Rapid and simple detection of Escherichia coli by loop-mediated isothermal amplification assay in urine specimens

Background: To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method [LAMP] for detection of the Eschericia coli [E. coli] in urine samples, for the first time

Methods: Primers were designed to target the malB gene of Escherichia coli. LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms

Results: As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies

Conclusion: LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of E. coli infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment

effects of sodium selenite on mitochondrial DNA copy number and reactive oxygen species levels of in vitro matured mouse oocytes

Objective: The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite [SS] on oocyte mitochondrial DNA [mtDNA] copy number and reactive oxygen species [ROS] levels

Materials and Methods: In this experimental study germinal vesicle [GV], metaphase I [MI], and metaphase II [MII] stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were cultured and matured in the presence and absence of SS. Then in vivo and in vitro matured [IVM] oocytes were subjected to mitochondria staining by Mito Tracker green, ROS analysis, and mtDNA copy number determination using absolute real-time polymerase chain reaction [PCR]

Results: The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group [79.25%] compared to the control group [72.46%, P<0.05]. The intensity of mitochondrial staining was not different among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some aggregation pattern. The in vivo obtained MII oocytes had lower ROS levels and higher mtDNA copy numbers than IVM-MII oocytes [P<0.05]. The SS supplemented group had significantly lower ROS levels and higher mtDNA copy numbers than the non-treated group [P<0.05]

Conclusion: SS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence

effects of lysophosphatidic acid on the incidence of cell death in cultured vitrified and non-vitrified mouse ovarian tissue: separation of necrosis and apoptosis border

Objective: The aim of the present study was to examine whether lysophosphatidic acid [LPA] could decrease cell death and improve in vitro culture [IVC] conditions in cultured vitrified mouse ovarian tissue

Materials and Methods: In this experimental study, we collected and randomly divided 7-day-old mouse ovarian tissues into vitrified and non-vitrified groups. The ovaries were cultured in the presence and absence of LPA for one week. Morphology and follicular development were evaluated by hematoxylin and eosin [H and E] and Masson's trichrome [MTC] staining. The incidence of cell death was assessed by flow cytometry using annexin V/propidium iodide [PI] and a caspase-3/7 assay in all studied groups

Results: The vitrified groups had a significantly decreased follicle developmental rate compared to the non-vitrified groups [P<0.05]. Overall, qualitative and quantitative results showed prominent follicular degeneration in the vitrified groups compared with the respective non-vitrified groups. Both LPA treated groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups [P<0.05]. groups had a significantly higher proportion of preantral follicles compared to the non-LPA treated groups [P<0.05]. Flow cytometry analysis results showed significantly greater early and late apoptotic cells in all groups [17.83 +/- 8.80%] compared to necrotic cells [7.97 +/- 0.92%, P<0.05]. The percentage of these cells significantly increased in the vitrified groups compared with non-vitrified groups. LPA treated groups had a lower percentage of these cells compared to non-LPA treated groups [P<0.05]. The lower enzyme activity was observed in non-vitrified [especially in the LPA+ groups] cultured ovaries compared to the vitrified group [P<0.05]

Conclusion: Both vitrification and IVC adversely affected cell survival and caused cell death. We postulated that LPA supplementation of culture medium could improve the developmental rate of follicles and act as an anti-cell death factor in non-vitrified and vitrified ovarian tissues. It could be used for in vitro maturation of ovarian tissue

Morphological and molecular aspects of in vitro culture of preantral follicles derived from vitrified ovarian tissues using a two-step culture

Objective: This study aimed to evaluate the expression of the genes related to folliculo-genesis after vitrification of mouse ovarian tissues using a two-step in vitro culture

Materials and Methods: In this experimental study, vitrified and non-vitrified ovaries from 7- day old [neonate] female mice were cultured using alpha-Minimum Essential Medium [alpha-MEM] supplemented with 5% fetal bovine serum [FBS] for 7 days. Morphology, surface area of ovaries and percentage of normal follicles were evaluated and compared in both groups. After one-week culture, in non-vitrified group, preantral follicles of cultured ovaries were isolated and cultured in a three-dimensional alginate culture system for 12 days. Then, the collected metaphase [M] II oocytes were inseminated with capacitated spermatozoa derived from 7-8-week old [adult] male NMRI mice. Follicular diameter, oocyte maturation, fertilization, embryo development and the expression of genes related to follicular development [Pcna, Fshr and Cyp17a1,] using real time reverse transcription-polymerase chain reaction [RT-PCR] were assessed at the end of last culture period in both groups

Results: The ovarian area in vitrified group [162468.20 703.78] was less than non-vitrified group [297211.40 6671.71], while the percentage of preantral follicles in vitrified group [18.40%] was significantly lower than those of non-vitrified group [24.50%] on day 7 of culture [P<0.05]. There were no significant differences between the two groups in terms of follicular diameter, expression of genes related to development of follicles, oocyte maturation, fertilization, as well as embryo development [P>0.05]

Conclusion: The results of this study showed that vitrification of ovarian tissue following in vitro culture had negative impact on the survival and development of follicles within the tissue. However, no significant alterations were observed in development, gene expression and hormonal production of in vitro culture of isolated follicles derived from vitrified ovarian tissues as compared to the non-vitrified samples

Expression of folliculogenesis-related genes in vitrified human ovarian tissue after two weeks in vitro culture

Objective: This study was designed to evaluate the effects of vitrification and in vitro culture of human ovarian tissue on the expression of oocytic and follicular cell-related genes

Materials and Methods: In this experimental study, ovarian tissue samples were obtained from eight transsexual women. Samples were cut into small fragments and were then assigned to vitrified and non-vitrified groups. In each group, some tissue fragments were divided into un-cultured and cultured [in alpha-MEM medium for 2 weeks] subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin [H and E] staining. Expression levels of factor in the germ line alpha [FIGLA], KIT ligand [KL], growth differentiation factor 9 [GDF-9] and follicle stimulating hormone receptor [FSHR] genes were quantified in both groups by real-time reverse transcriptase polymerase chain reaction [RT-PCR] at the beginning and the end of culture

Results: The percentage of normal follicles was similar between non-cultured vitrified and non-vitrified groups [P>0.05], however, cultured tissues had significantly fewer normal follicles than non-cultured tissues in both vitrified and non-vitrified groups [P<0.05]. In both cultured groups the rate of primary and secondary follicles was significantly higher than non-cultured tissues [P<0.05]. The expression of all examined genes was not significantly altered in both non-cultured groups. Whiles, in comparison with cultured tissues non-cultured tissues, the expression of FIGLA gene was significantly decreased, KL gene was not changed, GDF-9 and FSHR genes was significantly increased [P<0.05]

Conclusion: Human ovarian vitrification following in vitro culture has no impairing effects on follicle normality and development and expression of related-genes. However, in vitro culture condition has deleterious effects on normality of follicles

Short term organ culture of mouse ovary in the medium supplemented with bone morphogenetic protein 15 and follicle stimulating hormone: a morphological, hormonal and molecular study

Background: Bone morphogenetic protein 15 [BMP15] is a growth factor derived from oocyte and is essential for in vivo ovarian follicular growth and in this study, its effects on the improvement of growth and development of follicles during in vitro culture of neonatal mouse ovaries was investigated

Methods: Two week old mice were cultured for 7 days in the basic culture media with or without follicle stimulating hormone [FSH] and BMP15 as four experimental groups; FSH[-]/BMP15[-], FSH[+]/BMP15[-], FSH[-]/BMP15[+] and FSH[+]/BMP15[+]. The ovarian follicles at different developmental stages in paraffin embedding sections of cultured and non-cultured ovaries were counted and compared. The 17-beta estradiol [E2] and progesterone [P4] levels were analyzed in collected culture media. The expression ratio of developmental genes [PCNA, BMPR-IB, BMPR-II, FSH-R, CYP17 and ZP3] to housekeeping gene [GAPDH] was analyzed by real time PCR [RT-PCR] in comparison with non-cultured control ovaries. The data was compared by independent t-test and one-way ANOVA [with Tukey's Post Hoc test]. The p<0.05 was considered significant

Results: The percentage of antral follicles, ovarian size, concentration of E2 and P4 and the expression ratio of PCNA and ZP3 genes in the ovaries cultured in medium supplemented with BMP15 and FSH increased significantly in comparison with other cultured groups [p<0.05]. The BMPR-IB, BMPR-II and FSH-R mRNA level was significantly lower [p<0.05] and CYP 17 mRNA level did not change in the FSH[+]/ BMP15[+] group than other cultured groups

Conclusion: This study demonstrated a favorable effect of BMP15 in combination with FSH on in vitro development of small size mouse follicles to antral stage

Identification of Reliable reference genes for quantification of MicroRNAs in serum samples of sulfur mustard-exposed veterans

Objective: In spite of accumulating information about pathological aspects of sulfur mustard [SM], the precise mechanism responsible for its effects is not well understood. Circulating microRNAs [miRNAs] are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims

Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction [qRT-PCR], we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war [1980-1988] and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle [Cq] method were employed to find the least variable reference gene

Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that

Conclusion: We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers

Effect of human ovarian tissue vitrification/warming on the expression of genes related to folliculogenesis

Ovarian tissue cryopreservation is an alternative strategy to preserve the fertility of women predicted to undergo premature ovarian failure. This study was designed to evaluate the expression of folliculogenesis-related genes, including factor in the germline alpha [FIGLA], growth differentiation factor-9 [GDF-9], follicle-stimulating hormone receptor [FSHR], and KIT LIGAND after vitrification/warming of human ovarian tissue. Human ovarian tissue samples were collected from five transsexual women. In the laboratory, the ovarian medullary part was removed by a surgical blade, and the cortical tissue was cut into small pieces. Some pieces were vitrified and warmed and the others were considered as non-vitrified group [control]. Follicular normality was assessed with morphological observation by a light microscope, and the expression of FIGLA, KIT LIGAND, GDF- 9, and FSHR genes was examined using real-time RT-PCR in both the vitrified and non-vitrified groups. Overall, 85% of the follicles preserved their normal morphologic feature after warming. The percentage of normal follicles and the expression of FIGLA, KIT LIGAND, GDF-9, and FSHR genes were similar in both vitrified and non-vitrified groups [P > 0.05]. Vitrification/warming of human ovarian tissue had no remarkable effect on the expression of folliculogenesis-related genes

Human ovarian tissue vitrification/warming has minor effect on the expression of apoptosis-related genes

In this study, we evaluated the incidence of apoptosis at the ultrastructural levels and expression of some apoptosis-related genes in vitrified human ovarian tissue just after warming. Human ovarian tissue biopsies from 23 women after caesarean section were transported to the laboratory within 2 hours, and then they were cut into small pieces. Some pieces were vitrified and warmed and the other samples were considered as control. Apoptosis was assessed by a transmission electron microscope and also by molecular analysis of pro-apoptotic [Fas, FasL, Bax, p53, caspase8, and caspase3] and antiapoptotic [Bcl-2 and BIRC5] genem RNA levels using real-time RT-PCR before and after vitrification. No sign of apoptosis was shown ultrastructurally in vitrified samples. The level of FasL, Bcl-2, Bax, p53, and caspase3 mRNA and Bax:Bcl-2 ratio were similar in non-vitrified and vitrified groups; however, the expression of Fas and caspase8 genes was higher and BIRC5 was lower in vitrified samples compared to non-vitrified group [P<0.05]. The fine structure of human vitrified ovarian tissue was well preserved; moreover, vitrification was shown to affect the expression of some apoptosis-related genes. However, additional study is needed to confirm this observation

[Optimization of extraction and amplification of microRNAs [mir-21, mir-141 and mir-205] in urine samples of patients with bladder cancer]

The current methods for bladder cancer diagnosis suffer from low sensitivity and specificity. Therefore, finding a novel tumor markers with high specificity and sensitivity is of great interest. MicroRNAs [miRNA, miR] are small endogenously-produced, non-coding RNAs with an important role in regulating gene expression. Recent studies show that miRNAs expression profiles represent significant tumor-specific changes that are unique for most cancers. The aim of this study was to optimize miRNA containing total RNA extraction from urine and use it as a reliable and repeatable technique for miRNA detection in urine of patients with bladder cancer. Total RNA was extracted from the urine of patients with bladder cancer and normal individuals using RNX and Trizol solutions with and without modifications of original protocols. Real-time quantitative RT-PCR was then used to detect miRNAs with a potential link to bladder tumorigenesis. RNX and the modified Trizol are practical methods for RNA extraction from urine samples. The mir-21 amplification of the extracted RNAs using modified Trizol method was more efficient than that of RNX method. It is noteworthy that, the levels of miRNAs expression were much higher in the frozen urines compared to the fresh ones. We have succeeded to set-up a protocol to easily amplify miRNAs in urine samples. Based on the data, microRNAs seem to be good biomarkers for early detection and screening of bladder cancer