[Mesenchymal stem cells proliferation exposed to hypoxia]

Background and Objective: Some problems such as low viability and apoptosis after injection to the body because of exposure to toxic factors such as hypoxia, thermal stress, oxidative stress and food deprivation are encountered with stem cell application. It is suggested that preconditioning of the cells with cytotoxic factors before injection could enhance their efficiency. This study was done to determine the mesenchymal stem cell proliferation exposed to hypoxia by cobalt chloride

Methods: In this experimental study, Mesenchymal stem cells were isolated from rat bone marrow and cultured at least for four times. The cells were cultured in 96 well plates and treated with different concentration [0, 5, 10, 20, 50, 70, 90, 100, 120, 150 and 200 microM] of cobalt chloride for 6, 12, 24 and 46 hours. Cell proliferation was detected by MTT assay [3-[4,5-Dimethylthiazol-2-Yl]-2,5- Diphenyltetrazolium Bromide]

Results: The cells isolated from bone marrow were propagated easily in culture condition. The cells morphology was not altered after exposure to cobalt chloride. Preconditioning of mesenchymal stem cells with 120 microM for 6 hours, 20 microM for 12 and 24 hours and 5 microM for 48 hours significantly improved cell proliferation after hypoxia in cell culture [P<0.05]

Conclusion: Hypoxia preconditioning increases proliferation of mesenchymal stem cell

Histomorphological evaluation of fresh ovarian tissue transplanted into back muscles of Balb/C mice

Today, different methods for maintaining reproductive capability in young women with cancer are being considered. One of the most prominent of these methods is ovarian tissue transplant. Despite the relative success of this method, the appropriate location and methods of transplantation is still a matter of discussion. The present study evaluated the histomorphology of fresh ovarian tissue transplantation by two methods, inter muscular and intra muscular, in Balb/C mice. The study was conducted at Hamedan University of Medical Sciences in 2009. Fresh ovarian tissues from 12-14 day old Balb/C mice were transplanted into back muscles of ovarectomized 6 week old Balb/C mice both intermuscularly and intramuscularly. All transplanted mice received intra-peritoneal injections of a unit of rFSH for 4 weeks, every other day. At the end of the tenth week, all transplant recipient mice were killed and the transplanted ovarian tissues were removed. All samples were assessed for the angiogenesis and viability of follicles. Data were analyzed using SPSS software, using independent t-test. In intermuscular transplanted group, the transplanted tissues were rejected in two cases. In the sections prepared from the other cases, in spite of the presence of some small necrotic areas, the majority of ovarian tissues had a healthy appearance within the primordial, primary, secondary and antral follicles. Apart from a significant reduction in the number of follicles and smaller size of follicles in the transplanted tissue in comparison with control group, no other major differences in morphology, histology, and the process of maturation of ovarian follicles were observed between the transplanted and control groups. Fresh ovarian tissue transplantation into muscles of the back area without basic vascular pedicle has new angiogenesis capabilities, appropriate survival and development of primordial follicles and significant natural growth of maturing follicles. Degeneration of transplanted tissue in the intra muscular area indicates that it is an inappropriate site for transplantation

[ in vitro assessment of biochemical factors in hepatocyte like cells derived from umbilical cord blood stem cells]

Umbilical cord blood [UCB] is a source of Hematopoietic Stem Cells [HSC] and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood [UCB] have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood [UCB] derived stem cells to functional hepatocytes. The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood [UCB] was obtained from Fatemieh hospital [Hamadan, Iran]. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum [FBS] supplemented with 20 ng/mL Hepatocyte Growth Factor [HGF], 10 ng/mL basic Fibroblast Growth Factor [bFGF] and 20 ng/mL Oncostatin M [OSM].The medium was changed every 3 days and stored for Albumin [ALB], Alpha Fetoprotein [AFP], Alkaline Phosphatase [ALP], and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Measurement of biochemical factors in different days showed that concentration of albumin [ALB], alpha fetoprotein [AFP], alkaline phosphatase [ALP], and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Stem cell-derived from human umbilical cord blood [HUCB] is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells

[Induction of differentiation of bone marrow mesenchymal stem cells of rat into hepatocyte and study of their biochemical factors]

Bone marrow mesenchymal stem cells [MSCs] are multi potential and capable of differentiating into specialized tissues. The aim of this study was to investigate the induction of differentiation of the bone marrow MSCs of rat into hepatocytes by use of fibroblast growth factor-2 [bFGF], hepatocyte growth factor [HGF] and oncostatin M [OSM] in order to find a suitable source of hepatoecytes for bioartificial liver or liver transplantation. In this research MSCs were obtained from the bone marrow of rat and cultured in DMEM-LG medium supplemented with 15% FBS. These cells were treated with differential medium supplemented with HGF, bFGF and OSM for 24 days. Morphology, RT-PCR and Biochemical assays were used to identify the differentiation of stem cells into hepatic cells. Result: MSCs took a round shape after differentiation, while undifferentiated cells had fibroblast-like morphology. Albumin, urea and alpha-fetoprotein [AFP] were detected in differentiated cells. Alkaline phosphatase activity was confirmed by biochemical tests. The mRNA expression of CK-18 and tyrosine amino transferase [TAT] in differentiated cells was demonstrated by RT-PCR after induction. The rat MSCs from bone marrow can differentiate into hepatocyte-like cells in the presence of HGF, bFGF and OSM in vitro. Cytokines may play an important role in differentiation of bone marrow MSCs of rat into hepatocytes. Bone marrow derived MSCs are a new source of cell types which can be used for cell transplantation in hepatic diseases

[Effects of carnitine on sperm parameters of infertile males with idiopathic asthenospermia]

Studies confirm that a number of nutritional and environmental factors may negatively affect spermatogenesis and cause male infertility. Carnitine is an important factor for sperm motility. Carnitine deficiency decreases sperm motility and may cause male infertility. The aim of this study was to assess the effects of carnitine on sperm parameters in infertile males with idiopathic asthenospermia. This study is a before and after clinical trial performed on 40 asthenospermia men who were treated with 750 mg per/day carnitine in Fatemieh infertility research center in years 2006-2007. Sperm parameters were assessed before and after treatment. The obtained data were analyzed using SPSS10 and paired T-test. The results showed a significant increase in sperm concentration, morphology, sperm total motility and rapid progressive motility after treatment by carnitine [p<0.05]. Carnitine supplementation has a significant effect on sperm parameters in men with idiopathic asthenospermia

Analysis of prognostic factors for successful outcome in patients undergoing intrauterine insemination

Intrauterine insemination [IUI] has been widely used for the treatment of infertility. Several prognostic factors for IUI outcome have been proposed, including the endometrial thickness and follicle numbers, etiology and duration of infertility and morphology, type and motility of sperms. A total of 463 IUI cycles in which clomiphene citrate and/or human menopausal gonadotrophin [HMG] were used for ovarian stimulation analyzed retrospectively to identify prognostic factors regarding treatment outcome. The overall pregnancy rate was 13% per cycle. Logistic regression analyses were done on 14 sets of data, including age, Duration of infertility, Type of infertility, The etiology of infertility, Sperm count, Sperm motility before and after processing, The method of ovarian stimulation, Endometrial thickness, Type of catheter, Use of tenaculum, Season of IUI performing, The number of dominant follicle and cycle number. Logistic regression analysis revealed two predictive variables as regards pregnancy: number of the dominant follicles [P = 0.003] and the thickness of endometrium [P = 0.001]. The odds ratios for number of the dominant follicles and thickness of endometrium were 1.41 and 1.78 respectively. The results indicate that controlled ovarian hyperstimulation [COH] and IUI achieves the best results with increased number of preovulatory follicles and endometrial thickness

L-carnitine level in seminal plasma of fertile and infertile men

To determine any correlation between infertility and semen quality with concentrations of L-carnitine in human seminal plasma. This case-control study performed in Fatemieh Hospital in Hamadan, Iran. Seminal plasma of 72 infertile men and 80 men with proved fertility as a control group was investigated and L-carnitine level was determined using UV enzymatic test at 340 nm. The concentration of L-carnitine was significantly lower in the infertile group compared with control group [80.59 +/- 56.43 mg/l versus 108.43 +/- 42.26 mg/l; P= 0.0009]. There was also a statistically significant positive correlation between seminal plasma L-carnitine concentration, total sperm count, and the percentage of motile sperm [P= 0.0009, and P= 0.0000, respectively]. These finding suggest that the determination of seminal plasma L-carnitine level may be a useful test in evaluation of male infertility

[ effect of vitamin E in prevention of developmental defects of diabetic rats embryos]

Introduction: Now days it is proven that maternal hyperglycemia increases production of free radicals and decreases antioxidants in the embryonic cells and through this it injures the embryos of diabetic mothers. Therefore it seems that use of antioxidants can decrease deleterious effects of hyperglycemia on embryos

Objective: This study aims to investigate the effect of vitamin E in control of embryonic developmental disorders in diabetic rats

Materials and Methods: In this experimental study, diabetes was induced by intraperitoneal injection of 65-mg/kg streptozotocin in Wister female rats with 250-300g weights and was placed with healthy male rats in the same cage for mating. Then they were divided into two groups. One group received 150 mg/kg Vitamin E per day from the first day of pregnancy. Animals were killed on day 13 of gestation and embryos were collected and examined for number, size and incidence of malformations in cerebral hemispheres, cranial and caudal neuropors, upper and lower limb buds, otic and lens placodes using an stereomicroscope. Then, One Way ANOVA compared the means of length, number of reabsorbed and achieved embryos in three groups. The incidences of malformations in the groups were compared by Chi-Square test

Results: Findings show a significant difference between control group and experimental diabetic group when achieved and reabsorbed embryos counted. Frequencies of malformed embryos [at least one diagnosed malformation] in control group were 1% and in diabetic and treated diabetic groups were 46.3% and 11.6% respectively. Result showed that the incidence of embryonic malformation significantly decreased in diabetic rates, that received vitamin E and there was a significant difference in diabetes and treated diabetic groups [P<0.001]

Conclusion: þThe results shows that Vitamin E supplementation in early stages of pregnancy in diabetic mothers can prevent deleterious effects of diabetes

Nitric oxide level in seminal plasma of fertile and infertile males and its correlation with sperm parameters

Nitric oxide [NO] is a free radical molecule, produced by most cells and tissues in the body. The effect of NO on cells is concentration dependent. Low concentration of NO is essential in biology and physiology of most of cells, but high amounts of NO is toxic and has detrimental effects on cells. The role of NO in biology of male and female genital systems is under investigation. In the present study, the nitric oxide concentration was measured in the seminal plasma of both fertile and infertile males and compared with spermatogram parameters. For this purpose, semen samples were collected from 45 patients and 70 healthy donors. After analysis of samples, the stable metabolites of nitric oxide [nitrite and nitrate] were measured by Griess assay. The results indicated that the nitric oxide concentration in the seminal plasma of infertile males was significantly higher than controls. There was a significant negative correlation between the nitric oxide concentration and sperm motility and viability in infertile males. In conclusion, this study demonstrated that the level of nitric oxide in seminal plasma of infertile men was higher than that of fertile men. The increasing level of nitric oxide concentration in seminal plasma leads to the decrease in sperm motility and viability and affects fertility

Effects of L-arginine on development of mouse pre-implantation embryos in culture media with high level of glucose

It is accepted that maternal hyperglycemia causes delay in early embryonic development, spontaneous miscarriage and malformations. According to various studies, some of these problems occur in earlier stages of embryonic developmen especially pre-implantation stage. It seems that elevated glucose level of blood can have important role in this regard as potential teratogen factor. One of cases, which can be related to racousnesses resulting from glucose effects is Nitric Oxide [NO] system disorder in hyperglycemic condition. Some evidences show at first in hyperglycemic condition, L-arginine uptake of media by embryo increases and therefore leads to decrease amount of available L-arginine and since L-arginine is essential substrate for NO production, so it's decrease inhibits NO production. To examine this hypothesis, 2-cell embryos of mice were cultured in media of high concentration of glucose [30mM] and different concentrations of L-arginine [5, 10, 20 mM] and their growth and development were assessed and at the end, embryos were stained by Hoechst 33254 color and the number of their blactocysts were counted by use a Fluorescence microscope. Comparison of embryos culture in HTF culture media with different concentration of glucose and L-arginin showed in high glucose media up to 30 mM affects growth and development of embryos totally and decrease their blactocysts numbers, but addition of 5-10 mM L-arginine to this media significantly improves this condition. On the contrary addition of L-NAME [an antagonist of L-arginine] significantly inhibits the development of pre-implantation embryos. It seems that reduction in NO production in diabetes is due to decreases in amount of available L-arginine, because increase in L-arginine concentration in high glucose media up to 10 mM partially improves high glucose embryo toxicity. Base on acquired result, it seems use of L-arginine or material which cause NO release in media, can have important role in prevention of high glucosis embryo toxicity