The Effect of Wharton's Jelly-Derived Conditioned Medium on the In Vitro Maturation of Immature Oocytes, Embryo Development, and Genes Expression Involved in Apoptosis.

Oocyte cytoplasmic maturation is a crucial process during in vitro maturation (IVM), and finding an appropriate IVM medium that promotes oocyte competence is very critical in assisted reproductive technology (ART). The aim of this study was to investigate the effects of umbilical cord Wharton's jelly (WJ-MSCs)-derived conditioned media on the maturation of immature oocytes and their developmental potential in humans after IVM, as well as apoptotic gene expression. A total of 392 germinal vesicle (GV) oocytes were collected from 207 women aged 25-35 years and divided into two IVM groups: (1) control group, which was cultured in CleavTM medium, and (2) experimental group, which was cultured in supernatants of umbilical cord Wharton's jelly as a conditioned medium (CM). First, WJ-MSCs were isolated, and their purity was analyzed. The immunophenotypes of WJ-MSCs were analyzed by flow cytometry. The quantitative expression of BCL2, BAX, and BAG1 in matured oocytes and embryos was evaluated through quantitative real-time polymerase chain reaction (qRT-PCR). Our findings showed that WJ-MSCs have a high proliferating capacity. The purity of the isolated cells was further validated by immunophenotyping, which revealed that their surface antigen expression had phenotypical properties similar to WJ-MSCs. When compared to CD34 and CD45 surface markers, the enlarged cells were positive for CD90, CD105, and CD44. There were significant differences in cytoplasmic maturation of oocytes and embryo quality between the two groups. The mRNA expression levels of BCL-2, BAG1, and BAX in matured oocytes and embryos were also significantly different between the two groups. Therefore, WJ-MSCs medium indicated potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and genes expression of BAX, BCL-2, and BAG1.

The Detection of CatSper1 and CatSper3 Expression in Men with Normozoospermia and Asthenoteratozoospermia and Its Association with Sperm Parameters, Fertilization Rate, Embryo Quality.

CatSper affects sperm function and male fertilization capacity markers, including sperm motility and egg penetration. The study has aimed to evaluate the mRNA expression of CatSper1, and CatSper3 in the spermatozoa of men with normozoospermia and Asthenoteratozoospermia, and to assess the correlation between genes expression and sperm parameters, fertilization rate, and embryo quality in intracytoplasmic sperm injection (ICSI). Reverse transcription-polymerase chain reaction was utilized to evaluate the mRNA expression of CatSper1 and CatSper3 in sperm in two patient groups: Normozoospermia (NOR; n = 32), and Asthenoteratozoospermia (AT; n = 22). In all patients receiving intracytoplasmic sperm injection, the fertilization rate and embryo quality were evaluated. CatSper1, and CatSper3 mRNA expression in sperm was significantly lower in AT males than in NOR (P < 0.05). Levels of these genes demonstrated a significant positive correlation with sperm motility, mitochondrial membrane potential (MMP), capacitation, fertilization rate, cleavage rate, and embryo quality (P < 0.05) following ICSI. However, a negative correlation was found between mRNA expression of CatSper1, 3 and sperm DNA fragmentation (P < 0.05). Findings indicate low levels of CatSper1 and CatSper3 mRNA expression in men with Asthenoteratozoospermia, which resulted in poor sperm quality and impaired embryo development following ICSI therapy.

Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression.

Background: Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs). Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days. Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed. Conclusion: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.

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.

Association Between Single Nucleotide Polymorphisms (rs1484215 and rs6495096) in CYP11A1 Gene in Iranian Women with Polycystic Ovary Syndrome.

Background: Genetic factors are significantly have important role in the etiology of the polycystic ovary syndrome (PCOS). This study examined the possible relation of rs1484215 and rs6495096 polymorphisms of CYP11A1 gene in Iranian women with PCOS. Methods: The population of the case-control research included 100 women presenting with PCOS and 100 women as a control group who were referred to Infertility Center in Qom, Iran. The genotypes of rs1484215 and rs6495096 polymorphisms in CYP11A1 gene were detected with the tetra-ARMS PCR method. The independent segregation of alleles was tested for the Hardy-Weinberg equilibrium (HWE). Differences in quantitative traits were assessed between each group with a single PCOS feature and control group using Mann-Whitney U test for categorical variables and student's t-test for continuous variables. Statistical analysis of allele and genotype frequencies between women with PCOS and control was performed using the chi-square test. Significance level was defined as p<0.05. Results: There was a significant association of C with G alleles in rs6495096 polymorphism and susceptibility to PCOS (p=0.001), but no significant relation was found between C and T alleles in rs1484215 polymorphism and susceptibility to PCOS. Also, GG genotype of rs6495096 was significantly associated with the waist-to-hip ratio (WHR) and infertility duration compared to CG and CC (p<0.01). However, rs1484215 showed no association with these variables. Conclusion: The results from the research indicated that rs6495096 polymorphism of CYP11A1 gene is related to the PCOS risk in Iranians women.

Effect of Myo-inositol on sperm quality and biochemical factors in cryopreserved semen of patients with Asthenospermia.

In this study, the influence of myoinositol (MYO) as an antioxidant on the inhibition of the negative impacts of cryopreservation on sperm quality in men with Asthenospermia was investigated. In this prospective study, each semen sample from 25 cases was separated into three groups: Fresh, Control (with freezing medium), Myoinositol (2 mg/ml). According to the World Health Organization criteria (WHO) (2010), total motility, progressive sperm motility, viability, normal morphology, and DNA integrity were assessed. In addition, the hypo-osmotic swelling (HOS) test and mitochondrial membrane potential (MMP) were used. Total antioxidant capacity (TAC), malondialdehyde (MDA), and antioxidant enzyme activity were determined by the ELISA method. In contrast to the fresh samples, lipid peroxidation, DNA integrity damage, DNA fragmentation, HOST, and MMP had significant enhancement in the control samples. Sperm quality was significantly decreased (p < 0.05). Mean percentage viability, normal morphology, total motility, progressive motility, and DNA integrity were significantly enhanced in the MYO group in comparison to the control group (p < 0.05). The MDA and TAC levels and DNA damage in the MYO group were significantly lower compared to the control group (p < 0.05). The findings confirm that sperm quality in patients with Asthenospermia is improved by the administration of 2 mg/ml of myoinositol together with the freezing medium after sperm cryopreservation.

Structure and Antibacterial Activity of Chitosan from the American Cockroach, the German Cockroach and the Mealworm Beetle.

Background: Owing to chitosan properties such as biocompatibility and antimicrobial activities, and several applications in biomedical field, some physicochemical and anti-bacterial properties, and the level of chitosan from three species of American cockroach, Periplaneta americana (Dictyoptera: Blattidae), the German cockroach, Blattella germanica (Dictyoptera: Ectobiidae) and the Mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae) were investigated. Methods: The cuticle of adults derived from specimens was dried and grounded. The powders were demineralized as well as deproteinized after deacetylation via NaOH. At last, the chitosan yields from insects were studied for antibacterial activity on Gram-positive bacteria (Proteus mirabilis, Klebsiella pneumoniae), and Gram-negative bacteria (Enterococcus faecalis and Staphylococcus epidermidis). The Fourier transform infrared spectroscopy (FTIR) was used to analyze the chitosan composition. Results: The chitosan ratios of the American and German cockroaches and the mealworm beetle were 5.80, 2.95, and 1.70% per 3 g of the dried bodies respectively. The chitin DD's for the American cockroach, the German cockroach and the mealworm beetle were 36.8%, 31.5% and 27.3%, respectively. The bactericidal activity of chitosan obtained from the American cockroach at a concentration of 1% had the greatest effect on P. mirabilis compared to other concentrations, while chitosan obtained from the German cockroach at a concentration of 0.01% had the greatest effect on K. pneumoniae compared to other concentrations. Conclusion: According to the results, the anti-bacterial influence of the chitosan is based upon the insect species and chitosan concentration. Probably, the variation relates to the changes in the chitin structure among the three insect species.

Mesenchymal stem cells as professional actors in gastrointestinal cancer therapy: From Naïve to genetically modified.

Considering the high incidence and mortality rate of gastrointestinal cancers (GIs) worldwide and partial success of the current available GI cancer treatments, there is a necessity to discover more effective approaches in cancer therapy. The failure in conventional therapies seems to be related to the resistance of cancer cells to chemotherapy, inability to target tumor cells especially in metastatic cancers, deficient drug concentrations in tumor sites, and unfavorable effects on pivotal non-malignant bodily tissues following systemic administration. In this context, we need an appropriate carrier for the delivery of therapeutic agents specifically to the GI cancer site. Mesenchymal stem cells (MSCs), a prominent cell-based strategy for cancer treatment, overcome various cancer therapy limitations and could be used as vehicles to deliver many anticancer agents such as therapeutic genes (DNA or interference RNA), oncolytic viruses, and chemotherapeutic or nanoparticle drugs. Moreover, secreted molecules of unmodified MSCs lead to deregulation of several proteins and different signaling pathways eradicating cancer cells. In the present review, at first, we overview the characteristics and utility of MSCs in cancer therapy, secondly, we discuss the application of naïve MSCs and utilization of MSCs in the delivery of therapeutic agents in GI cancer therapy and, finally, more information about harnessing of genetically modified MSCs in GI cancer treatment will be presented.

The effect of Quercetin on the quality of sperm parameters in frozen-thawed semen of patients with Asthenospermia.

The study has aimed to investigate the effect of Quercetin, as a potent antioxidant, on preventing the negative effects of freezing process on sperm quality of patients with Asthenospermia. Semen sample from 25 patients was randomly divided into three groups; fresh, control and Quercetin (50 µM). Seven days after freezing, samples were thawed at ambient temperature. Total motility, progressive sperm motility, normal morphology, viability and DNA integrity were evaluated according to WHO criteria, Papanicolaou, eosin- nigrosine and acridine orange staining respectively. In addition, the health of sperm membrane and mitochondrial membrane potential was assessed by HOS test and rhodamine staining. MDA and antioxidant enzyme activity were also evaluated using ELISA method. In contrast to the fresh group, the mean level of MDA and DNA damage had significant increase in the control group and decreased significantly sperm quality (p ≤ 0.001). The mean percentage of total motility and progressive motility, normal morphology, viability and antioxidant enzyme activity had significant increase in the Quercetin group than the control group. In the Quercetin group, the MDA level and DNA damage also had significant reduction in comparison with the control group (p ≤ 0.001). Therefore, the Quercetin supplementation improves the quality of cryopreserved human semen.

Association of heat shock protein A2 expression and sperm quality after N-acetyl-cysteine supplementation in astheno-terato-zoospermic infertile men.

In infertile men, reduced expression of heat shock protein A2 (HSPA2) is related to reduced sperm quality and function. The present study has aimed to investigate the effects of N-acetyl-cysteine (NAC) supplementation on expression of heat shock protein A2 (HSPA2). In this study in continuation of previous study, semen samples from 50 astheno-terato-zoospermic men who have received NAC (600 mg/day) orally for three months were evaluated for expression HSPA2 using RT-PCR, and Western blot analysis. In addition, semen samples of these individuals were assessed for sperm parameters, DNA fragmentation (TUNEL), protamine deficiency (CMA3), lipid peroxidation index (MDA) and total antioxidant capacity (TCA). All assessment was carried out before and after NAC treatment. In addition to improved sperm parameters and aforementioned functional parameters, the presented results revealed the significant increase in relative expression levels of HSPA2 was obtained after using NAC treatment (p < .05). Correlation analysis also demonstrated that HSPA2 expression is significantly related to most of the assessed parameters. NAC may directly or indecently impose its beneficial effect through increased expression of HSPA2, which plays a potential role in proper folding of element needed to counteract stress condition in infertile individuals.

The protective effect of Kombucha against silver nanoparticles-induced toxicity on testicular tissue in NMRI mice.

Silver nanoparticles (SNPs) can pass from the cell membrane and testicular blood barrier due to their small size, and by increasing oxidative stress they cause disorder in the male reproductive system. Kombucha is a traditional fermented drink with detoxification and potent antioxidant properties. We aimed to examine the protective effect of Kombucha against the damages due to SNPs on the testis tissue. In this experimental study, NMRI mice were randomly separated into four groups (n = 6), namely control (distilled water), SNPs (500 mg/kg), Kombucha extract (9 ml/kg) and SNPs + Kombucha, and were treated with gavage for 35 days. A significant decrease in testosterone level and total antioxidant capacity, and a significant increase in malondialdehyde concentration was observed in the SNPs group in comparison with the control group. Histological studies on the testis of mice treated with SNPs showed vacuolation, decrease in generational epithelium thickness, seminiferous tubules diameter, testis volume and the number of spermatozoa in lumen of the seminiferous tubule and increase in the volume of interstitial space while the mentioned parameters were improved in the SNPs + Kombucha group compared to the SNPs group. Kombucha reduces the adverse effects of SNPs on testis tissue and improves the function of the male reproductive system.

N-Acetylcysteine Compared to Metformin, Improves The Expression Profile of Growth Differentiation Factor-9 and Receptor Tyrosine Kinase c-Kit in The Oocytes of Patients with Polycystic Ovarian Syndrome.

BACKGROUND: Paracrine disruption of growth factors in women with polycystic ovarian syndrome (PCOS) results in production of low quality oocyte, especially following ovulation induction. The aim of this study was to investigate the effects of metformin (MET), N-acetylcysteine (NAC) and their combination on the hormonal levels and expression profile of GDF-9, BMP-15 and c-kit, as hallmarks of oocyte quality, in PCOS patients. MATERIALS AND METHODS: This prospective randomized, double-blind, placebo controlled trial aims to study the effects of MET, NAC and their combination (MET+NAC) on expression of GDF-9, BMP-15 and c-kit mRNA in oocytes [10 at the germinal vesicle (GV) stage, 10 at the MI stage, and 10 at the MII stage from per group] derived following ovulation induction in PCOS. Treatment was carried out for six weeks, starting on the third day of previous cycle until oocyte aspiration. The expression of GDF9, BMP15 and c-kit were determined by quantitative real time polymerase chain reaction (RT-qPCR) and western blot analysis. Data were analyzed with one-way ANOVA. RESULTS: The follicular fluid (FF) level of c-kit protein significantly decreased in the NAC group compared to the other groups. Significant correlations were observed between the FF soluble c-kit protein with FF volume, androstenedione and estradiol. The GDF-9 expression in unfertilized mature oocytes were significantly higher in the NAC group compared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups. The c-kit expression in unfertilized mature oocytes were significantly lower in the NAC group compared to the other groups (P<0.001). Similar difference was not observed between the MET, NAC+MET and control groups (Registration number: IRCT201204159476N1). CONCLUSION: We concluded that NAC can improve the quality of oocytes in PCOS.

Aluminium-Induced Oxidative Stress, Apoptosis and Alterations in Testicular Tissue and Sperm Quality in Wistar Rats: Ameliorative Effects of Curcumin.

BACKGROUND: Reproductive toxicity is a major challenge associated with aluminum (Al) exposure. No studies have evaluated the possible effects of curcumin (CUR) on Al-induced reproductive dysfunction. Therefore, this study investigated the effects of CUR treatment on Al-induced reproductive damage. MATERIALS AND METHODS: In this experimental study, 40 male Wistar rats were allocated to the five groups (n=8) based on the treatment they received: no treatment (control), solvent [dimethyl sulfoxide (DMSO) or distilled water], CUR 10 mg/kg body weight (BW), Al chloride 10 mg/kg BW, and CUR+Al chloride (10 mg/kg BW/each alone). Treatments were performed by intraperitoneal (IP) injections for 28 days. The left testis was assessed for histopathological analysis as well as the incidence of germ cell apoptosis. One-way analysis of variance (ANOVA) followed by the Tukey's test was used. P<0.05 was considered significant. RESULTS: Significant reductions in body and testis weight; plasma testosterone and luteinizing hormone levels; sperm count, motility, morphology, and viability; germinal epithelium thickness; seminiferous tubules diameter; as well as, superoxide dismutase activity were observed in rats treated with Al. Moreover, Al exposure caused significant increments in the lumen diameter of tubules, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and malondialdehyde (MDA) levels compared to the control group. However, in rats receiving CUR+Al, CUR significantly reversed the adverse effects of Al on testis and sperm quality. No significant differences in follicle-stimulating hormone (FSH) levels and nuclear diameter of spermatogonia were detected among all groups. CONCLUSION: It can be concluded that Al causes reproductive dysfunction by creating oxidative damage. CUR, on the other hand, reduces the toxic effects of Al and improves the antioxidant status and sperm quality in male rats.

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.

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).