The effect of kisspeptin on the maturation of human ovarian follicles in culture following vitrification-thawing processes.

OBJECTIVE: Ovarian cryopreservation is one of the effective methods to preserve fertility for cancer patients. Still, this approach has some problems, namely ROS, resulting in adverse effects on oocytes and ovarian follicles. Kisspeptin as an antioxidant to control ovarian function, directly or indirectly. In this study, the effect of kisspeptin on follicle maturation was evaluated in culture following ovarian cryopreservation. METHODS: Ovarian tissue samples of women between 20 and 35 years old (n=12) were laparoscopically collected. The samples were randomly divided into four groups: 1) control, 2) vitrification, 3) vitrified+1µM kisspeptin, and 4) vitrified+10µM kisspeptin. After vitrification and thawing processes, the tissues were cultured in DMEM medium for 7 days. H&E staining for histological evaluation, Real-Time PCR for GDF9 and BMP15 gene expression, and immunohistochemical staining for GDF9 and BMP15 protein expression were performed. RESULTS: In the vitrification group, ovarian tissue morphology was incoherent, and more primordial follicles than other follicle types were found. The expression of GDF9 and BMP15 genes and proteins were significantly decreased in this group compared with other groups (p<0.05). In the vitrification groups with kisspeptin (1 and 10 µM), the number of primary and secondary follicles was more than in the vitrification group. Besides, the expression of these genes and proteins was dramatically elevated in the vitrification groups with kisspeptin compared to the vitrification group alone (p<0.05). CONCLUSIONS: It seems that kisspeptin is an effective substance to improve the quality of the human ovarian cryopreservation medium by improving follicle maturation.

Sildenafil citrate ameliorates the adverse effects of cryopreservation on sperm quality in asthenozoospermic men.

In this study, the effects of Sildenafil Citrate on the sperm quality during cryopreservation in the asthenozoospermic patients were investigated for the first time. Thirty semen samples were collected from asthenozoospermic patients and each sample was divided into 3 groups: Control (fresh), Freeze and Freeze + Sildenafil. In each groups the sperm parameters, DNA fragmentation, acrosome integrity, protamine deficiency, mitochondrial membrane potential, plasma membrane integrity, the expression of Bcl-2 and HSP70 genes, as well as the level of Tumor necrosis factor-alpha, Malondialdehyde and antioxidants (Catalase, Glutathione, and Superoxide dismutase) in sperm were assessed. Data were analyzed statistically using Repeated Measure Analysis. The level of Malondialdehyde and Tumor necrosis factor-alpha, morphological abnormalities, DNA fragmentation, protamine deficiency and the expression of Bcl-2 and HSP70 genes increased significantly in the Freeze group compared to the Control, while the level of sperm parameters and antioxidants, plasma membrane integrity, mitochondrial membrane potential and acrosomal integrity significantly decreased. In the Freeze + Sildenafil group, compared to the Freeze group, all the mentioned parameters were significantly reversed except for the acrosomal integrity (decreased even more) and the expression of Bcl-2 (increased even more) and HSP70 genes (with no change). Although adding Sildenafil to the freezing medium decreased the adverse effects of freezing on the sperm of asthenozoospermic patients and improved sperm quality, but it also caused premature acrosome reaction. Therefore, we suggest the consumption of Sildenafil along with another antioxidant, to benefit from the favorable effects of Sildenafil as well as to maintain the integrity of the sperm acrosome.

Comparative evaluation of metformin & sitaformin in classic PCOS patients undergoing intracytoplasmic sperm injection: A randomized controlled pilot study.

Background & objectives: Studies have shown that insulin resistance and hyperinsulinaemia play a major role in the pathogenesis of polycystic ovary syndrome (PCOS). Therefore, the use of insulin sensitizing drugs in the treatment of PCOS has attracted the attention of medicine and researchers. The aim of this study was to investigate the effects of sitaformin (sitagliptin/metformin) and metformin on the quality of oocyte and embryo in classic PCOS patients undergoing intracytoplasmic sperm injection (ICSI). Methods: Sixty patients of PCOS (25-35 yr) were randomly allocated into three groups (n=20, each group): a metformin-treated group (administered metformin 500 mg twice daily), a sitaformin-treated group (administered sitaformin 50/500 mg twice daily) and a placebo group. Participants in all the groups received the drug two months prior to the start of the ovulation cycle and treatment continued until the day of the oocyte aspiration. Results: Serum insulin and total testosterone levels decreaseed significantly after treatment in both the treatment groups as compared to the placebo (P<0.05). A significant decrease in the number of immature oocytes [MI + germinal vesicle (GV) stage] was observed in metformin and sitaformin groups as compared to the placebo. In addition, sitaformin group when compared to the metformin group showed a significant decrease in the number of immature oocytes (P<0.05). The number of mature and normal MII oocytes increased significantly in both the treatment groups compared to the placebo group (P<0.05). The number of mature and normal oocytes increased in sitaformin group in comparison to the metformin group, but the difference was not significant. There was a significant increase in the number of grade I embryos, fertilization and cleavage rates in the sitaformin group compared to the other groups (P<0.05). Interpretation & conclusions: This is the first study to compare the impact of sitaformin with metformin on oocyte and embryo quality in women with PCOS undergoing a gonadotropin-releasing hormone (GnRH) antagonist cycle. In conclusion, sitaformin can be more effective in decreasing immature oocytes and increasing the quality of embryos than the use of metformin.

The therapeutic effect of co-administration of pentoxifylline and zinc in men with idiopathic infertility.

Pentoxifylline is a derivative of methylxanthine that affects sperm motility. Also, zinc is an antioxidant that is involved in the activation of antioxidant enzymes. This study aimed to evaluate the effect of co-administration of pentoxifylline, and zinc in men with idiopathic infertility. In the present study, men with idiopathic infertility were identified and randomly divided into four groups: pentoxifylline, zinc, pentoxifylline + zinc, and placebo. According to the grouping, the patients received pentoxifylline and zinc for 3 months. Then, sperm parameters, biochemical factors, reproductive hormones, inflammatory factors, and DNA damage were evaluated before and after intervention. Data analysis was performed using SPSS software. Pentoxifylline and zinc were significantly effective in improving biochemical parameters, inflammatory factors, concentration, and motility of sperm. Pentoxifylline did not affect sperm morphology and reproductive hormones. However, in the zinc and zinc + pentoxifylline groups, a significant increase in normal morphology and reproductive hormones was observed. In the pentoxifylline group, sperm DNA fragmentation increased significantly, while in the zinc and zinc + pentoxifylline group, DNA fragmentation reduced significantly. Because of the role of zinc in protecting sperm chromatin, it is recommended that zinc and pentoxifyllinebe prescribed simultaneously. Clinical trial code: NCT05156684.

Sitagliptin/metformin improves the fertilization rate and embryo quality in polycystic ovary syndrome patients through increasing the expression of GDF9 and BMP15: A new alternative to metformin (a randomized trial).

The aim of this study was to investigate the effects of sitagliptin/metformin (sitaformin), metformin and sitagliptin in PCOS patients. PCOS is a hormonal disorder and therefore the use of treatments that modulate hormone levels Like AMH, testosterone, insulin, leptin and especially FAI and HOMA-IR can reclaim the symptoms of PCOS. PCOS also increases oxidative stress and lipid peroxidation. Therefore, in clinical and research trials, the level of these factors is usually examined to reduce patients' symptoms. Participants were randomly assigned to receive metformin, sitagliptin, sitaformin or placebo Treatment was carried out 2 months before the start of the ovulation cycle and continued until the day of ovum pick up. The serum levels of HOMA-IR and FAI decreased significantly in the treated groups compared to the placebo. The serum and the FF levels of leptin also decreased significantly in the sitaformin group when compared to the metformin and sitagliptin groups. Moreover, the serum and FF levels of AMH and MDA had a significant decrease in the sitaformin and sitagliptin group compared to the placebo. The mRNA expression and protein levels of GDF9 and BMP15 in the cumulus cells increased significantly in the sitaformin compared to metformin and sitagliptin groups. The expression level of GDF9 and BMP15 mRNA were positively correlated with the fertilization rate and grade I embryos. Sitaformin improves levels of GDF9 and BMP15 in PCOS compared to metformin and sitagliptin, which can increase the rate of fertilization and grade I embryos.

Taurine improves follicular survival and function of mice ovarian grafts through increasing CD31 and GDF9 expression and reducing oxidative stress and apoptosis.

Ischemia-reperfusion (IR) injury is a major limitation of ovarian transplantation which threatens the follicular and graft survival. Taurine as a potent anti-oxidant, anti-apoptotic and anti-inflammatory agent, can prevent graft damages due to IR. We aimed to investigate the effect of taurine on the follicular survival and function of autotransplanted mouse ovaries. Female mice (4-5 weeks old) were divided into: control, autograft and autograft + taurine (200 mg/kg/day). The level of CD31 expression was evaluated two days (48 h) post transplantation. In addition, on day 7 post transplantation the serum levels of malondialdehyde (MDA) and the total antioxidant capacity (TAC) were assessed. Also, 28 days post transplantation; ovaries were studied stereologically and the percentage of apoptotic follicles, level of GDF9 expression and the serum concentrations of progesterone and estradiol were measured. Data were analyzed using one-way ANOVA and Tukey's test and the means were considered significantly different at P < 0.05. The total volume of the ovary (P < 0.01), volume of the cortex (P < 0.01) and medulla (P < 0.04), total number of different types of follicles, expression of GDF9 and CD31 and also the levels of progesterone, estradiol and TAC increased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). The MDA level and apoptosis rate decreased significantly in the autograft + taurine group compared to the autograft group (P < 0.001). Taurine could significantly improve follicular survival and the function of grafted ovaries by accelerating the angiogenesis and reducing oxidative stress and apoptosis.

Utilizing platelet-rich fibrin bioscaffold at the graft site improves the structure and function of mice ovarian grafts.

Aim: The effect of platelet-rich fibrin (PRF) bioscaffold on the structure and function of mice-autotransplanted ovaries was investigated. Materials & methods: Mice were divided into three groups: control, autografted and autografted + PRF bioscaffold. Angiogenesis, ovary histology and serum biochemical factors were assessed. Results: The total volume of the ovary, the number of follicles and the level of superoxide dismutase activity, total antioxidant capacity, IL-10, progesterone and estradiol were significantly higher in the autografted + PRF bioscaffold group compared with the autografted group. In the autografted + PRF bioscaffold group, angiogenesis was accelerated and apoptosis rate, IL-6, TNF-α, malondialdehyde concentrations were significantly lower compared with the autografted group. Conclusion: PRF bioscaffold improves the structure and function of mice-autografted ovary.

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.

Differentiation of stem cells from the apical papilla into osteoblasts by the elastic modulus of porous silk fibroin scaffolds.

In addition to traditional approach for differentiation (biochemical factors), physical properties (elastic modulus) of the matrix will be becoming an important factor in differentiation of stem cells into different lineages. The porous scaffolds were prepared from the silk fibroin solutions 4%, 5%, 6%, 7% and 8.4% weight per volume (w/v) by freeze drying which produced scaffolds with the minimal changes in the pore size (96-160 µm) but with a ≈ 8-fold range of modulus (16 -131 kPa) in the wet condition. After isolation and characterization, stem cells of the apical papilla (SCAP) were seeded on the scaffolds and the osteogenic differentiation was evaluated by alizerin red staining, alkaline phosphatase test, real time PCR and immunohistochemistry. Results demonstrated modulus-dependent osteogenic differentiation of the apical papilla stem cells from 4% w/v (16 kPa) to 7% w/v (83 kPa) and then a sudden decrease of differentiation was observed in the 8.4% w/v (131 kPa). The highest differentiation was happened in the 7 % w/v (83 kPa). The stem cells can respond to elasticity ranged from 0.1 to 100 kPa and then differentiate into different lineages in terms of the amount of elastic modulus. This is probably the reason why bone differentiation potential of the SCAP decreases in the modulus over 100 kPa.

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.

Auto-fluorescence of a silk fibroin-based scaffold and its interference with fluorophores in labeled cells.

Silk fibroin is increasingly emerging as an important biomaterial for tissue engineering applications. The ability to fluorescently image silk matrices under a microscope would be helpful in differentiating embedded labeled cells from background signal, critical for the study of silk-based engineered tissues. In this study, we fabricated a scaffold using freeze drying and confirmed its structure by X-ray diffraction and Fourier transform infrared spectroscopy. We then examined the fluorescence of the silk fibroin scaffold using confocal microscopy, both before and after cell seeding and fluorescent labeling. We subsequently investigated the fluorescent signature of the silk fibroin scaffold chemically. Fluorophore-labeled cells seeded into the scaffold showed the same fluorescent color as the scaffold itself when excited by the same wavelength of light. UV-Vis and fluorescence spectroscopy of a silk fibroin solution indicated absorption and emission maxima at 277 and 345 nm, respectively, which is a typical protein-derived signal. HPLC and GC-MS were used to detect quercetin and quercetin derivatives, without success. We therefore conclude that unlike silk cocoons, the fluorescent behavior of silk fibroin scaffolds does not derive from quercetin and its derivatives but from the intrinsic fluorescence of fibroin protein. We also find that the fluorescent signals deriving from a scaffold and from labeled cells embedded in it can be distinguished when the different optical channels are merged.

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.

Cadmium chloride toxicity suppresses osteogenic potential of rat bone marrow mesenchymal stem cells through reducing cell viability and bone matrix mineralization.

BACKGROUND AND OBJECTIVES: Cadmium an environmental pollutant, exert several risks to human health. In this study we investigated the effect of cadmium chloride (CdCl 2 ) on Viability, morphology and bone Matrix Miniralization of Rat Bone Marrow Mesenchymal Stem Cells (rMSCs). MATERIALS AND METHODS: rMSCs were cultured in DMEM containing 15% FBS and pen-strep. After 21 days of treatment with the selected doses of 750 and 2000 nM of CdCl 2 viability, colony forming unit, population doubling number, DAN breakage and the morphology of the cells were studied. Also to study the effects of CdCl2 on differentiation property, the morphology and bone matrix mineralization via estimation of intracellular calcium concentration and quantitative alizarin red were also evaluated in the cells using Hoechst, Acridine orange and Alizarin red staining. Data was analyzed using one-way ANOVA and Tukey ' s test and the means difference was considered significant at P<0.05. RESULTS: The mean viability, colony forming unit, population doubling number and also the mean bone matrix mineralization of the rMSCs treated with CdCl 2 significantly reduced in a dose dependent manner. Nuclear fragmentation and cytoplasm shrinkage was also seen in the treated cells. CONCLUSION: CdCl 2 can reduce the viability and bone matrix mineralization of rMSCs even at low doses.