Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment

BACKGROUND: Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation. METHODS: Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen-thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy. RESULTS: No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage. CONCLUSIONS: Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.     

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.     

Comparison of conditions for cryopreservation of testicular tissue from immature mice

BACKGROUND: Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. METHODS: Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. RESULTS: DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. CONCLUSIONS: Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.     

Xenotransplantation of testicular tissue into nude mice can be used for detecting leukemic cell contamination

BACKGROUND: Xeno-grafting of testicular tissue may allow viable gamete maturation. This would be beneficial for prepubertal cancer patients in that it may allow restoration of fertility without the risk of a cancer relapse. However it is unknown whether cancer cells in the testicular graft can transmit the malignancy into the host animal and also if gametes can be retrieved from testicular grafts that are contaminated with malignant cells. METHODS: Rat T-cell leukemia was employed as the source of leukemic lymphoblasts and testicular tissue. This was injected i.p. (lymphoblasts) or grafted s.c. (fresh or cryopreserved testicular tissue) into the back skin of intact nude mice. To simulate clinical autografting, testicular tissue was also transplanted into healthy piebald variegated (PVG) rats. RESULTS: 50-70% of the mice, receiving 200 or 6000 leukemic lymphoblasts, developed terminal leukemia. All mice, grafted with either fresh or cryopreserved testicular tissue from leukemic donor, developed generalized leukemia and/or local tumors. All syngenic PVG rats, treated in the same manner, died of generalized leukemia. In all of the retrieved leukemic grafts, rat spermatogenesis was destroyed and only leukemic infiltration was detected. CONCLUSIONS: Grafting testicular tissue contaminated with leukemic cells led to tumor growth at the injection site without potential to differentiate germline stem cells into gametes. Xenografting could provide a novel functional strategy for simultaneous detection of malignant cell contamination and spermatogonial potential in testicular xenografts collected for fertility preservation.     

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants

BACKGROUND: Cryopreservation of testicular tissue is an optionin fertility preservation for pre-pubertal boys who will losespermatogenic cells as a result of chemotherapy. We comparedthree different protocols and cryoprotectants in cryopreservationof testicular tissue. METHODS: Testicular tissue obtained from16 infertile men was evaluated by light microscopy(LM), immunostainingagainst MAGE-A4, transmission electron microscopy (TEM) andorgan culture. Seminiferous tubules (1312) from non-frozen (n=16)and frozen–thawed samples (n=34) were studied followingcryopreservation using protocols with either 1,2-propanediol(PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants.RESULTS: Normal structure was seen in 86±6% (mean ±SD)of the fresh tissue. After freezing with DMSO, 70±6%and after PrOH, 37±3% of the tubules were judged to begood. When glycerol was used, the structure of the basal compartmentof the tubules was severely damaged. The ultrastructure of thecryopreserved samples as revealed by TEM and MAGE-positive spermatogoniaconfirmed the findings. Cryopreserved Leydig cells maintainedtheir morphology and ability to release testosterone in culture.CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration)proved to maintain the structure of testicular tissue, especiallyspermatogonia, after cryopreservation better than PrOH or glycerol.     

Spermatogonial survival after cryopreservation and short-term orthotopic immature human cryptorchid testicular tissue grafting to immunodeficient mice

BACKGROUND: Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days. METHODS AND RESULTS: Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting. CONCLUSIONS: Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.     

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.     

Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination

BACKGROUND: Fertility protection is an urgent clinical problem for prepubertal male oncology patients who undergo either chemotherapy or radiotherapy. As these patients do not have mature sperm to be frozen, there is as yet no effective method to preserve their fertility. METHODS AND RESULTS: Single pieces of immature mouse (1.5 x 1.5 x 1.5 mm) or rabbit (2.0 x 2.0 x approximately 3.0 mm) testis were cryopreserved, thawed and transplanted into mouse testes. Histological techniques were used to determine the presence of spermatogenesis, which was restored in both mouse and rabbit testicular pieces, and led to the production of mature sperm after both cryopreservation and syngeneic or xenogeneic transplantation into mouse testes. Using sperm developed in the frozen-thawed transplants, mouse offspring were born after in-vitro microinsemination. Furthermore, rabbit offspring were obtained using rabbit sperm that developed in fresh transplants in a xenogeneic surrogate mouse. CONCLUSIONS: This approach of 'testicular tissue banking' is a promising technique for the preservation of fertility in prepubertal male oncology patients. Xenogeneic transplantation into immunodeficient mice may provide a system for studying spermatogenic failure in infertile men.     

Autologous spermatogonial stem cell transplantation in man: current obstacles for a future clinical application

Fertility preservation is becoming an important issue in the management of the quality of life of prepubertal boys undergoing cancer treatment. At present, the only theoretical option for preservation of fertility in these boys is the preservation of the spermatogonial stem cells for autologous intratesticular stem cell transplantation. In animal models, this technique has shown promising results. However, before translation to the clinic, some major concerns should be evaluated. Improving the efficiency of the technique is one of the first goals for further research, besides evaluation of the safety of the clinical application. Also, the cryopreservation of the spermatogonial stem cells needs extra attention, since this first step will be crucial in the success of any clinical application. Another concern is the risk of malignant contamination of the testicular tissue in childhood cancer patients. Extensive research in this field and especially on the feasibility of decontaminating the testicular tissue will be inevitable. Another important, though overlooked, issue is the prevention of damage to the testicular niche cells. Finally, xenografting and in vitro proliferation/maturation of the spermatogonia should be studied as alternatives for the transplantation technique.     

Transplantation in reproductive medicine: previous experience, present knowledge and future prospects

The use of transplantation in reproductive medicine has beenconsidered by physicians and scientists alike for many years.Despite being side-tracked into futile pursuits of rejuvenationin the early days, the possibility of usefulness remains, particularlyfor preserving fertility in patients undergoing ablative chemo-or radiotherapy. These aims have been enhanced by advances intissue cryopreservation. When isolated primordial folliclesare transferred in the mouse, or ovarian tissue slices are graftedinto sheep, it is possible to obtain follicular survival withsubsequent maturation and oestrogen secretion and even restorefertility to sterilized hosts. For preservation of fertility,autografts avoid both the immunological problems of allograftsand the ethical dilemmas when using donor tissue. In the male,the concept of spermatogonial cell transfer after isolationand frozen storage of cells recovered from a testicular biopsyis most attractive, since it may provide another option forrescuing fertility in cancer patients, and provide a much neededone in children. Recent results demonstrate that gonocytes fromimmature mice injected into the tubules of sterilized hostsrestore spermatogenesis and produce fertile spermatozoa. Furthermore,the gonocytes can be stored frozen prior to transfer and stillproduce fertile tubules. This review presents a broad historyof transplantation in the male and female genital tracts aswell as attempts to anticipate possible future developments.     

Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries

BACKGROUND: Ovarian tissue cryopreservation and transplantation can be used to restore fertility to sterile females. A question that warrants further investigation is whether the follicular content is affected by the freeze-thawing and grafting procedure, and if so, to what extent and by what mechanism. METHODS AND RESULTS: Intact newborn mouse ovaries were allografted under the kidney capsule or were cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant prior to grafting. Estrogenic activity of ovariectomized recipient mice, as revealed by vaginal cytology, resumed after 11 days of transplantation. At 14 days after transplantation, ovarian grafts were recovered and processed histologically for follicle number counting. The follicular content of grafts of fresh ovaries was 58% of that from ovaries of age-matched 14 day old mice. In frozen-thawed ovarian grafts, the follicular content was only 9% lower than that of fresh grafted ovaries. Apoptosis of follicular cells was investigated by DNA nick end labelling. We observed a marked increase in the staining of fragmentation of DNA shortly after transplantation (2-12 h) of fresh newborn mouse ovaries. CONCLUSIONS: The results of the present study indicate that transplantation rather than cryopreservation accounts for the major and early loss of primordial follicles in grafted newborn mouse ovaries.     

Restoring Fertility with Cryopreserved Prepubertal Testicular Tissue: Perspectives with Hydrogel Encapsulation,Nanotechnology, and Bioengineered Scaffolds

New and improved oncological therapies are now able to cure more than 80% of cancer-affected children in Europe. However, such treatments are gonadotoxic and result in fertility issues, especially in boys who are not able to provide a sperm sample before starting chemo/radiotherapy because of their prepubertal state. For these boys, cryopreservation of immature testicular tissue (ITT) is the only available option, aiming to preserve spermatogonial stem cells (SSCs). Both slow-freezing and vitrification have been investigated to this end and are now applied in a clinical setting for SSC cryopreservation. Research now has to focus on methods that will allow fertility restoration. This review discusses different studies that have been conducted on ITT transplantation, including those using growth factor supplementation like free molecules, or tissue encapsulation with or without nanoparticles, as well as the possibility of developing a bioartificial testis that can be used for in vitro gamete production or in vivo transplantation.     

Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation

BACKGROUND: Cryopreservation of ovarian tissue for fertility preservation is becoming increasingly common. Treatment of diseases that may deprive the ovaries of follicles is often performed at local hospitals that are without the necessary facilities and expertise to cryopreserve ovarian tissue. The aim of the present study was to evaluate whether primordial follicles of ovarian cortex survive transport for up to 4 h prior to cryopreservation. METHODS: Immediately after recovery of one ovary from each of four patients, the cortex was roughly isolated, placed in IVF culture medium, kept on ice and transported for 3-4 h to the centre where final dissection and cryopreservation took place. Transplantation of pieces of thawed ovarian cortex under the skin of ovariectomized immunodeficient mice for a period of 4 weeks was used to assess the survival of primordial follicles. RESULTS: After transplantation, ovarian tissue from each of the four patients contained surviving follicles. CONCLUSIONS: Transport of roughly isolated ovarian cortex cooled on ice for a period of up to 4 h allows survival of primordial follicles following cryopreservation and transplantation to immunodeficient mice.     

Live births after autologous transplant of cryopreserved mouse ovaries

The fertility of mice after autologous transplantation of ovaries, before or after cryopreservation, was investigated in this study. Female mice were randomly assigned to either sham-operated (n = 14), ovariectomized (n = 11), fresh (n = 12) or cryopreserved (n = 11) ovarian transplant groups. Ovaries were cryopreserved in 1.4 M dimethyl sulphoxide (DMSO) by cooling to -55 degrees C at 0.5 degree C/min (ice nucleation at -7 degrees C), plunged in liquid nitrogen and then thawed at room temperature. Oestrous cyclicity was observed 7 days after sham operation or 15 days after fresh or cryopreserved ovarian transplant. Ovariectomized animals did not demonstrate oestrous cyclicity but were mated, and no pregnancies resulted. Live births were recorded from all sham-operated, all fresh transplant, and 8/11 (73%) cryopreserved transplant animals. Overall mean +/- SEM litter sizes from fresh (4.32 +/- 0.44) and cryopreserved (4.71 +/- 0.57) transplant groups were smaller (P < 0.05) than those of sham-operated animals (12.54 +/- 0.44), although the sizes were not significantly different (P > 0.05) from each other. Animals were mated at least four times, with four litters of live pups from 4/4 sham-operated, 1/10 fresh and 1/9 cryopreserved ovarian transplant animals. Litter sizes from pups of sham-operated and transplant animals were not significantly different from each other. Following autologous transplantation of mouse ovaries, before or after cryopreservation, offspring appeared normal, with high rates of fertility.      

Cryopreservation of intact testicular tissue from boys with cryptorchidism

BACKGROUND: Boys with cryptorchidism often face fertility problems in adult life despite having orchiopexy performed at a very young age. During this operation, a biopsy of the testis is normally taken in order to evaluate their infertility potential and the presence of malignant cells. This study evaluated the morphology and functional capacity of cryopreserved testes biopsies and their possible use in fertility preservation. METHODS: Biopsies from 11 testes (eight boys) were obtained. Each biopsy was subdivided into six pieces and two pieces were frozen in each of two different cryoprotectants. One fresh and two cryopreserved pieces were cultured for 2 weeks. All pieces were prepared for histology. Used culture media were analysed for testosterone and inhibin B concentrations. RESULTS: The morphology of the fresh and frozen-thawed samples was similar, with well-preserved seminiferous tubules and interstitial cells. A similar picture appeared after 2 weeks of culture, but a few of the cultured biopsies contained small necrotic areas. The presence of spermatogonia was verified by c-kit-positive immunostaining. Production of testosterone and inhibin B (ng/mm(3) testis tissue) in the frozen-thawed pieces was on average similar to that of the fresh samples. CONCLUSIONS: Intact testicular tissue from young boys with non-descended testes tolerates cryopreservation with surviving spermatogonia and without significant loss of the ability to produce testis-specific hormones in vitro. It may be an option to freeze part of the testis biopsy, which is routinely removed during the operation for cryptorchidism, for fertility preservation in adult life.     

Cryoloop vitrification of rabbit oocytes

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.     

Cryopreservation of ovarian tissue and in vitro matured oocytes in a female with mosaic Turner syndrome: Case Report

We report a novel approach of fertility preservation in a young woman with mosaic Turner syndrome. A 16-year-old female with 20% 45XO and 80% 46XX karyotype underwent laparoscopic ovarian wedge resection. Before performing ovarian tissue cryopreservation, all visible follicles on the ovarian surface were aspirated. We recovered 11 immature germinal vesicle stage oocytes, which were subjected to in vitro maturation (IVM). Eight oocytes that matured (73% maturation rate) were cryopreserved by vitrification. The combination of ovarian tissue cryobanking and immature oocyte collection from the tissue followed by IVM and vitrification of matured oocytes represent a promising approach of fertility preservation for young women with mosaic Turner syndrome.     

人骨软骨组织体外冷冻保存的效果

背景：异体骨软骨移植是治疗关节软骨缺损的有效方法,然而由于移植物体外有效保存时间短,限制了临床应用以及移植物的利用效率。 目的：观察梯度降温冷冻保存同种异体关节软骨的保存效果。 方法：将关节软骨块经体积分数10%二甲基亚砜预处理后,应用程序冷冻仪以-1 ℃/min行梯度降温至-4 ℃,-10 ℃,     -20 ℃,-40 ℃各30 min,最后至-80 ℃冷冻保存。 结果与结论：保存1,3,6个月及1年后检测发现,与新鲜组相比,冷冻后软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平下降(P < 0.05）,且软骨细胞存活率、细胞活性以及浅、中、深层的蛋白多糖水平随冷冻保存时间延长而降低(P < 0.05）,提示冷冻保存可有效地延长骨软骨移植物存活时间。     

自制多脏器保存液对大鼠睾丸一氧化氮合酶的影响

背景：在睾丸移植过程中,低温保存和缺血可导致睾丸产生氧自由基而损伤睾丸组织。 目的：观察自制多脏器保存液对低温保存大鼠睾丸一氧化氮合酶的影响。 方法：采用自制多脏器保存液和UW液低温保存大鼠睾丸,分别于保存24,48,72 h时切取睾丸,测定睾丸组织内的总抗氧化能力和一氧化氮合酶活性。 结果与结论：自制多器官保存液低温保存各时点大鼠睾丸一氧化氮合酶活性和总抗氧化能力与UW液组比较差异均无显著性意义(P > 0.05)。表明自制多脏器保存液能明显减轻低温保存大鼠睾丸氧自由基损伤,其作用与经典的美国威斯康星大学UW保存液基本相当。     

Reducing ischaemic damage in rodent ovarian xenografts transplanted into granulation tissue

BACKGROUND: Anti-cancer therapies frequently lead to ovarian damage and impaired fertility. To preserve fertility, cryopreservation and subsequent transplantation of the ovaries have been suggested. One of the challenges in ovarian graft transplantation is overcoming the initial ischaemic damage that depletes a significant fraction of the oocyte pool. METHODS AND RESULTS: Follicular survival in ovarian grafts was examined by magnetic resonance imaging (MRI) and fluorescence microscopy in a model system in which rat ovaries were transplanted into nude mice. Transplantation into angiogenic granulation tissue created during wound healing shortened the ischaemic period by 24 h and significantly increased the pool of healthy primordial follicles and the perfused area of the transplanted grafts. Functional blood vessels were detected within the grafts as early as 2 days after transplantation. Gain of function was demonstrated both by growth of the grafts and by the hormonal influence on the host uteri. CONCLUSION: Implantation of ovarian grafts into an angiogenic granulation tissue improved graft vascularization and follicular survival. This procedure/treatment may be used for reducing the ischaemic damage in ovarian transplants, thus prolonging graft functionality and increasing the yield of oocytes that can be easily recovered for fertilization.