Abstract
Purpose
The purpose of this study was to investigate whether slow‐rate freezing or vitrification is better for cryopreservation of ovary tissues pretreated with gonadotropin‐releasing hormone agonist.
Methods
In this nonclinical study performed in rats, leuprorelin acetate was administered to female Wistar rats, aged 6–8 weeks. After confirming arrest of the estrous cycle by examination of vaginal smears, ovarian tissue was cryopreserved by vitrification and slow‐rate freezing prior to thawing and autotransplantation. The time required for estrous cycle recovery was assessed from vaginal smears in each group starting from day 1 of transplantation. Estradiol levels were also monitored after transplantation.
Results
The estrous cycle recovered after transplantation of ovarian tissue frozen by either method, but recovery was significantly faster after transplantation of vitrified tissue. The estradiol level also recovered by 10 days after transplantation.
Conclusions
Ovarian function was restored after transplantation of tissue preserved by either vitrification or slow‐rate freezing after pretreatment with leuprorelin acetate. This method may be applicable for patients scheduled to undergo cryopreservation of ovarian tissue before chemotherapy.
Keywords: Fertility preservation, GnRH analogue, Rat, Slow freezing, Vitrification
Introduction
In recent years, the incidence of breast cancer and ovarian cancer among young women has been increasing. In 2006, the United States recorded a total of 1,399,790 new cancer patients over the previous year, and approximately half of them (48.6%, 679,540) were women [1]. About 8% of the female cancer patients were less than 40 years old [2]. Cancer is treated by surgery, chemotherapy, and/or radiation therapy. However, chemotherapy and radiation therapy also affect normal cells, and the quality of life (QOL) of young women with cancer can be markedly impaired by loss of fertility due to ovarian failure induced by anticancer therapy. The cytotoxic effects of anticancer agents are reversible in the case of the bone marrow or digestive tract mucosa due to the high regenerative capacity of these tissues, but they are often irreversible in the ovaries. Ovarian failure induces oligomenorrhea, amenorrhea, and anovulatory menstruation, which is known as chemotherapy‐related amenorrhea, and it has an incidence of 20–100% [3]. Preservation of ovarian function after chemotherapy in young female cancer patients is not only required for preservation of fertility, but also for maintenance of QOL in general. Development of methods for removal of eggs or ovaries from patients before the start of chemotherapy for cryopreservation is being targeted by both basic and clinical studies worldwide [2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13].
In 2004, Donnez et al. [8] reported a successful delivery after cryopreservation of resected ovarian tissue in a patient with non‐Hodgkin's lymphoma, and Meirow et al. [11] also reported successful delivery by a patient with non‐Hodgkin's lymphoma. Many problems still need to be solved with regard to ovarian cryopreservation, including ethical issues, patient selection, and elimination of cancer cells from the transplanted tissue, but achieving pregnancy and delivery is now technically possible. It has also been reported that concomitant administration of a gonadotropin‐releasing hormone agonist (GnRHa) with chemotherapy reduces blood flow to the ovaries and protects these organs against the toxicity of anticancer agents [14, 15, 16, 17]. Matsuo et al. compared 6‐week‐old rats receiving a platinum agent alone or combined with a GnRHa, and they found that loss of follicles was inhibited in the GnRHa group. Toxicity of platinum agents has been reported to preferentially affect mature follicles [16].
In preparing to conduct the present study, we took into account the concept of preemptive administration of GnRHa before treatment with anti‐cancer agents in young women with cancer, with the aim of preserving ovarian function. Thus, in this study, rat ovaries were cryopreserved with two different freezing methods, slow‐rate freezing and vitrification, to investigate whether it was possible to cryopreserve ovaries that had been pretreated in the same way as ovaries that had not been pretreated and whether the damage from freezing could be inhibited. These ovaries were then thawed and autografted, after which ovarian function recovery was compared and investigated.
Materials and methods
Animals
Female Wistar rats (SLC Japan, Shizuoka, Japan), aged 6–8 weeks, were housed in the Institute of Experimental Animals at St. Marianna University School of Medicine in a room controlled at a constant temperature (23 ± 1°C) and humidity (55 ± 5%) with daytime lighting (0600–1800 hours). They were provided with commercially available animal feed and tap water ad libitum. After intraperitoneal injection of 40 mg/kg of pentobarbital sodium (Nembutal Injection; Dainippon Pharmaceutical, Osaka, Japan), the ovaries were resected and placed in Medium 199 solution (Gibco, Grand Island, NY). Thin slices (5 mm) of the ovaries were prepared under a stereomicroscope and assigned to a non‐frozen group (n = 6), a vitrification group (n = 3), and a slow‐rate freezing group (n = 4). Fresh or thawed ovarian slices in semen straws (Fujihira Industry, Tokyo, Japan) were grafted subfascially into the backs of the rats. Experiments were performed after obtaining the approval of the Experimental Animal Committee of the Institute of Experimental Animals.
Vitrification method
The resected ovaries were immersed in tissue preservative at 37°C, extraneous tissue was removed, and thin slices, about 5 mm thick, were cut under a stereomicroscope. These ovarian slices were soaked in buffer at 37°C for 30 min and then placed in vitrification fluid at 4°C. Next, the tissue slices were sealed in semen straws (Fujihira Industry), left for 5 min on ice, and placed in liquid nitrogen for cryopreservation. For thawing, the semen straws were removed from the liquid nitrogen and transferred to a water bath at 35°C. Then, the straws were left to stand for 3 min in diluent at 37°C, washed twice with washing liquid, and placed in tissue preservative at 37°C until grafting. The composition of the solutions used was as follows. The buffer was Medium 199 with 4% (vv) ethylene glycol (Sigma, St. Louis, MO) and 15% (v/v) serum substitute supplement (SSS; IS Japan, Saitama, Japan). Vitrification fluid was Medium 199 with 35% (vv) ethylene glycol, 5% (w/v) polyvinylpyrrolidone (Sigma), 0.4 M sucrose (Wako Pure Chemical Industries, Osaka, Japan), and 20% (v/v) SSS. The diluent was Medium 199 with 0.3 M sucrose and 20% (v/v) SSS, the washing liquid was Medium 199 with 20% (v/v) SSS, and the tissue preservative was Medium 199 with 0.1% polyvinylpyrrolidone (Sigma).
Slow‐rate freezing method
The resected ovaries were immersed in phosphate‐buffered saline (PBS; Wako Pure Chemical Industries) with 20% (v/v) SSS at 37°C, and extraneous tissue was removed under a stereomicroscope. Then, the tissues were transferred to PBS at 37°C containing 1.5 M propanediol (Sigma) and 20% (v/v) SSS. After soaking for 15 min, the tissues were transferred to PBS at 37°C containing 1.5 M propanediol, 0.1 M sucrose, and 20% (v/v) SSS, and soaked for another 15 min. Next, the thin sections of the ovaries were sealed in semen straws and placed in a programmed freezer (Asahi Life Science, Saitama, Japan). The program consisted of cooling from room temperature to −7°C at 1.0°C/min, cooling after ice crystal seeding to −30°C at 0.3°C/min, and then cooling to −180°C at 50°C/min. After freezing, the straws were placed in liquid nitrogen for cryopreservation. For thawing, the straws were removed from the liquid nitrogen and thawed in a vacuum. Then, the straws were placed in PBS at 37°C containing 1.0 M propanediol, 0.1 M sucrose, and 20% (v/v) SSS, and left to stand for 5 min. Subsequently, the straws were transferred to PBS at 37°C containing 0.5 M propanediol, 0.1 M sucrose, and 20% (v/v) SSS, and left to stand for another 5 min, followed by transfer to PBS at 37°C containing 0.1 M sucrose and 20% (v/v) SSS. Finally, the straws were placed in Medium 199 until grafting.
Drugs
For GnRHa therapy, leuprorelin acetate (Leuplin; Takeda Pharmaceutical, Tokyo, Japan) was injected subcutaneously into 6‐week‐old female rats at a dose of 3.75 mg/kg. Then, it was confirmed that the estrous cycle did not recover over a 2‐week period. The serum estradiol level was measured by enzyme‐linked immunosorbent assay (Neogen, Lexington, KY) on days 10, 20, and 30 after grafting of the ovarian tissue.
Ultrastructural examination
After thawing, ovarian tissues preserved by vitrification and slow‐rate freezing were prepared for ultrastructural examination. Fresh ovaries were used as a control. The tissue specimens were fixed in 2% glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 4°C, and then fixed in 1% glutaraldehyde in 0.1 M PBS for 1–7 days at 4°C. After being rinsed in PBS, the tissues were post‐fixed with 1% osmium tetroxide in PBS for 2 h at 4°C, dehydrated in an ethanol series for 2 h, immersed in propylene oxide overnight for solvent substitution, and embedded in Epon. Semi‐thin sections were cut and stained with 0.1% toluidine blue, while ultra‐thin sections (80 nm) were stained with aqueous lead citrate and alcoholic uranyl acetate. Examination and photography were done with a transmission electron microscope (JEM‐1220; JEOL, Tokyo, Japan).
Assessing recovery of the estrous cycle after different cryopreservation methods
Three groups of rats were investigated: one group underwent autografting of ovarian tissue slices on the same day (non‐frozen group, n = 6), the second group underwent autografting of tissue frozen by vitrification and preserved for 1 week in liquid nitrogen (vitrification group, n = 4), and the third group received tissue that had been preserved after slow‐rate freezing (slow‐rate freezing group, n = 7). The number of days from postoperative day 1 until recovery of the estrous cycle was investigated in each group by assessing vaginal smears. Blood samples were obtained from the vitrification and slow‐rate freezing groups on days 10 and 20 after grafting to measure estradiol levels, and the results were compared with the t test.
Assessing recovery of the estrous cycle after different cryopreservation methods with GnRHa pretreatment
Leuprorelin acetate (3.75 mg/kg) was administered subcutaneously to female Wistar rats aged 6–8 weeks, and cessation of the estrous cycle was confirmed by examining vaginal smears. Then, a non‐frozen group (n = 7), a vitrification group (n = 6), and a slow‐rate freezing group (n = 5) were formed as in the above‐mentioned experiment. Blood samples were obtained from the vitrification and slow‐rate freezing groups on days 10, 20, and 30 after grafting to measure estradiol levels, and data were compared with the t test.
Statistics
Student's t test was used for comparisons of the three groups. In all analyses, P < 0.05 was considered to indicate statistical significance.
Results
Recovery of the estrous cycle after different cryopreservation methods without GnRHa pretreatment
The estrous cycle recovered in all three groups. The mean number of days until recovery was 10.7 ± 5.5 days in the non‐frozen group (n = 6), 22.8 ± 5.1 days in the vitrification group (n = 4), and 15.1 ± 2.3 days in the slow‐rate freezing group (n = 7). Significant differences were found between the non‐frozen and vitrification groups, as well as between the vitrification and slow‐rate freezing groups (Fig. 1A‐1).
Figure 1.
Recovery of the estrous cycle and changes in estradiol after grafting of cryopreserved ovarian tissue with or without GnRHa treatment. A‐1 Mean number of days to recovery of the estrous cycle in rats. The estrous cycle recovered in all three groups. The mean number of days until recovery was 10.7 ± 5.5 days in the non‐frozen group (n = 6), 22.8 ± 5.1 days in the vitrification group (n = 4), and 15.1 ± 2.3 days in the slow‐rate freezing group (n = 7). Significant differences were found between the non‐frozen and vitrification groups, and between the vitrification and slow‐rate freezing groups (P < 0.05). A‐2 Mean number of days until recovery of the estrous cycle in rats with GnRHa. The estrous cycle recovered in all three groups. The mean number of days until recovery was 34 ± 2.0 days in the non‐frozen group (n = 7), 27.3 ± 0.5 days in the vitrification group (n = 6), and 48 ± 13.4 days in the slow‐rate freezing group (n = 7). There was a significant difference between the vitrification and slow‐rate freezing groups (P < 0.05). B‐1 Changes in estradiol levels after cryopreservation. The mean estradiol levels after grafting in the vitrification and slow‐rate freezing groups were 22.0 ± 9.2 and 55.5 ± 27.9 pg/ml, respectively, on day 10, and 57.0 ± 26.8 and 43.1 ± 26.3 pg/ml, respectively, on day 20; there were no significant differences between the two groups. B‐2 Changes in estradiol levels after cryopreservation with GnRHa. The mean estradiol levels after grafting in the vitrification and slow‐rate freezing groups were: 44.9 ± 23.7 and 31.7 ± 21.9 pg/ml, respectively, on day 10; 22.6 ± 0.9 and 29.4 ± 9.9 pg/ml, respectively, on day 20; and 38.2 ± 10.1 and 38.4 ± 6.9 pg/ml, respectively, on day 30. No significant differences were observed between the groups
The mean estradiol level on day 10 after grafting was 22.0 ± 9.2 pg/ml in the vitrification group and 55.5 ± 27.9 pg/ml in the slow‐rate freezing group. On day 20 after grafting, these values were 57.0 ± 26.8 and 43.1 ± 26.3 pg/ml, respectively. No significant differences were observed between the two groups (Fig. 1B‐1).
Recovery of the estrous cycle after different cryopreservation methods with GnRHa pretreatment
The estrous cycle recovered in all three groups. The mean number of days until recovery was 34 ± 2.0 days in the non‐frozen group (n = 7), 27.3 ± 0.5 days in the vitrification group (n = 6), and 48 ± 13.4 days in the slow‐rate freezing group (n = 5). A significant difference was found between the vitrification and slow‐rate freezing groups (Fig. 1A‐2).
The mean estradiol level on day 10 after grafting was 44.9 ± 23.7 pg/ml in the vitrification group and 31.7 ± 21.9 pg/ml in the slow‐rate freezing group. On day 20 after grafting, the values were 22.6 ± 0.9 and 29.4 ± 9.9 pg/ml, respectively, and on day 30, the values were 38.2 ± 10.1 and 38.4 ± 6.9 pg/ml, respectively. No significant differences were observed between the two groups (Fig. 1B‐2). In rats pretreated with GnRHa, the estrous cycle was confirmed even 120 days after grafting.
Electron microscopic observation
After preservation by the vitrification and slow‐rate freezing methods, thawed ovarian tissues from rats with GnRHa pretreatment were evaluated by electron microscopy. Fresh ovaries of rats with cessation of the estrous cycle after GnRHa treatment were used as controls. Oocytes in the control ovarian tissues formed a monolayer surrounded by squamous cells. The organelles of these oocytes were clustered in the cytoplasm (Fig. 2A‐1). There was a membrane between the squamous pre‐granulosa cells and the oocytes, and there was close contact (Fig. 2A‐2). Many rod‐shaped mitochondria and Golgi bodies were present in the cytoplasm of the oocytes (Fig. 2A‐3, A‐4). After preservation by the vitrification method and the slow‐rate freezing method, oocytes also formed monolayers surrounded by squamous cells, and their organelles were clustered in the cytoplasm. A membrane was also present between the squamous pre‐granulosa cells and oocytes, and there was close contact (Fig. 2B‐1, B‐2, C‐1, C‐2). There was more marked vacuolization of the mitochondria in oocytes preserved by the slow‐rate freezing method compared with the vitrification method (Fig. 2B‐3, B‐4, C‐3, C‐4).
Figure 2.
Electron microscopy of ovaries cryopreserved and thawed by each method with GnRHa pretreatment. A: control‐1 Oocytes in control ovarian tissue form a monolayer surrounded by squamous cells. The organelles are clustered in the cytoplasm of the oocytes. A‐2 A membrane is present between the squamous pre‐granulosa cells and oocytes, and there is close contact. A‐3 and A‐4 Many rod‐shaped mitochondria and Golgi bodies are seen in the cytoplasm of the oocytes. B: vitrification‐1, B‐2, C: slow‐rate freezing‐1, and C‐2 Ovarian tissue cryopreserved by the vitrification and slow‐rate freezing methods. In both cases, oocytes form a monolayer surrounded by squamous cells. Organelles are clustered in the cytoplasm of the oocytes. B‐3, B‐4, C‐3, and C‐4 There is a membrane between the squamous pre‐granulosa cells and oocytes, and there is close contact. There is more severe vacuolization of the mitochondria with the slow‐rate freezing method than with the vitrification method
Discussion
This study demonstrated that the functionality of grafted ovarian tissue was maintained, because there was recovery of the estrous cycle in all groups of rats. Although the estrous cycle recovered most rapidly in the non‐frozen group, it also recovered in the groups that received transplantation of ovarian tissue after cryopreservation. There was a difference in the time until recovery of the estrous cycle between the vitrification and slow‐rate freezing groups, as well as between the non‐frozen group and the vitrification group, suggesting that slow‐rate freezing causes less damage to the follicles than vitrification. Comparison of estradiol levels showed no significant differences on day 10 after grafting, but the estradiol level of the slow‐rate freezing group was higher. On day 20, the estradiol level of the vitrification group showed an increase, but the level of the slow‐rate freezing group increased from before day 10, which also supported the idea that less damage to the follicles was caused by the slow‐rate freezing method compared with the vitrification method. Wang et al. [18] studied ovarian tissue after cryopreservation/thawing by the slow‐rate freezing method or the vitrification method, and they reported that the percentage of primordial follicles, primary follicles, and secondary follicles maintaining a normal morphology showed no significant differences between the two methods. Given such reports, histological observations of the frozen/thawed tissue were not performed with each freezing method. Nonetheless, though tissue may have normal morphology histologically, this does not necessarily mean that it will also have normal function. Without having the rats become pregnant and give birth after thawing and grafting ovaries that have been frozen with each method, one cannot say with complete assurance that function has been preserved. However, we did not go that far in this study.
With the slow‐rate freezing method, ice crystals are formed extracellularly from seed crystals during slow cooling, while formation of intracellular ice crystals is inhibited by dehydration of the cells, concentration of the extracellular fluid, and depression of the freezing point. As dehydration progresses, however, freezing becomes more rapid, and cellular damage occurs. Unlike the slow‐rate freezing method, the vitrification method does not require strict control of the freezing rate. Instead, very rapid freezing is achieved in the presence of high concentrations of protective agents after dehydration of cells in the protective solution and intracellular penetration of the protective agents. When water is frozen very rapidly or frozen with a high solute concentration, the formation of ice crystals is inhibited. With the vitrification method, ice crystals are not formed either inside or outside the cells, and the water is frozen into a glass‐like solid. When dehydration and penetration of the protective agents are insufficient, however, damage can occur due to ice crystals, and the toxicity of the protective agents at the high concentrations required to suppress ice crystal formation is also a problem. In comparison with the cryopreservation of single cells such as embryos, the cryopreservation of ovarian tissue is much more difficult because it consists of various sizes of cells, and the ovaries are organs of some size in which it is difficult to achieve uniform concentrations of the protective agents. In this study, sooner recovery of the estrous cycle was observed after transplantation of ovarian tissue with cryopreservation by the slow‐rate freezing method than by the vitrification method. However, no severe damage to primordial follicles was found in cryopreserved ovarian tissues by both methods, suggesting that the slow‐rate method induced less damage to more mature follicles.
It has been reported that live birth was achieved by patients with Hodgkin's disease who had their ovaries removed, cryopreserved, grafted after chemotherapy, and then stimulated with superovulation [19]. The protective effect of GnRHa treatment against damage to reproductive function by chemotherapy has also been reported, suggesting that it would be possible to administer a GnRHa prior to or in parallel with chemotherapy before cryopreservation of ovarian tissue to avoid ovarian failure in young women with cancer. If cryopreservation can be performed after GnRHa treatment in the same way as for ovarian tissue of untreated patients, and damage due to cryopreservation can be inhibited, this should result in stronger protection of ovarian function. Therefore, we administered GnRHa and assessed recovery of the estrous cycle after transplantation of ovarian tissue preserved by the slow‐rate freezing method or the vitrification method compared with control fresh tissue. In the present study, the function of ovarian tissue grafted after GnRHa pretreatment was maintained in the same way as that of untreated ovarian tissue, but the mean number of days to recovery of the estrous cycle was significantly shorter in the vitrification group. When vitrification was done with or without GnRHa pretreatment, there was no significant difference of the mean time to recovery of the estrous cycle, while recovery was significantly slower after slow‐rate freezing with GnRHa pretreatment than without GnRHa pretreatment. GnRHa treatment inhibits follicular development and decreases large follicles in the ovaries [16]. Thus, when cryopreservation was performed by the vitrification method using ovarian tissue without large follicles due to GnRHa administration, effective inhibition of damage was achieved by appropriate concentrations of protective agents for the uniform small follicles. With slow‐rate freezing, however, small follicles were still damaged (as was the case without GnRHa), and recovery of estrous was slower. In addition, although no significant difference was seen in estradiol levels by number of days after grafting, in cases when GnRHa was not administered, higher estradiol levels were seen on the 10th day after grafting in rats with slow‐rate freezing than in those with vitrification, and a recovery trend was seen. On the 20th day after grafting, recovery was seen in estradiol levels in the vitrification group as well. Conversely, when GnRHa was administered, rats in the vitrification group showed higher estradiol levels on day 10 after grafting, and although there was a temporary decline on day 20, high levels were seen on day 30. In the slow‐rate freezing group, estradiol levels had not sufficiently recovered on days 10 and 20 after grafting, but high levels were finally seen on day 30. These changes in estradiol levels indicate that, since small follicles were damaged in the slow‐rate freezing group, the estradiol levels recovered soon after grafting when GnRHa was not administered. When GnRHa was administered, estradiol levels were slow to recover because of the large number of small follicles. In the vitrification group, more time was needed for the follicles to grow, since large follicles were damaged, and it took time for hormone levels to recover after grafting when GnRHa was not administered. When GnRHa was administered, there were fewer large follicles to be damaged, and so it is thought that recovery of estradiol levels occurred sooner. However, in the future, it will be necessary to conduct further studies with each combination of slow‐rate freezing and vitrification with and without administration of GnRHa to investigate the stage at which follicles are damaged or whether they are preserved. It will also be necessary to investigate how long the hormone cycle will continue when estradiol levels recover—in other words, the level of remaining ovarian function. On evaluation by electron microscopy after GnRHa pretreatment, the morphology of oocytes and granulosa cells was maintained with both cryopreservation methods, but more marked morphological abnormalities of the cellular organelles were found with the slow‐rate freezing method. Therefore, if ovaries are cryopreserved after GnRHa pretreatment, it seems possible that the vitrification method could be superior to the slow‐rate freezing method.
In conclusion, it was possible to maintain ovarian function by cryopreservation of ovarian tissue with both the vitrification method and the slow‐rate freezing method after GnRHa administration, suggesting the possible clinical use of this procedure before starting chemotherapy in women of reproductive age.
Acknowledgments
This research was supported by a grant for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 20591933) (NS).
References
- 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C et al. Cancer statistics. CA Cancer J Clin, 2006, 56, 106–130 10.3322/canjclin.56.2.106 [DOI] [PubMed] [Google Scholar]
- 2. Donnez J, Martinez‐Madrid B, Jadoul P, Langendonckt AV, Demylle D, Dolmans M. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update, 2006, 12, 519–535 10.1093/humupd/dml032 [DOI] [PubMed] [Google Scholar]
- 3. Bines J, Oleske M, Cobleigh MA. Ovarian function in premenopausal women treated with adjuvant chemotherapy for breast cancer. J Clin Oncol, 1996, 14, 1718–1729 [DOI] [PubMed] [Google Scholar]
- 4. Kagabu S, Makita T, Mamba K, Ishida T. Transplantation of rat ovary into ovarian bursa after freezing and thawing. J Mamm Ova Res, 1988, 5, 89–97 [Google Scholar]
- 5. Parrot DMV. The fertility of mice with orthotopic ovarian grafts derived from frozen tissue. J Reprod Fertil, 1960, 1, 230–241 10.1530/jrf.0.0010230 [DOI] [PubMed] [Google Scholar]
- 6. Salha O, Picton H, Balen A, Rutherford A. Cryopreservation of human ovarian tissue. Hosp Med, 2001, 62, 222–227 [DOI] [PubMed] [Google Scholar]
- 7. Callejo J, Vilaseca S, Ordi J, Cabre S, Lailla JM, Balasch J. Heterotopic ovarian transplantation without vascular pedicle in syngeneic Lewis rats: long‐term evaluation of effects on ovarian structure and function. Fertil Steril, 2002, 77, 396–402 10.1016/S0015‐0282(01)02970‐3 [DOI] [PubMed] [Google Scholar]
- 8. Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet, 2004, 346, 1405–1410 10.1016/S0140‐6736(04)17222‐X [DOI] [PubMed] [Google Scholar]
- 9. Arav A, Revel A, Nathan Y, Bor A, Gactitua H, Yavin S et al. Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary. Hum Reprod, 2005, 20, 3554–3559 10.1093/humrep/dei278 [DOI] [PubMed] [Google Scholar]
- 10. Yeoman RR, Wolf DP, Lee DM. Coculture of monkey ovarian tissue increases survival after vitrification and slow‐rate freezing. Fertil Steril, 2005, 83, 1248–1254 10.1016/j.fertnstert.2004.11.036 [DOI] [PubMed] [Google Scholar]
- 11. Meirow D, Levron J, Elder‐Geva T, Hardan I, Fridman E, Zalel Y et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med, 2005, 353, 318–321 10.1056/NEJMc055237 [DOI] [PubMed] [Google Scholar]
- 12. Maltaris T, Dragonas C, Hoffmann I, Mueller A, Beckmann MW, Dittrich R. Simple prediction of the survival of follicles in cryopreserved human ovarian tissue. J Reprod Dev, 2006, 52, 577–582 10.1262/jrd.18012 [DOI] [PubMed] [Google Scholar]
- 13. Ino M, Saito J, Taniuchi A, Takahashi N, Tsuda C, Suzuki N et al. Cryopreservation and heterotopic autotransplantation of ovarian tissue in mouse. St Marianna Med J, 2007, 35, 177–189 [Google Scholar]
- 14. Ataya K, Rao LV, Lawrence E, Kimmel R. Luteinizing hormone‐releasing hormone agonist inhibits cyclophosphamide‐induced ovarian follicular depletion in rhesus monkeys. Bio Reprod, 1995, 52, 365–372 10.1095/biolreprod52.2.365 [DOI] [PubMed] [Google Scholar]
- 15. Asaga S, Ando J, Arai T, Fujii H. Adjuvant chemotherapy of premenopausal breast cancer with LH‐RH analogue for ovarian protection—a case report. Jpn J Cancer Chemother, 2005, 32, 1069–1072 [PubMed] [Google Scholar]
- 16. Matuo G, Usijima K, Shinagawa A, Takahashi S, Fujiyoshi N, Takemoto S et al. GnRH agonist acts as ovarian protection in chemotherapy induced gonadotoxicity: an experiment using a rat model. Kurume Med J, 2007, 54, 25–29 10.2739/kurumemedj.54.25 [DOI] [PubMed] [Google Scholar]
- 17. Blumenfeld Z. How to preserve fertility in young women exposed to chemotherapy? The role of GnRH agonist cotreatment in addition to cryopreservation of embryos, oocytes, or ovaries. Oncologist, 2007, 12, 1044–1054 10.1634/theoncologist.12‐9‐1044 [DOI] [PubMed] [Google Scholar]
- 18. Wang Y, Xiao Z, Li L, Fan W, Li S. Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Hum Reprod, 2008, 23, 2256–2265 10.1093/humrep/den255 [DOI] [PubMed] [Google Scholar]
- 19. Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease. Oncologist, 2007, 12, 1437–1442 10.1634/theoncologist.12‐12‐1437 [DOI] [PubMed] [Google Scholar]