KC, and FP groups recovered their morphologic integrity on Day 7, and only the SC group presented a significant decrease in G1 follicle ratios. On Day 21, the G1 follicle ratios of the FP and KC groups were greater than the sham control group. The proportion of apoptotic follicles of the four OT graft groups was higher than in the sham group on Day 2, followed by a significant decrease in the KC group and an increase in the SC group on Day 7. The serum follicle-stimulating hormone levels were significantly increased in all grafting groups on Day 2. On Day 7, only the SC group showed the high follicle-stimulating hormone level compared with the other groups. The mean numbers of oocytes from OT grafts were the highest in the KC group, except in the control group, and the lowest in the SC group. The ratios of mature oocytes were also significantly greater in the sham and KC groups. However, the ratios of normal spindle did not differ among the five groups. In conclusion, the KC was the optimal site for OT transplantation in this murine model, whereas the SC site was unfavorable for this procedure. In this study, we confirmed that the different grafting sites influenced the outcomes of transplantation.

Effect of preoperative simvastatin treatment on transplantation of cryopreserved-warmed mouse ovarian tissue quality

After the ovarian tissue (OT) transplantation, the ischemia-reperfusion injury causes depletion and apoptosis of follicle. Recent reports stated that simvastatin reduces ischemic damage. Therefore, we used the mouse whole ovarian vitrification and autotransplantation models to investigate the effects of simvastatin. Five-week-old B6D2F1 mice were randomly divided into four groups. Three groups were given simvastatin orally (5 mg/kg) before ovariectomy, either 2 hours before (2H Tx) or once a day for 3 or 7 days. The control group was given saline 2 hours before ovariectomy. All ovaries were cryopreserved by vitrification, held in liquid nitrogen for 1 week before being warmed, and autotransplanted. The grafts were collected for analysis on 2, 7, or 21 days after transplantation. Ovarian follicle morphology and apoptosis were assessed by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Vessel integrity in ovary was evaluated by immunohistochemistry using anti-CD31 antibody. Serum FSH level was measured to estimate the transplanted ovarian reserve. The proportion of morphologically normal (G1) follicles at 7 and 21 days and the percentage of CD31 (+) tissue at 21 days was significantly higher in the 2H Tx group than that in the control group. In addition, the 2H Tx group showed a significantly increased intact primordial follicle ratio at 2 and 21 days after OT transplantation. Administration of simvastatin 2 hours before ovariectomy could improve the quality after transplantation of cryopreserved mouse OT.

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.

Effect of necrostatin on mouse ovarian cryopreservation and transplantation

OBJECTIVE To investigate the effects of necrostatin-1 (Nec-1) supplementation on vitrification, warming and transplantation of ovarian tissue. STUDY DESIGN Ovaries from 4-week-old ICR mice were vitrified using a two-step procedure; ovaries were suspended in equilibration solution for 10min, and then mixed with vitrification solution for 5min. Ovaries were divided at random into three groups and 0 (control), 25 or 100μM Nec-1 was added to the vitrification solution. After warming, follicular morphology and apoptosis were assessed. For each group, a sample of vitrified, warmed ovaries was autotransplanted. The same dose of Nec-1 that was added to the vitrification solution was added to each warming solution and injected intraperitoneally. Follicular morphology and apoptosis of transplanted ovaries were assessed after 2 weeks. RESULTS After vitrification and warming, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1-treated groups compared with the control group (control, 45.1%; 25μM Nec-1, 51.7%; 100μM Nec-1, 57.9%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 11.2%; 25μM Nec-1, 8.5%; 100μM Nec-1, 7.2%). After transplantation of the vitrified, warmed ovaries, morphological analysis revealed that the intact follicle ratio was significantly higher in the Nec-1 treated groups compared with the control group (control, 43.1%; 25μM Nec-1, 60.6%; 100μM Nec-1, 70.7%). The rate of apoptosis was lower in the Nec-1 treated groups compared with the control group (control, 5.3%; 25μM Nec-1, 2.5%; 100μM Nec-1, 2.0%). CONCLUSIONS Nec-1 supplementation during vitrification, warming and transplantation has beneficial effects on the survival of ovarian tissue. These results can help to improve ovarian tissue vitrification and transplantation protocols for fertility preservation.

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.

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.