Effective dosage of growth differentiation factor-9ß in folliculogenesis and angiogenesis in the sheep ovarian tissues grafted onto chick embryo chorioallantoic membrane.

AIM: Scientists have tried to culture and transplant the ovarian tissues (OT), to preserve fertility in cancer patients. However, one of the main limitations to the applicability of this technique is the folliculogenesis disruption after transplantation. Due to the roles exerted by growth differentiation factor-9ß (GDF9ß), we decided to determine the most effective dose of GDF9ß on promotion of folliculogenesis and angiogenesis in sheep OT grafted onto the chick chorioallantoic membrane (CAM). METHODS: Fresh sheep OT were grafted onto the CAM for 5 days, and divided into four groups based on the addition of increasing doses of GDF9ß (0, 150, 200 and 250 ng/mL). Following culture, histological (hematoxylin and eosin [H&E] staining) and immunohistological studies (Ki-67) were done. Fibrotic and necrotic regions were measured using MICROVISIBLE software. For comparing the follicle development rates between the groups as well as differences in the Ki-67-positive follicles, analysis of variance was applied. RESULTS: In both 200 and 250 ng/mL GDF9ß groups, significantly higher rates of intermediary and primary follicles were observed, also the numbers of good quality follicles increased in the aforementioned groups and the rates of fibrotic and necrotic areas decreased. Moreover, in the 200 and 250 ng/mL GDF9ß groups, the number of capillaries and the proliferative activity increased. The lower dose of GDF9ß (150 ng/mL) neither activated the primordial follicles nor lead to an increase in the number of growing follicles. CONCLUSION: Addition of high dosages of GDF9ß to the OT, grafted onto the CAM resulted in higher folliculogenesis and better transplantation features due to improvement in angiogenesis.

GDF9-ß promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs.

PURPOSE: Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-ß expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-ß can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue. METHODS: Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-ß to the grafted tissue. After 5 days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software. RESULTS: Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-ß cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-ß was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-ß cultured group. CONCLUSIONS: GDF9-ß, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.

Embryo Cryopreservation Following In-Vitro Maturation for Fertility Preservation in a Woman with Mullerian Adenosarcoma: A Case Report.

In-vitro maturation (IVM) of the immature oocytes recovered from the surgically removed ovarian tissue has been considered as a process for fertility preservation in patients with cancer. Fertility preservation for a woman with Mullerian adenocarcinoma. A 37-year-old woman with Mullerian adenocarcinoma was a candidate for ovarian resection. The immature oocytes were retrieved after ovarian resection from a 37-year-old woman with Mullerian adenocarcinoma. The oocytes underwent IVM and were fertilized using intracytoplasmic sperm injection (ICSI). Two healthy embryos were cryopreserved for future use. The immature oocytes from the ovarian tissue can be matured with IVM for generation of embryos after ICSI. The embryos can be vitrified using routine methods for fertility preservation in young women with cancer.

In-Vitro Application of Pentoxifylline Preserved Ultrastructure of Spermatozoa After Vitrification in Asthenozoospermic Patients.

PURPOSE: To evaluate the effect of in vitro application of pentoxifylline (PX) on sperm parameters and ultrastructure after vitrification in asthenozoospermic patients. MATERIALS AND METHODS: A total of 30 asthenozoospermic semen samples (aged 25-45 years) were divided into four groups before vitrification, after vitrification, control (without PX) and experimental (with PX). In experimental group, each sample was exposed for 30 min to 3.6mmol/l PX and the control group without any treatment apposing in 370C for 30 min. After incubation, the samples were washed and analyzed again. Vitrification was done according to straw method. Eosin-nigrosin and Papanicolaou staining were applied for assessment of sperm viability and morphology, respectively. The samples without PX and post treatment with PX were assessed by transmission electron microscopy (TEM). RESULTS: A significant decrease in sperm motility (P ≤ .001), morphology (11.47 ± 2.9 versus 6.73 ± 2.01) and viability (73.37 ± 6.26 versus 54.67 ± 6.73) was observed post vitrification, but sperm motility (19.85 ± 4.75 versus 32.07 ± 5.58, P ≤ .001) was increased significantly following application of PX. This drug had no significant (P >.05) detrimental neither negative effect on ultrastructure acrosome, plasma membrane and coiled tail statues of spermatozoa. CONCLUSION: Vitrification had detrimental effects on sperm parameters, but PX reversed detrimental effects on sperm motility. However, PX had no alteration on ultrastructure morphology of human spermatozoa after vitrification.

Transplantation of differentiated umbilical cord mesenchymal cells under kidney capsule for control of type I diabetes in rat.

Nowadays, stem cells have been introduced as an appropriate source of regenerative medicine for treatment of type I diabetes. Human umbilical cord matrix-derived mesenchymal cells (hUCMC) have successfully been differentiated into insulin producing cells. The isolated hUCM cells were characterized by the expression of stem cell surface markers and by differentiation into adipocytes and osteocytes. The hUCMCs were cultured with different concentrations of neural conditional medium (NCM) and were induced to differentiate into insulin producing cells (IPCs). As 60% NCM concentration resulted in higher nestin and PDX1 expression, the cells were first exposed to 60% NCM and were then induced for IPCs differentiation. PDX1 and insulin gene expression was evaluated in the treated cells. Also, the secretion capacity of the IPCs was assessed by glucose challenge test. IPCs were transferred under the rat kidney capsule. Blood glucose level, weight gain and immunohistochemistry assessments were done in the treated animals. hUCMC expressed mesenchymal cell surface markers and successfully differentiated into adipocytes and osteocytes. Higher NCM concentration resulted in higher PDX1 and nestin expression. The IPCs expressed insulin and PDX1. IPCs were detectable under the kidney capsule 2 months after injection. IPCs transplantation resulted in a sharp decline of blood sugar level and less weight loss. Differentiated hUCM cells could alleviate the insulin deprivation in the rat model of type I diabetes. In addition, higher NCM concentration leads to more differentiation into IPCs and more nestin and PDX1 expression. Kidney capsule can serve as a suitable nominee for IPCs transplantation.