Abstract
ObjectiveTo evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation.MethodsTwenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed.ResultsThe viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site.ConclusionThe vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.
Highlights
Infertility is considered a reproductive system disease that consists of an absence of clinical pregnancy after 12 months of unprotected intercourse (Jose-Miller et al, 2007)
The testes were removed by a scrotal incision; and after the surgical procedure the testes were fragmented in pieces of approximately 3mm and used as autologous graft into the periauricular region, immediately after the orchiectomy (Group 3) or were subjected to vitrification protocols (Groups 1 and 2)
Protocol 2 presented inferior results in maintaining cell viability and this difference in viability recovery between the two tested protocols may be explained by the cryoprotectants concentration and the warming protocol, which were performed at different times
Summary
Infertility is considered a reproductive system disease that consists of an absence of clinical pregnancy after 12 months of unprotected intercourse (Jose-Miller et al, 2007). There are several causes for male infertility, which include gonadotoxic cancer treatments, such as chemotherapy and radiotherapy. One potential alternative for fertility preservation of these boys is testicular tissue cryopreservation, as fragments (Brinster, 2007) or cell suspensions (Yango et al, 2014) It has been suggested the grafting of cryopreserved testicular fragments, where the germ cells could differentiate and eventually produce spermatozoa (Brinster, 2007). This technique is still considered experimental and with varied results in testicular tissue samples. Orthotopic and heterotopic transplantations were successfully performed on ovarian fragments, with reports of baby births after tissue cryopreservation and transplantation (Sánchez-Serrano et al, 2010; Silber, 2012)
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