Vitrification as an alternative means of cryopreserving ovarian tissue.

Because of the simplicity of vitrification, many authors have investigated it as an alternative to slow freezing for cryopreserving ovarian tissue. In the last decade, numerous studies have evaluated vitrification of ovarian tissue from both humans and animals.Different vitrification solutions and protocols, mostly adapted from embryo and oocyte vitrification, have been applied. The results have been discrepant from species to species and even within the same species, but lately they appear to indicate that vitrification can achieve similar or even superior results to conventional freezing. Despite the encouraging results obtained with vitrification of ovarian tissue from humans and different animal species, it is necessary to understand how vitrification solutions and protocols can affect ovarian tissue, notably preantral follicles. In addition, it is important to bear in mind that the utilization of different approaches to assess tissue functionality and oocyte quality is essential in order to validate the promising results already obtained with vitrification procedures. This review summarizes the principles of vitrification, discusses the advantages of vitrification protocols for ovarian tissue cryopreservation and describes different studies conducted on the vitrification of ovarian tissue in humans and animal species.

Can prepubertal human testicular tissue be cryopreserved by vitrification?

OBJECTIVE: To assess vitrification of prepubertal human testicular tissue in vitro. DESIGN: Case report. SETTING: Academic research unit. PATIENT(S): Two patients (6 and 12 years of age) who were to start gonadotoxic treatment for chronic granulomatous disease and acute lymphoblastic leukemia. INTERVENTION(S): Long-term (10-day) organotypic culture performed immediately after vitrification and warming. Fresh tissue and tissue cryopreserved by slow-freezing were used as control samples. MAIN OUTCOMES MEASURE(S): Spermatogonial cell survival (MAGE-A4) and proliferation (Ki67) were evaluated by immunohistochemistry (IHC) and tubular integrity by light microscopy. RESULT(S): Qualitative analysis revealed that histologic characteristics of spermatogonia and Sertoli cells were preserved, as were cell-cell cohesion and cell adhesion to the basement membrane, in vitrified tissue as well as in frozen and fresh control samples. Survival of spermatogonia and their ability to proliferate as evidenced by IHC was also confirmed in cultured fresh, slow-frozen, and vitrified tissue. CONCLUSION(S): Vitrification, having the advantage of being a faster and more convenient method, shows promise as an alternative strategy to slow-freezing in the emerging field of immature testicular tissue cryopreservation.

Cryopreservation of prepubertal mouse testicular tissue by vitrification.

OBJECTIVE: To compare cryopreservation of prepubertal testicular tissue after vitrification (V) and slow-freezing (SF). DESIGN: Prospective experimental study. SETTING: Academic research unit. ANIMAL(S): Six-day-old mice. INTERVENTION(S): After cryopreservation, viability tests (n = 10) and short-term culture (1 and 3 days) (n = 5) were performed. A comparison was made with fresh (FR) and noncultured controls (FR Ctrl). MAIN OUTCOMES MEASURE(S): Tissue viability was assessed by lactate dehydrogenase release assay. Apoptosis (caspase-3) and proliferation (Ki67) were evaluated by immunohistochemistry, and tubular diameter, integrity, and cell density by light microscopy. RESULT(S): Lactate dehydrogenase release was greater after SF than V (54.6% vs. 26.7%), whereas the mean number of apoptotic cells/tubule was higher after V than SF (2.13 vs. 0.07). On day 1, a decrease in cell density was noted in both cryopreserved groups, but this difference was not subsequently observed. On day 3, an increase in proliferation was seen in the SF and V groups versus FR tissue, and similar tubular diameter, integrity, and cell density were found in all cultured groups. CONCLUSION(S): This study shows that both SF and V protocols preserve survival, development, and integrity of prepubertal mouse testicular tissue in short-term organotypic culture. Additional investigation should now be conducted to assess tissue functionality.

Options for fertility preservation in prepubertal boys.

BACKGROUND: Fertility in adult life may be severely impaired by gonadotoxic therapies. For young boys who do not yet produce spermatozoa, cryopreservation of immature testicular tissue (ITT) is an option to preserve their fertility, albeit still experimental. This paper covers current options for ITT cryopreservation and fertility restoration. METHODS: Relevant studies were identified by an extensive Medline search of English and French language articles. Search terms were: gonadotoxicity, cytoprotection, cryopreservation, ITT, spermatogonia, testicular transplantation, testicular grafting and in vitro maturation (IVM). RESULTS: Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell protection in humans, current evidence supports the feasibility of ITT cryopreservation before gonadotoxic treatment with a view to fertility preservation. Controlled slow freezing with dimethyl sulfoxide allows survival and proliferation of human spermatogonia after xenotransplantation, but only partial differentiation. Animal data look promising, since healthy offspring have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and IVM have proved efficient and safe in humans as yet. CONCLUSION: While additional evidence is required to define optimal conditions for ITT cryopreservation with a view to transplantation or IVM, the putative indications for such techniques, as well as their limitations according to disease, are outlined.

Bone marrow transplantation or hydroxyurea for sickle cell anemia: long-term effects on semen variables and hormone profiles.

Ten male subjects affected by sickle cell anemia (SCA) were studied to evaluate the long-term effects of therapies on their fertility. Their ages ranged from 18 to 34 years (median: 32 years). Four subjects were treated by hydroxyurea (HU) and 6 by hematopoietic stem cell transplantation (HSCT). The median follow-up after HU initiation and HSCT was 10.5 years (range: 8-15 years) and 15.5 years (range: 8-21 years), respectively. Three of the 6 in the HSCT group and two of the 4 in the HU group were azoospermic. One HSCT subject had normal semen and hormone variables, showing that normal fertility can occasionally be expected after transplantation in SCA. The remaining 4 patients (2 HSCT and 2 HU) were oligozoospermic. With regard to HU, semen impairment appears to be related to the duration of treatment. To draw general conclusions, further research with a large number of patients treated since childhood with HU or HSCT is warranted.

Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue.

BACKGROUND: Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. METHODS: Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. RESULTS: Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered. CONCLUSIONS: The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.

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.