L-Carnitine improves follicular survival and function in ovarian grafts in the mouse.

CONTEXT: Ovarian tissue transplantation is performed to preserve fertility in patients undergoing chemotherapy and radiotherapy. However, the ischemia-reperfusion injury which occurs after the ovarian tissue transplantation causes follicular depletion and apoptosis. l -Carnitine has antioxidant and anti-inflammation properties. AIMS: Therefore, we aimed to investigate the beneficial effect of l -carnitine on mouse ovaries following heterotopic autotransplantation. METHODS: Mice were randomly divided into three groups (six mice per group): control, autografted and autografted+l -carnitine (200mg/kg daily intraperitoneal injections). Seven days after ovary autografting, the serum levels of malondialdehyde (MDA), total antioxidant capacity, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10 were measured. Ovary histology, serum concentrations of progesterone and estradiol were also measured 28days after autotransplantation. Data were analysed using one-way analysis of variance (ANOVA) and Tukey test, and the means were considered significantly different at P Key results: In the autografted+l -carnitine group, the total volume of the ovary, the volume of the cortex, the number of follicles, the serum concentrations of IL-10, estradiol and progesterone significantly increased compared to the autografted group. In the autografted+l -carnitine group, serum concentrations of IL-6, TNF-α and MDA were significantly decreased compared to the autografted group. CONCLUSIONS: Our results indicated that l -carnitine can ameliorate the consequences of ischemia-reperfusion on the mice ovarian tissue following autotransplantation. IMPLICATIONS: l -carnitine improves the structure and function of transplanted ovaries.

L-Carnitine improves endocrine function and folliculogenesis by reducing inflammation, oxidative stress and apoptosis in mice following induction of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is related to low levels of serum l-carnitine, which has antioxidant, anti-inflammatory and antiapoptotic properties. The aim of this study was to investigate the effect of l-carnitine on folliculogenesis in mice following induction of PCOS. PCOS was induced by daily injections of testosterone enanthate (1mg per 100g, s.c., for 35 days). NMRI mice (21 days old) were divided into four groups (n=6 per group): Control, Control+l-carnitine, PCOS and PCOS+l-carnitine. Mice were treated with 500mgkg-1, i.p., l-carnitine every second day for 28 days. Ovaries were studied stereologically and serum concentrations of FSH, LH, testosterone, interleukin (IL)-6 and tumour necrosis factor (TNF)-α were determined using ELISA kits. Serum concentrations of malondialdehyde (MDA) and the ferric ion reducing antioxidant power (FRAP) were also analysed. Apoptosis of follicles was evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL). CD31 was assessed immunohistochemically. Data were analysed using one-way analysis of variance (ANOVA) and Tukey's test, differences considered significant at P<0.05.The total volume of the ovary, cortex volume, oocyte volume, zona pellucida thickness and the number of antral follicles increased significantly, whereas the number of primary and preantral follicles decreased significantly, in the PCOS+l-carnitine versus PCOS group. In the PCOS+l-carnitine group, serum concentrations of FSH and FRAP increased significantly, whereas there were significant decreases in serum concentrations of testosterone, LH, MDA, IL-6 and TNF-α, as well as in the percentage of TUNEL-positive apoptotic cells, compared with the PCOS group. l-Carnitine improves folliculogenesis and is therefore suggested as a therapeutic supplement in the treatment of PCOS.

Diabetic sera disrupted the normal exosome signaling pathway in human mesenchymal stem cells in vitro.

Human mesenchymal stem cells were exposed to diabetic sera for 7 days. Cell viability and apoptosis rate were detected by MTT and flow cytometry assays. The expression of key genes such as CD63, Alix, Rab27a, Rab27b, and Rab8b was monitored by real-time PCR. We also measured acetylcholinesterase activity and size and zeta potential of exosomes in the supernatant form diabetic cells and control. The cellular distribution of CD63 was shown by immunofluorescence imaging and western blotting. Any changes in the ultrastructure of cells were visualized by electron microscopy. Data showed a slight decrease in survival rate and an increased apoptosis in diabetic cells as compared to control (p < 0.05). By exposing cells to diabetic sera, a significant increase in the level of all genes CD63, Alix, Rab27a, Rab27b, and Rab8b was observed (p < 0.05). Flow cytometry analysis and immunofluorescence imaging confirmed increasing CD63 protein content upon treatment with diabetic sera (p < 0.05). We found an enhanced acetylcholinesterase activity in a diabetic condition which coincided with the increasing size of exosomes and decrease in zeta potential (p < 0.05). The fatty acid profile was not significantly affected by diabetic sera. Ultrastructural examination detected more accumulated cytoplasmic lipid vacuoles in diabetic cells.

Improvement of the folliculogenesis by transplantation of bone marrow mesenchymal stromal cells in mice with induced polycystic ovary syndrome.

BACKGROUND: Many studies have reported that inflammation and oxidative stress are involved in the pathogenesis of polycystic ovary syndrome (PCOS). Bone marrow mesenchymal stromal cells (BM-MSCs) have anti-oxidant and anti-inflammation properties. In this study, we investigate the beneficial effect of stem cell therapy on folliculogenesis in mice with induced PCOS METHODS: Mouse model of PCOS was performed through daily injection of testosterone enanthate (1 mg/100 g/body weight subcutaneous (s.c).) for a period of 5 weeks. Naval Medical Research Institute (NMRI) mice (21 days old) were divided into three groups: control, PCOS and PCOS + BM-MSCs. BM-MSCs were labeled with Hoechst 33342 (0.5 µg/mL) and then injected into the mice (106/animal, via the tail vein) at 1 and 14 days after PCOS confirmation. Mice were humanely killed at 2 weeks after last transplantation. Ovarian stereological studies were done. Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), testosterone, interleukin (IL)-6 and tumor necrosis factor (TNF)-α serum levels were measured. The levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum were analyzed. Apoptotic index for ovarian follicles was assessed using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). CD31 expression in ovarian vessels was assessed with the immunohistochemistry. RESULTS: There was a significant increase in the total volume of ovary, cortex, number of antral follicles, volume of oocyte and zona pellucida thickness, and there was a significant decrease in the primary and preantral follicles number in the PCOS + BM-MSCs group compared with the PCOS group. There was a significant increase in the serum level of FSH and TAC and a significant decrease in the serum level of testosterone, LH, MDA and percentage of TUNEL-positive apoptotic cells in the PCOS + BM-MSCs group in comparison with the PCOS group. DISCUSSION: BM-MSC transplantation improves folliculogenesis in mice with induced PCOS. BM-MSC therapy can be an operative treatment for PCOS via anti-inflammatory, anti-oxidant and anti-apoptotic properties.

N-Acetylcysteine improves oocyte and embryo quality in polycystic ovary syndrome patients undergoing intracytoplasmic sperm injection: an alternative to metformin.

Polycystic ovary syndrome (PCOS) is associated with low-quality oocytes. The aim of the present study was to investigate the effects of metformin (MET), N-acetylcysteine (NAC) and their combination on follicular fluid parameters, oocytes and embryo quality in PCOS patients. A prospective randomised placebo-controlled pilot study on 60 Iranian women with PCOS (aged 25-35 years) undergoing intracytoplasmic sperm injection (ICSI) was designed. Women were divided into four groups (n=15 in each): (1) an MET, administered 1500mg day(-1) MET; (2) an NAC group, administered 1800mg day(-1) NAC; (3) an NAC + MET group; and (4) a placebo group. Drugs were administered from the 3rd day of previous cycle until the day of oocyte aspiration (6 weeks treatment in total). Data were analysed by one-way ANOVA, with significance set at P<0.05. The number of immature and abnormal oocytes decreased significantly in the NAC compared with placebo group, with a concomitant increase in the number of good-quality embryos in the NAC group (P<0.05). Malondialdehyde levels decreased significantly in the NAC and NAC + MET groups compared with the placebo-treated group (P<0.02). In addition, there were significant decreases in leptin levels in the NAC, MET and NAC + MET groups compared with the placebo group (P<0.001). Insulin and LH levels were significantly lower in the MET and NAC groups compared with the placebo-treated group (P<0.02). We concluded that NAC improves oocyte and embryo quality and could be administered as an alternative to MET.

Sitagliptin/Metformin: A New Medical Treatment in Polycystic Ovary Syndrome.

Metformin has long been used in the treatment of polycystic ovarian syndrome (PCOS). Recently, sitagliptin has been reported to improve ovarian cycles and ovulation in PCOS. We suggest that a combination of sitagliptin and metformin can be more effective than either treatment alone in improving different aspects of PCOS.

Co-Administration of Metformin and N-Acetyl Cysteine Fails to Improve Clinical Manifestations in PCOS Individual Undergoing ICSI.

BACKGROUND: Studies have demonstrated the efficacy of metformin (MTF ) in reducing insulin resistance and N-acetyl cysteine (NAC) in inhibiting oxidative stress which are involved in the pathogenesis of polycystic ovarian syndrome (PCOS). We aimed to compare the effects of MTF and NAC combination on serum metabolite and hormonal levels during the course of ovulation induction in PCOS individual candidates of intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: In this prospective randomized clinical trial, placebo con- trolled pilot study, 80 patients of polycystic ovarian syndrome at the age of 25-35 years were divided into 4 groups (n=20): i. NAC=treated with N-acetyl cysteine (600 mg three times daily), ii. MTF=treated with metformin (500 mg three times daily), iii. MTF+NAC=treated with N-acetyl cysteine plus metformin (the offered doses) and iv. placebo (PLA). A total number of 20 patients (6 from MTF group, 4 from NAC group, 6 from MTF+NAC group and 4 from PLA group) were dropped of the study. The drugs were administrated from day 3 of menses of previous cycle until ovum pick-up. RESULTS: Serum levels of luteinizing hormone (LH), total testosterone, cholester- ol and triglyceride, insulin and leptin significantly reduced in the MTF and NAC groups compared to the placebo (p<0.01). But levels of LH, total testosterone, cholesterol and triglyceride had no significant reduction in the MTF+NAC groups compared to the placebo. The serum levels of malonyldialdehyde (MDA), insulin and leptin reduced significantly after treatment in the MTF+NAC group compared to the placebo (p<0.05). CONCLUSION: CONSIDERING THE ADVERSE EFFECT OF COMBINATION THERAPY, WE PROPOSED THE CONADMINISTRATION MIGHT HAVE NO BENEFICIAL EFFECT FOR PCOS PATIENT DURING COURSE OF OVULATION INDUCTION OF ICSI (REGISTRATION NUMBER: IRCT201204159476N1).

Caspase-mediated apoptosis in sensory neurons of cultured dorsal root Ganglia in adult mouse.

OBJECTIVE: Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG. MATERIALS AND METHODS: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 µM) and immunohistochemistry for activated caspase-3 were used. RESULTS: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours. CONCLUSION: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

Para-nonylphenol impairs osteogenic differentiation of rat bone marrow mesenchymal stem cells by influencing the osteoblasts mineralization.

OBJECTIVES: Para-Nonylphenol (p-NP) is used in many industries and our previous study showed that p-NP causes a reduction in rats bone marrow mesenchymal stem cells (MSCs) viability. The aim of this study was to investigate the effect of p-NP on osteogenic differentiation of MSCs. MATERIALS AND METHODS: MSCs were isolated and expanded to 3rd passage, then cultured in DMEM supplemented with osteogenic media as well as 0.5 or 2.5 µM of p-NP. After 5, 10, 15, and 21 days, the viability and the level of mineralization was determined using MTT assay and alizarin red, respectively. In addition, morphology and nuclear diameter of the cells were studied with the help of fluorescent dye. Furthermore, calcium content and alkalinphosphatase activity were also estimated using commercial kits. Data were statistically analyzed and the P<0.05 was taken as the level of significance. RESULTS: The viability and mineralization of the cells treated with 2.5 µM of p-NP reduced significantly after day 10 in comparison with the control group and administration of 0.5 µM. Moreover, chromatin condensation, reduction of nuclei diameter, and cytoplasm shrinkage was observed in the cell treated with 2.5 µM. The calcium concentration and alkalinphosphatase activity of the cells decreased significantly with 2.5 µM of p-NP when compared with 0.5 µM and control group. CONCLUSION: Adverse effect of p-NP was observed on osteogenic differentiation of MSCs at 2.5 µM due to disruption of mineralization. We strongly suggest more investigations on this chemical with respect to other stem cells, especially skin stem cells as p-NP is used in the formulation of cosmetics.