rahim beheshti

 Premature ovarian insufficiency (POI) is a challenging issue in terms of reproduction biology. In this study, therapeutic properties of bone marrow CD146+ mesenchymal stem cells (MSCs) and CD144+ endothelial cells (ECs) were separately investigated in rats with POI.

 POI rats were classified into control POI, POI + CD146+ MSCs, and POI + CD144+ ECs groups. Enriched CD146+ MSCs and CD144+ ECs were directly injected into ovarian tissue (15 × 104 cells/10 μL) in relevant groups. After 4 weeks, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were measured in blood samples. Ovarian tissues were collected and subjected to Hematoxylin-Eosin and Masson’s trichrome staining. The expression of angp-2, vegfr-2, smad-2, -4, -6, and tgf-β1 was studied using qRT-PCR analysis. Histopathological examination indicated an increased pattern of atretic follicles in the POI group related to the control rats (P<0.0001).

 Data indicated that injection of POI + CD146+ MSCs and CD144+ ECs in POI rats reduced atretic follicles and increased the number of normal follicles (P<0.01). Along with these changes, the content of blue-colored collagen fibers was diminished after cell transplantation. Besides, cell transplantation in POI rats had the potential to reduce increased FSH, and LH levels (P<0.05). In contrast, E2 content was increased in POI + CD146+ MSCs and POI + CD144+ ECs groups compared to control POI rats, indicating restoration of follicular function. CD144+ (smad-2, and -4) and CD146+ (smad-6) cells altered the activity of genes belonging TGF-β signaling pathway. Unlike POI + CD146+ MSCs, aberrant angiogenesis properties were significantly down-regulated in POI + CD144+ ECs related to the control POI group (P<0.05).

 The transplantation of bone marrow CD146+ and CD144+ cells can lead to the restoration of ovarian tissue function in POI rats via modulating different mechanisms associated with angiogenesis and fibrosis

Cell-based therapies with certain cell types are touted as novel and hopeful therapeutic intervention in the clinical setting. Here, we aimed to assess the regenerative potential of c-Kit+ cells in the rejuvenation of ovarian tissue and fertility rate in rat model of premature ovarian failure (POF).

Rats were treated with 160 mg/kg/BW of 4-vinylcyclohexene dioxide for 15 days. Freshly enriched rat bone marrow-derived c-Kit+ (MACS) and c-Kit- cells (4×105 cells/10 µL) were transplanted into the ovaries of treatment and control animals. Prior to transplantation as well as 2, 4, 6, and 8 weeks post-transplantation, randomly-selected rats were euthanized and ovarian tissues were subjected to pathophysiological examinations and real-time PCR analyses.

POF status was confirmed by the presence of pathological features and a decreased number of immature and mature follicles compared with the control group (P < 0.05). Histological examination revealed a substantial reduction of atretic follicles in POF rats receiving c-Kit+ cells in comparison with POF rats that did not receive these cells (P < 0.05). Compared with the control samples, angiogenesis-related genes, Angpt2 and KDR, showed increased and decreased expressions in POF ovaries, respectively (P < 0.05). c-Kit+ cells had potential to restore angiogenesis in the ovarian tissue within normal ranges. Systemic levels of FSH did not significantly change in pre- or post-transplantation time points for any group (P > 0.05). Notable reduction of collagen deposition was found in c-Kit-treated rats. Transplantation of c-Kit+ cells also restored the reduced fertility rate (P < 0.05).

The administration of c-Kit+ cells can modulate angiogenesis and pathological changes, leading to the rejuvenation of ovarian function of a rat model of premature menopause.

Many attempts have been made to preserve fertility by improving the cryopreservation of the ovarian tissue. This current studyaimed to improve of direct cover vitrification (DCV) protocol on follicular preservation and angiogenesis in autografted ovarian tissue.

In this experimental study, sixty five female Balb/c mice (5-6 week-old) were anesthetized and their ovaries were dissected. The left ovaries were vitrified by DCV solution, thawed by descending concentrations of sucrose, and then autografted subcutaneously. The right ovaries were autografted with no vitrification procedure prior to transplantation. The animals were sacrificed under anesthesia on the 7th day after transplantation to obtain ovarian tissue. Follicular quality was assessed by histological and ultrastructure observations, and angiogenesis was examined by immunohistochemical staining and real-time polymerase chain reaction (PCR) analysis.

The histological and ultrastructure features of the follicles preserved well after vitrification of the ovarian tissue by 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO). Revascularizationwas manifested prominently in the DCV1-vitrified/grafted ovaries by von Willebrand factor (vWF) and alpha smooth muscle actin (α-SMA) immunostaining. The ovarian tissue vitrified in DCV1 protocol had higher expression levels of angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) 7 days after autotransplantation (P<0.01).

These findings suggest that DCV with 10% of both EG and DMSO, is an effective cryopreservation solution for preservation of good quality follicles as well an upregulation of angiogenic factors after ovarian tissue transplantation.

Cryopreservation of mammalian ovaries has been reported with different levels of success. Cryopreservation of ovarian tissue may be a potential alternative for treatment of infertility and many attempts have been done to improve the efficiency of ovarian cryopreservation. The objective of the present study was to compare the direct cover vitrification (DCV) with ethylene glycol (EG), dimethyl sulfoxide (DMSO) and EG plus DMSO. Eighty five mice were sacrificed by cervical dislocation and their ovaries were cryopreserved in the presence of 5% EG or DMSO alone or as mixture, 10% EG or DMSO alone or as mixture and a group with ascending concentrations of cryoprotectants. After toxicity testing and vitrification warming, the ovaries were fixed for histological and ultrastructural studies. In addition, the viability of mechanically isolated follicles was studied by trypan blue staining. All data were compared by ANOVA (p<0.05). Ovarian tissues frozen in EG plus DMSO in ascending concentrations retained a higher percentage of morphologically normal and or viable follicles than tissues frozen in 10 M EG plus DMSO or in either concentration of EG and DMSO alone (p<0.001). Ultrastructural analysis of ovarian tissues frozen in ascending concentrations of EG plus DMSO showed that these follicles were well preserved and it was very similar to the control group. Cryopreservation of ovarian tissue in EG plus DMSO is the most effective method for preserving the structural integrity of follicles within the ovary.

This study was designed to investigate the effect of cysteamine and vitamin E on post thaw buffalo bull's sperm quality. For this purpose, 20 ejaculates from four buffalo bulls possessing more than 70% visual sperm motility were diluted at 37◦C in BioXcell® extender. The diluted semen was cooled to 4◦C within 2 hours, equilibrated at 4◦C following the addition of (0.75, 1.5, 2 and 5 mM) of vitamin E and (7.5, 12.5, 15 and 20 mM) cysteamine per 90 ml, filled in 0.5 ml French straws and were subjected to cooling condition before being plunged into liquid nitrogen. Semen was thawed at 37◦C for 40 seconds after 72 hours of storage inside liquid nitrogen .Post-thaw sperm motility and some qualitative parameters of each frozen semen sample were assessed by using computer assisted semen analyzer (CASA). In general, the results showed that the addition of 1.5 mM vitamin E and 20 mM cysteamine in the commercial diluents BioXcell extender for freezing buffalo semen increased the motility of spermatozoa and some qualitative parameters to post-thawed buffalo sperm.

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen–thawed semen quality of buffalo. Ejaculated semen was extended in a Tris–citrate egg yolk and bioexcell extenders containing various concentrations of BHT (0. 5، 1. 0، 2. 0 and 3. 0 mM). After cooling at 5 ºC، semen samples were frozen in liquid nitrogen vapor and plunged into liquid nitrogen for storage. Straws from each treatment were thawed to assess the semen quality in terms of sperm motility، viability، plasma membrane integrity and acrosomal integrity at 0 and 6 h after thawing. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability، plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining، hypo-osmotic swelling test and normal acrosomal reaction، respectively. Adding BHT in two extenders conferred better cryopreserving ability to spermatozoa compared to control groups. The highest (P < 0. 05) value was achieved by addition of 2. 0 mM BHT upon thawing and 1. 0 mM at 6 h after thawing. However، higher concentration of BHT (3. 0 mM) reduced the some evaluated parameters compared to other concentration used.