A comparison between a new vitrification protocol and the slow freezing method in the cryopreservation of prepubertal testicular tissue.

OBJECTIVE: This study aimed to compare a new vitrification protocol with reduced cryoprotectant exposure to the slow freezing method in the cryopreservation of prepubertal rat testicular tissue. METHODS: Five sexually immature male Wistar rats were submitted to bilateral orchiectomy. Tissue samples from each testicle were fragmented into small pieces and randomly assigned to three groups: Group A, fresh tissue (control); Group B, slow programmable freezing (SPF); and Group C (vitrification). Frozen/thawed, vitrified/warmed, and fresh testicular tissue were histologically compared. A pathologist blinded to the procedures assessed the morphology (cell differentiation, nuclei, and epithelium) of 10 seminiferous tubules from each testicle (100 tubules per Group). RESULTS: Sertoli and spermatogonial stem cells were easily differentiated, and the nucleoli were easily viewed in the tubules assessed in all three groups. Small alterations in tissue architecture were observed in the control group as a result of tissue handling. Moderate alterations of the epithelium with the formation of small gaps and cell detachment from the basement membrane were observed in 28% of the frozen and 9% of the vitrified tubules. Condensed nuclei involving a small proportion of cells were observed in six and three tubules of the frozen and vitrified group, respectively. Despite the alterations, 97% of the frozen and 99% of the vitrified tubules were considered well preserved. CONCLUSIONS: The findings indicate that the vitrification protocol tested in this study adequately preserved the morphological integrity of prepubertal testicular tissue in a rat model. Further studies are required to confirm testicular tissue function after grafting.

Viability of homologous and heterologous subcutaneous transplantation of fresh germ cells in rabbits.

OBJECTIVE: This study aimed to compare heterologous to homologous transplantation of fresh ovarian germ cells in rabbits. METHODS: Twelve female white New Zealand rabbits (Oryctolagus cuniculus) were randomly numbered and submitted to bilateral oophorectomies. The ovaries from the six odd-numbered rabbits were dissected and cortical germinal tissue was digested in collagenase type 1 to obtain six solutions containing stromal and germ cells, which were injected in the abdominal region of the odd-numbered rabbits themselves (homologous transplantation) and of the even-numbered rabbits (heterologous transplantation) off immunosuppression. Sixty days after transplantation, the tissue around the transplanted region was excised, processed and sent to histological analysis with hematoxylin-eosin staining and Bcl-2 immunohistochemistry to verify the presence and viability of the transplanted cells. RESULTS: The analyzed specimens contained ovarian stroma, while follicular cells were found in 66.6% of the homologous and in 60% of the heterologous transplant specimens. Mild inflammatory reaction was observed in all heterologous specimens, and in only one (16.7%) of the homologous specimens. However, this inflammatory reaction was not so intense as to cause the death of the implanted cells. Except for the specimens from rabbits 7 and 8, all specimens were stained for Bcl-2, indicating that most of them were viable. CONCLUSIONS: The results of this study supported the viability of heterologous transplantation of fresh ovarian germ cells. However, more studies are required to further our understanding and improve the germ cell separation technique.

Vitrification of Human Oocytes and its Contribution to In Vitro Fertilization Programs.

OBJETICVE: To study the cumulative pregnancy outcome, particularly in terms of live births, with the consecutive transfer of embryos from fresh and vitrified/warmed oocytes to infertile patients in a routine infertility program. METHODS: Patients were initially submitted to in vitro fertilization embryo transfer with fresh embryos, while surplus oocytes were vitrified with the Vitri-Ingá method. Patients who did not succeed to carry their gestation to term underwent a new cycle with embryos from their own warmed oocytes. Some of the patients participating in the first warming cycle, who still possessed surplus oocytes, underwent a second warming cycle. Clinical and pregnancy outcomes obtained with fresh and warming cycles were compared using the chi-square test at a level of significance of 5%. RESULTS: Of the 211 participating patients, 97 (46%) got pregnant with fresh embryo transfer, and 69 (32.7%) carried their pregnancies to term. Of the patients participating in the first and second warming cycles, 32/100 (32%) and 6/20 (30.0%) resulted in live births, respectively. Thus, of the 211 participating patients, 107 carried their pregnancies to term, representing a cumulative live birth rate of 50.7%. No statistically significant differences between the use fresh and vitrified oocytes were found for any of the variables studied. CONCLUSIONS: Oocyte vitrification offered the possibility of gestation in more than one attempt after just one controlled hyperstimulation. Apart from alleviating the financial burden on patients, vitrification of oocytes may result in a feasible solution for the problems generated by abandoned frozen embryos.

The First Ovarian Tissue Transplant between Monozygotic Twin Sisters Discordant for Ovarian function in Latin America.

Ovarian tissue transplant is an alternative to the cryopreservation of oocytes and embryos for the recovery of fertility and natural hormonal activity. The objective of this paper is to report on the first fresh ovarian tissue transplant between monozygotic twin sisters discordant for ovarian function, using the subcortical implant technique of ovarian tissue fragments, to take place in Latin America. A strip representing approximately a quarter of the cortical tissue was removed from the right ovary of the donor sister, cleaned, cut into small fragments and sent to adjacent room, where the receptor sister was concomitantly being prepared to receive the tissue graft. The ovarian fragments were placed under the cortical tissue onto a vascularized bed of the right ovary of the receptor sister. From 90 days postoperatively, the menstrual cycles of the receptor patient became regular with increased flow and longer periods, demonstrating normal hormonal activity and improved endometrial development. Attempts at spontaneous pregnancy, and the recovery of an oocyte followed by fertilization have not yet been successful. However, the ovarian tissue transplant between monozygotic sisters reported here clearly highlights the potential of the technique as a therapeutic option for the preservation of fertility.

A comparison of the effects of oral glutamine dipeptide, glutamine, and alanine on blood amino acid availability in rats submitted to insulin-induced hypoglycemia.

We compared the effects of oral administration of high-dose or low-dose glutamine dipeptide (GDP), alanine (ALA), glutamine (GLN), and ALA + GLN on the blood availability of amino acids in rats submitted to insulin-induced hypoglycemia (IIH). Insulin detemir (1 U/kg) was intraperitoneally injected to produce IIH; this was followed by oral administration of GDP, GLN + ALA, GLN, or ALA. We observed higher blood levels of GLN, 30 min after oral administration of high-dose GDP (1000 mg/kg) than after administration of ALA (381 mg/kg) + GLN (619 mg/kg), GLN (619 mg/kg), or ALA (381 mg/kg). However, we did not observe the same differences after oral administration of low-dose GDP (100 mg/kg) compared with ALA (38.1 mg/kg) + GLN (61.9 mg/kg), GLN (61.9 mg/kg), or ALA (38.1 mg/kg). We also observed less liver catabolism of GDP compared to ALA and GLN. In conclusion, high-dose GDP promoted higher blood levels of GLN than oral ALA + GLN, GLN, or ALA. Moreover, the lower levels of liver catabolism of GDP, compared to ALA or GLN, contributed to the superior performance of high-dose GDP in terms of blood availability of GLN.

Evaluation of liver glycogen catabolism during hypercortisolism induced by the administration of dexamethasone in rats.

BACKGROUND: The contribution of liver glycogen catabolism to hyperglycemia and glucose intolerance induced by pharmacological hypercortisolism were investigated. METHODS: For this purpose, adult male Wistar rats that received 1.0 mg/kg dexamethasone (DEX) ip at 8:00 a.m. (DEX group) or saline (CON group) once a day for 5 consecutive days were compared. RESULTS: Experimental hypercortisolism was confirmed by higher (p<0.05) glycemia, lower (p<0.05) body weight and glucose intolerance. In the fed state, the basal glycogen catabolism and the glucagon (1 nM) and epinephrine (2 µM) induced glycogen catabolism were similar between the groups. The activation of glycogen catabolism induced by phenylephrine (2 µM) and isoproterenol (20 µM) were increased (p<0.05) and decreased (p<0.05), respectively, in DEX rats. Furthermore, DEX rats exhibited higher (p<0.05) glycogen catabolism during the infusion of cAMP (3 µM). However, during the infusion of cAMP (15 µM), 6MB-cAMP (3 µM) or cyanide (0.5 mM), the intensification of glycogen breakdown was similar. Thus, in general, hypercortisolism does not influence the basal glycogen catabolism and the liver responsiveness to glycogenolytic agents in the fed state. In contrast with fed state, fasted rats (DEX group) showed a more intense (p<0.05) basal glycogen catabolism. CONCLUSION: The contribution of glycogen catabolism to hyperglycemia during hypercortisolism depends of the nutritional status, starting from a negligible participation in the fed state up to a significant contribution in the fasted state.