Fresh Ovarian Tissue Research Articles

Effects of supra-zero storage on human ovarian cortex prior to vitrification–warming

This study investigated the effects of supra-zero storage on ovarian cortex of 12 premenopausal patients before cryopreservation. Fresh ovarian tissue (control) was either vitrified immediately or stored at 4°C for 24 h or 48 h before vitrification. This study assessed malondialdehyde release during storage and the capacity to synthesize oestradiol in culture, follicle morphology and the proportion of apoptotic follicles subsequent to vitrification–warming. The malondialdehyde concentration in the storage medium increased significantly in a time-dependent manner (P = 0.029). Oestradiol release during tissue culture decreased statistically significantly in both storage groups compared with the immediate vitrification group (P = 0.043). The proportion of high-quality follicles was significantly different between the fresh group and the two storage groups (24 h, P < 0.01; 48 h, P < 0.001). The proportion of apoptotic follicles was significantly different between the fresh and immediate vitrification groups (P < 0.05). The 24-h and 48-h groups were not different compared with the other groups. This study provides evidence that storage at supra-zero conditions has detrimental effects on the human ovarian cortex. Storage duration should therefore be limited to a minimum to prevent potential damage.

Minimal residual disease detection of leukemic cells in ovarian cortex by eight-color flow cytometry

How can leukemic cells be detected in cryopreserved ovarian tissue? Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity. Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding. We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex. Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis. The tissue dissociation technique yield was 1.83 ± 1.49 × 10(6) viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10(-4). When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls. Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring. This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft. The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare.

Ovarian Tissue Cryopreservation by Vitrification in Olive Baboons (Papio Anubis): A Pilot Study

Background: Ovarian tissue cryopreservation by vitrification is an attractive technique for fertility preservation in women. However, this technique has not been optimized. The aim of this study was to evaluate the baboon as a model for the preclinical study of ovarian tissue cryopreservation by vitrification and thawing. Methods: Ovarian cortical tissues (1-mm cubes) were obtained surgically from adult female olive baboons (n = 9) maintained in captivity and vitrified using dimethyl sulphoxide and ethylene glycol protocol. The proportion of morphologically intact follicles (primordial, primary and secondary) in paired fresh and cryopreserved (vitrified-thawed) ovarian tissues was compared. Results: Overall, 67.1% of follicles were morphologically normal after vitrification. When compared to fresh ovarian tissue, vitrified-thawed ovarian tissue contained a comparable number of intact primordial follicles (48.9 vs. 52.9%), and a lower number of both primary (14.8 vs. 29.5%; p < 0.05) and secondary (2.0 vs. 0.7%; p < 0.05) follicles. Conclusion: After vitrification and thawing, baboon ovarian tissue retains about 67% of morphologically normal follicles, which is comparable to results for human ovarian tissue, and suggests that the olive baboon is a promising animal model for preclinical assessment of ovarian vitrification, thawing and autotransplantation studies.

Preservation of fertility

Cryopreservation of ovarian tissue is widely performed to preserve fertility in cancer patients, but its efficacy has been difficult to assess because most patients do not request transplantation of the thawed tissue for over a decade. Our objective was to evaluate the long-term results of transplantation of fresh ovarian versus frozen ovarian tissue.

Comparison Between Slow Freezing and Vitrification in Blastocyst Cryopreservation

Comparison Between Slow Freezing and Vitrification in Blastocyst Cryopreservation

Vitrified-warmed ovarian tissue autotransplantation into ovariectomized mice restores sufficient ovarian function to support full-term pregnancy.

Our previous study demonstrated that heterotopic autotransplantation of fresh ovarian tissue followed by transfer of blastocysts supported full-term pregnancy in the mouse. In the present study, to address whether vitrified-warmed ovarian tissue has the potential to support uterine preparation for implantation and subsequent pregnancy to full term, we examined vitrified-warmed ovarian tissue autotransplantation (VOAT) in mice. VOAT into kidney capsules was performed for sexual cycle-ceased mice after 7 days of ovariectomy. Uterine potential of decidualization was examined by oil infusion on day 4 of pseudopregnancy. Immunohistochemical analysis was performed to examine the potential in VOAT ovarian tissues. Blastocysts were transferred into uteri on day 4 of pseudopregnancy. In VOAT mice, uterine decidualization on day 8 of pseudopregnancy was the same as that in intact mice. Blastocyst transfer into the pseudopregnant VOAT mice showed the same rates of pregnancy and live birth pups as intact mice, while less steroidogenesis in the corpus luteum was detected in VOAT mice. The autotransplantation of vitrified-warmed ovarian tissues after 7 days of ovariectomy restored their sexual cycle and then supported their pregnancy and production of offspring.

Secretion profiles from in vitro cultured follicles, isolated from fresh prepubertal and adult mouse ovaries or frozen–thawed prepubertal mouse ovaries

In vitro folliculogenesis could be a new technology to produce mature oocytes from immature follicles that have been isolated from cryopreserved or fresh ovarian tissue. This technique could also be a tool for evaluation of oocyte quality and/or for determination of follicular parameters during follicular growth. Our objective was to characterize in mice the secretion profiles of follicles that had been isolated mechanically during in vitro follicular growth and in relation to the growth curve. Early preantral follicles from fresh prepubertal and adult mouse ovaries or frozen-thawed prepubertal mouse ovaries were cultured individually in microdrops under oil for 12 days. Each day, two perpendicular diameters of the follicles were measured. From day-3 to day-12 of culture, culture medium was collected and preserved for determination of inhibin B, anti-Müllerian hormone (AMH) and estradiol levels. At the end of the culture, after maturation, the status of the oocyte was evaluated. Follicular growth and their individual hormone production did not always correlate. Inhibin B was never secreted from follicles of less than 200 μm diameter, whether the follicles were examined when fresh or after freezing-thawing. Estradiol secretion was never observed in frozen-thawed follicles. AMH was mainly secreted between day-3 and day-9. Despite similar morphological aspects at the start of culture, follicles selected for in vitro folliculogenesis were found to be heterogeneous and differed in their ability to grow and to produce hormones, even if they had similar growth curves. Follicles from frozen-thawed ovaries developed slowly and produced fewer hormones than freshly collected follicles.

Cryopreservation of ovarian tissue for a decade in Denmark: a view of the technique

This paper presents the Danish 10-year experience (1999-2009) with cryopreservation (n=386) and autotransplantation of ovarian tissue (n=18). Before applying the technique to humans, the method was thoroughly tested and validated. The cryoprotectant solution was chosen after histological evaluation of mouse and human ovarian tissue after freezing with four different combinations of cryoprotectants. Viability was confirmed by transplantation of frozen-thawed human ovarian tissue (n=49) to oophorectomized Nude mice. Viability after transport of fresh tissue 4-5h prior to freezing had previously been validated. Overnight transport of fresh ovarian tissue prior to cryopreservation was evaluated when human ovarian tissue was kept on ice for 20h and then cryopreserved. The thawed ovarian tissue was transplanted to an oophorectomized Nude mouse and histology confirmed viability. In Denmark 12 women have received a total of 18 autotransplantations of ovarian tissue. All women resumed ovarian function and three healthy babies were born to two women. In both women, the tissue was transported on ice for 4-5h prior to cryopreservation. Ovarian tissue cryopreservation is an important method for fertility preservation; however, before applying the method clinically, each laboratory should perform thorough validation of their technique.

Restoration of ovarian function after allografting of ovarian cortex between genetically non-identical sisters

Aggressive chemotherapy generally results in the loss of both endocrine and reproductive functions. For some women, however, oocyte, embryo or ovarian tissue cryopreservation were not proposed at the time. For three such women, orthotopic allotransplantation of fresh ovarian tissue from their genetically non-identical sister was performed. Three women, aged 20, 15 and 12 years, respectively, underwent chemotherapy and total body irradiation before bone marrow transplantation (BMT), the donor in each case being their HLA-compatible sister. Years later, HLA group analysis revealed complete chimerism, and ovarian allografting was performed, with the ovarian tissue donor being the sister who had already donated bone marrow. No immunosuppressive therapy was administered. No sign of rejection was observed. Restoration of ovarian function occurred in all three cases, respectively, 6, 3.5 and 3.5 months after transplantation. The timing of the first estradiol peaks and the persistence of ovarian function were probably related to the primordial follicle density of donor ovarian tissue. Even in the absence of immunosuppressive therapy, ovarian allografting between genetically non-identical sisters allowed restoration of ovarian function in cases where previous BMT from the HLA-compatible sister resulted in full chimerism, avoiding the threat of rejection.

Morphological and Apoptotic Comparison of Primordial and Primary Follicles in Cryopreserved Human Ovarian Tissue

There are few reports which were designed to compare the survival rate of human primary follicles with primordial follicles after cryopreservation. This study was designed to evaluate whether such a difference occurs. Human ovarian biopsies were cryopreserved using dimethylsulphoxide/sucrose as the cryoprotectants. Fresh and cryopreserved ovarian samples were evaluated for viability differences between the two types of follicles using the endpoints of histology, ultrastructure and DNA fragmentation. In comparison with fresh ovarian tissue (83.9%+/-10.0%), the percentage of morphologically normal primordial follicles was not significantly different in cryopreserved tissue (73.9%+/-17.2%). However, a lower percentage of primary follicles with normal morphology was seen in the cryopreserved group (43.3%+/-25.7% vs 74.8%+/-19.4% for the fresh group). Transmission electron microscopy revealed that the cryopreservation did not appear to affect the structural integrity of primordial follicles; however, varying ultrastructural damage to the cytoplasm was observed in the majority of the cryopreserved primary follicles. Using a DNA fragmentation assay, the percentage of apoptotic primordial and primary follicles in the unfrozen (26.3% and 20%) and frozen (23.3% and 25%) ovarian tissue was similar. A higher proportion of primary follicles, compared to primordial follicles, suffer histological damage after slow freezing.

Fresh Ovarian Tissue and Whole Ovary Transplantation

A series of monozygotic (MZ) twin pairs discordant for premature ovarian failure presented an unusual opportunity to study ovarian transplantation. Ten MZ twin pairs requested ovarian transplantation and nine have undergone transplantation with cryopreservation of spare tissue. Eight had a fresh cortical tissue transplant, one of whom received a second frozen-thawed transplant after the first ceased functioning at 3 years. One had a fresh microvascular transplant. All recipients reinitiated ovulatory menstrual cycles and normal day 3 serum follicle-stimulating hormone levels by 77 to 142 days. Seven have already conceived naturally (three twice). Currently, seven healthy infants have been delivered out of 10 pregnancies. The oldest transplant ceased functioning by 3 years, but then she conceived again after a frozen-thawed secondary transplant. There was no apparent difference in return of ovarian function between the nine fresh ovarian grafts and the one frozen graft. Ovarian transplantation appears to restore ovulatory function robustly. Ten pregnancies, leading to seven healthy infants, including one after cryopreservation, bode well for application to fertility preservation.

Longevity of autologous ovarian transplants: extended experience in humans

OBJECTIVE: Currently no data are available on the long-term survival of autologous ovarian transplants (AOT). We performed this study to report on the extended experience of the oldest AOT program. DESIGN: A prospective follow up study of women who underwent AOT. MATERIALS AND METHODS: Between 1999 and 2008, we performed 5 AOTs with frozen banked (n=3) and fresh (n=2) ovarian tissue. Indications and patient characteristics are shown in table 1.Table 1Case NoYear of TransplantationIndication for Fertility PreservationAge at FreezingAge at TransplantationDuration of BankingType of Transplantation11999Probable Endometriosis28298 monthsOrthotopic (Pelvic sidewall)21999Cervical CancerFresh35N/AHeterotopic (s.c. forearm)32001Benign Ovarian CystFresh37N/AHeterotopic (s.c. forearm)42004Breast Cancer313772 monthsHeterotopic (s.c. lower abdomen)52005Hodgkin Lymphoma262929 monthsHeterotopic (s.c. lower abdomen) Open table in a new tab RESULTS: In all 5 cases, ovarian function returned within 3 months. In cases 1, 2, 4, and 5, ovarian stimulation was performed. Cases 2 and 4 underwent oocyte retrieval yielding GV or M-I oocytes, which had to be in vitro matured. From case 4, a 4-cell embryo was obtained but did not result in pregnancy. Same patient was also unable to conceive with two different egg donors, demonstrating poor embryo development. Because her partner was, due to past-chemotherapy, severely oligospermic, there was suspicion of a male etiology for poor embryo quality. Patient-5 spontaneously conceived twice, simultaneously with two consecutive ovulations from the heterotopic AOT and delivered a healthy child 3 years ago. The same patient recently underwent ovarian stimulation and percutaneous oocyte retrieval, resulting in retrieval of a M-II oocyte, which did not fertilize. Two months later the patient's FSH reached menopausal range. This was, however, the first case, where a mature oocyte was obtained from a frozen-banked AOT, placed into a subcutaneous location. In patients 1, 3, and 5, the graft function ceased 9, 17, and 41 months after AOT. Interestingly in patient 3, follicle development and menstruation returned 12 months later. Patient 2 experienced local cancer recurrence and, unfortunately, died 22 months after transplant while the graft was still functioning. In patient 4, the graft continues to function for > 4 years. CONCLUSIONS: AOT results in restoration of hormonal function in all cases. Longevity of the grafts is variable but appears to be shortened in comparison to in situ ovaries.

Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex

Frozen-thawed ovarian cortical fragments (1 mm(3)) were autotransplanted to the uterus of completely ovariectomized goats. The grafts developed preovulatory follicles, accompanied by estrous behavior and a rise in plasma E(2) levels, demonstrating successful cryopreservation and transplantation.

Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients

Storing ovarian tissue for fertility preservation in cancer patients carries the risk of the presence of malignant cells that could lead to recurrence of cancer after reimplantation. Methods to exclude presence of cancer cells were used to improve the safety of cryopreservation-reimplantation procedures. Fifty-eight patients with hematological malignancies were referred for the storage of ovarian tissue for fertility preservation. Investigation included preoperative imaging and histological evaluation of fresh ovarian tissue. After thawing markers to detect minimal residual disease (MRD) were used and compared with patient's disease used as positive control (five patients). Preoperative imaging detected disease in the ovaries (two patients). Conventional histology post-tissue harvesting did not disclose malignant cells (56 patients). MRD results post-thawing were negative in Hodgkin's disease (CD30 immunohistochemical staining), in T- and B-cell lymphoma (PCR for T-cell receptor and Ig clones, respectively) and in two chronic myelogenous leukemia patients (RT-PCR for BCR-ABL gene expression). However, highly sensitive real-time RT-PCR was positive in one CML patient and, this alarming result avoided tissue transplantation. Preoperative imaging prevented operations and storage of tissue with cancer. Evaluation of stored ovarian tissue for MRD using sensitive markers is essential to increase safety and to prevent reimplantation of tissue with malignant cells.

Response of cryopreserved ovarian tissue after autologous implantation in mouse stimulated with gonadotrophin

To investigate the response of cryopreserved ovarian tissue after autologous implantation in mouse stimulated with gonadotrophin. Thirty six female mice were randomly divided into three groups, with 12 mice in each group. In group of fresh ovarian tissue, fresh ovarian tissue was implanted into kidney capsule of mice; in group of cryopreserved ovarian tissue, ovarian tissue was implanted into kidney capsule of mice after cryopreserved by vitrification for two weeks. We investigated the response of ovarian tissue two weeks later after autologous implantation stimulated with gonadotrophin. Immunohistochemistry staining method was used to observe the expression of follicle stimulating hormone receptor. Before and after stimulation with gonadotrophin, the mature follicle rate of group of fresh ovarian tissue was 2.3% and 4.2%, that of group of cryopreserved ovarian tissue was 2.3% and 4.0%, and that of group of control was 2.6% and 5.8%. Regarding the percentages of mature follicle, there were significant differences after stimulation with gonadotrophin (P < 0.05). After stimulating with gonadotrophin the percentages of mature follicle were the same in the fresh tissue group, cryopreserved tissue group and control group (P > 0.05). The integrated optical density of follicle stimulating hormone receptor of fresh ovarian tissue in antrofollicle and pre-antrofollicle were 9408 +/- 2777 and 4531 +/- 1903, that of cryopreserved ovarian tissue were 9175 +/- 3093 and 4808 +/- 1386, and that of the control ovarian tissue were 8838 +/- 2064 and 5516 +/- 1136 respectively. There was no significant difference between any two groups (P > 0.05). The follicle stimulating hormone receptor is preserved by cryopreservation and transplantation, small pieces of ovarian tissue response to gonadotropin stimulation is normal.

Allograft of ovarian cortex between two genetically non-identical sisters: Case Report

Aggressive chemotherapy and radiotherapy generally result in the loss of both endocrine and reproductive functions. In 1990, a woman aged 20 years, presenting with beta-thalassemia major, underwent chemotherapy (busulfan and cyclophosphamide) and total body irradiation (TBI) before bone marrow transplantation (BMT), the donor being her 17-year-old HLA-compatible sister. The treatment resulted in premature ovarian failure. In 2006, after excision of ovarian cortical fragments from the HLA-compatible sister, these fragments were immediately sutured to the ovarian medulla of the patient. Both procedures were performed by laparoscopy. Six months after reimplantation, vaginal ultrasonography and hormone concentrations indicated recovery of ovarian secretion and function. From 6 to 11 months, the patient experienced menstrual bleeding and the development of a follicle concomitant with high estradiol levels. Eleven months after reimplantation, two follicles were detected and punctured under vaginal ultrasonographic control. Two mature oocytes were retrieved and inseminated by ICSI. Two embryos (2- and 3-cell) were obtained. Allotransplantation of fresh ovarian tissue was laparoscopically performed between two genetically non-identical sisters. Restoration of ovarian function was achieved after six months. Oocyte retrieval and embryo development were demonstrated.

Cyclin B1, D1 overexpression and its correlation with clinical prognostic factors in ovarian carcinoma

15067 Background: The change of cell cycle is one of the characteristics of cancer. The various proteins related to the cell cycle have been revealed and their expression in ovarian carcinoma has been demonstrated. Therefore, this study was conducted to determine the expression of cyclin B1, D1 and evaluate the relationship between cyclin B1, D1 and clinical prognostic factors in patients with ovarian carcinoma. Methods: 41 fresh ovarian tissue samples including 36 ovarian carcinomas and 5 normal ovarian tissues were surgically obtained at YUMC from May 2002 to February 2005. Cyclin B1, D1 expression were detected using the quantitative real-time RT-PCR and Western blot analysis. For clinical prognostic factors, age, stage, grade, histopathology, LN metastasis, residual tumor size, CA 125 and DNA flow cytometry were evaluated. Results: By quantitative real time RT-PCR, the mean 2−ΔΔCT value of cyclin B1 and D1 mRNA in ovarian carcinoma was 5.83 ± 12.03, 17.60 ± 22.20, slightly higher than that of the control. (p = 0.67, 0.07). The mean value of relative protein levels of cyclin B1 and D1 in Western blot analysis was also higher in ovarian carcinoma (1.30 ± 0.73, 1.81 ± 1.28, respectively) (p = 0.76, 0.06). No significant relationship of cyclin B1, D1 expression and clinical prognostic factors was observed. Conclusions: The expression of cyclin B1, D1 in ovarian carcinoma was higher than that of the control, although there was no statistical significance. This suggests that cyclin B1, D1 might be involved in the tumorigenesis and the progression of malignancy. Even though there was no significant correlation between cyclin expression and prognostic factors, further studies are needed assessing the relationship between cyclin expression and survival rate to elucidate the role of cyclin as a prognostic factor in ovarian carcinoma. No significant financial relationships to disclose.

Ovarian cortical transplantation may be an alternative to hormone therapy in patients with early climacterium

To discuss the 18-month follow-up results of fresh ovarian cortical tissue transplant. Case study. Academic medical center. A 46-year-old patient who was operated on for uterine fibroids, and who then received an ovarian tissue transplant in July 2003. Serum hormone levels were measured at 3-month intervals. Follicular development evident by ultrasound examination; serum hormone levels (FSH, LH, E2). Preoperative and 3rd-, 6th-, 9th-, 12th-, 15th-, and 18th-month hormone levels were, respectively, as follows: FSH: 9.06, 79.5, 13.7, 16.66, 51.91, 44.37, and 24.17 mIU/mL; LH: 5.91, 33.92, 8.78, 21.83, 38.31, 40.85, and 22.4 mIU/mL; E2: 166, 46, 48, 117, 31, 14.4, and 137.7 pg/mL. Folliculogenesis was confirmed by ultrasonography at the 6th, 9th, and 18th months during the follow-up period. Fresh ovarian autotransplantation may be a logical alternative for hormonal support for a specific patient group.

In vitro follicular development of cryopreserved mouse ovarian tissue

In a previous report, we showed that follicles isolated from frozen/thawed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The purpose of this study was to define a culture system with some technical modification for preantral follicles isolated from frozen/thawed ovarian tissue and to confirm cell injury. Ovaries obtained from three-week-old female mice were cryopreserved by the rapid freezing method. Preantral follicles isolated from frozen/thawed ovarian tissues were cultured for 12-16 days. The follicles were then stimulated with human chorionic gonadotropin. In vitro fertilization was performed on the released cumulus-oocyte complexes (COCs). Preantral follicle viability was assessed by supravital staining using Hoechst 33258. Using this stain cell death was found in part of the granulosa cells of a follicle obtained from frozen/thawed ovarian tissue. On the 14th and 16th days of culture, the diameters of follicles isolated from frozen/thawed ovaries were larger than on the 12th day of culture. The released COCs were fertilized and developed to the blastocyst stage in 15.8% (12/76) of the oocytes taken from the fresh group, and in 0% (0/73), 2.9% (2/69) and 19.1% (22/115) of the oocytes taken from the frozen/thawed group that had been cultured for 12, 14 and 16 days respectively. The preantral follicles isolated from frozen/thawed mouse ovarian tissues developed slowly compared with the freshly prepared preantral follicles. During prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage.

Blastocyst Development After Cryopreservation and Subcutaneous Transplantation of Mouse Ovarian Tissue

To investigate follicle survival and developmental potential with IVF of cryopreserved, subcutaneously transplanted mouse ovarian tissue. Fresh and frozen mouse ovarian tissue was autologously transplanted into subcutaneous tissue. Two weeks after the transplantation, the morphology and histology of the fresh and frozen grafts were compared. Superovulation and IVF was performed to evaluate the fertility potential of the frozen ovarian graft. Both fresh and frozen grafts of ovarian tissue survived in 14 of 16 mice (88%). Morphologically, both types of grafts resembled fresh ovarian tissue and contained follicles at all stages of folliculogenesis. A total of 73% of follicles in fresh grafts and 62% in frozen grafts survived after transplantation compared with fresh ovarian tissue. Sixteen ICR mice underwent superovulation. A total of 56 oocytes from antral follicles were recovered from the subcutaneously transplanted cryopreserved ovarian tissue. Fourteen (25%) oocytes were in metaphase II stage, 6 were fertilized by IVF, and 2 progressed to the blastocyst stage. Cryopreservation and subcutaneous transplantation of ovarian tissue provides a possible means of fertility preservation. The main loss of follicles occurred during grafting rather than during freezing and thawing.