Interplay of miR-542, miR-126, miR-143 and miR-26b with PI3K-Akt is a Diagnostic Signal and Putative Regulatory Target in HPV-Positive Cervical Cancer.

Human papillomavirus accounts for 99.7% of all cervical cancer cases worldwide. The viral oncoproteins alter normal cell signaling and gene expression, resulting in loss of cell cycle control and cancer development. Also, microRNAs (miRNAs) have been reported to play a critical role in cervical carcinogenesis. Especially these are not only appropriate targets for therapeutic intervention in cervical cancer but also early diagnostic signals. The given study tries to improve the sparse knowledge on miRNAs and their role in this physiological context. Deregulated miRNAs were identified by analyzing the raw data of the well-founded GSE20592 dataset including 16 tumor/normal pairs of human cervical tissue samples. The dataset was quantified by a conservative strategy based on HTSeq and Salmon, followed by target prediction via TargetScan and miRDB. The comprehensive pathway analysis of all factors was performed using DAVID. The theoretical results were subject of a stringent experimental validation in a well-characterized clinical cohort of 30 tumor/normal pairs of cervical samples. The top 31 miRNAs and their 140 primary target genes were closely intertwined with the PI3K-Akt signaling pathway. MiR-21-3p and miR-1-3p showed a prominent regulatory role while miR-542, miR-126, miR-143, and miR-26b are directly targeting both PI3K and AKT. This study provides insights into the regulation of PI3K-Akt signaling as an important inducer of cervical cancer and identified miR-542, miR-126, miR-143, and miR-26b as promising inhibitors of the PI3K-Akt action.

Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile.

Background and Objectives: Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Materials and Methods: Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Results: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Conclusion: Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

Functional analysis of a putative HER2-associated expressed enhancer, Her2-Enhancer1, in breast cancer cells.

HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339-39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.

Ethanol as a carotenoid production stimulator in Dunaliella salina CCAP 19/18.

Dunaliella salina is a rich source of carotenoids. Carotenoid production is induced under specific conditions, i.e., high light intensity, high salt concentration, nutrient limitation, and suboptimal temperatures in this microalga. The control of environmental factors is vital for high productivity of carotenoids. In this paper, the effect of different ethanol concentrations in combination with nitrogen deficiency was investigated to induce carotenoid production in D. salina CCAP 19/18. Also, some biochemical and molecular parameters were investigated in response to ethanol in the cells. It was shown that ethanol at 0.5% concentration increased cell number but, at 5% concentration, reduced cell viability compared to the control. The highest carotenoid production was achieved at 3% ethanol concentration, which was 1.46 fold higher than the nitrogen deficiency condition. Investigation of the 3 carotenoid biosynthesis genes revealed that their expression levels increased at 3% ethanol concentration, and the phytoene synthase gene was the most upregulated one. Lipid peroxidation increased at both 3% and 5% ethanol concentrations. At 3% concentration, the activity of catalase and superoxide dismutase increased, but no significant changes were seen at 5% ethanol concentration. Peroxidase activity reduced at both 3% and 5% concentrations. Moreover, proline and reducing sugar content increased at 3% concentration while decreased at 5% ethanol concertation. The results showed that at 3% ethanol concentration, higher carotenoid productivity was associated with an increase in other intracellular responses (molecular and biochemical). Ethanol as a controllable element may be beneficial to increase carotenoid production even under inappropriate environmental conditions in D. salina.

The expression level of mir-21-3p in platelet-rich plasma: A potential effective factor and predictive biomarker in recurrent implantation failure.

Recurrent implantation failure (RIF) is the most important complication associated with in vitro fertilization (IVF). Despite the good quality of the transferred embryo, the success rate is rather disappointing. Therefore, predictive biomarkers for implantation are critical to making decisions about transferring high-quality embryos or cryopreserving them for cycles with a higher chance of implantation. Recently, intrauterine infusion of autologous platelet-rich plasma (PRP) has been proposed to increase the endometrial receptivity in RIF patients. PRP is rich in both growth factors and microRNAs (miRNAs). We investigated the possible association of mir-21-3p, mir-21-5p, mir-494-3p, mir-145-5p, and insulin-like growth factor-I (IGF-I) levels in PRP and platelet-poor plasma (PPP) samples with the pregnancy outcomes in RIF patients. The miRNA expression level and IGF-I concentration were assessed using real-time PCR and chemiluminescence methods respectively. Mir-21-3p was upregulated in PRP samples of the pregnant group in comparison to the nonpregnant group. There was no difference in the expression of mir-21-3p in PPP samples of these groups. The concentration of IGF-I was higher in PRP and PPP samples of the nonpregnant in comparison to the pregnant group. Receiver-operating characteristic curve analysis showed that mir-21-3p can be a valuable biomarker for the prediction of pregnancy chance in RIF patients treated with PRP.

Lysophosphatidic acid supports the development of vitrified ovarian follicles by decreasing the incidence of cell death: An experimental study.

Background: Lysophosphatidic acid (LPA) contributes to follicular activation, oocyte maturation, in vitro fertilization, and embryo implantation. Objective: This study was designed to evaluate the effects of LPA to improve the development of isolated follicles derived from whole mouse cultured vitrified ovaries. Materials and Methods: In this experimental study, first, the 1-wk-old mouse ovaries in the non-vitrified and vitrified groups were cultured in the presence of 20 µM of LPA for 1 wk. Then, their isolated preantral follicles were cultured individually for 12 days in the presence or absence of 40 µM of LPA. The following evaluations were done for the cultured follicles: a viability test using Calcein AM staining, flow cytometry using annexin V/Pi, and analysis of the expression of genes by real-time reverse transcription polymerase chain reaction. The maturation rates of the oocytes were compared among groups and some of the released metaphase II oocytes were subjected to in vitro fertilization. Results: In all LPA treated groups, the rates of survival and follicular development were higher, and the incidence of cell death and expression of pro-apoptotic genes were lower, than in the non-LPA supplemented groups (p = 0.035). There was no significant difference between the vitrified and non-vitrified groups regarding follicular or oocyte development, but the expression of Bad and LPA receptors genes was significantly altered in the vitrified LPA supplemented group in comparison with the non-vitrified LPA supplemented group (p = 0.028). Conclusion: LPA improved the survival and developmental potential of the isolated follicles. Despite some alterations in the expression of apoptosis-related genes in the vitrified ovaries, LPA had positive effects on the survival and development of these follicles.

miR-21-5p, miR-141-3p, and miR-205-5p levels in urine-promising biomarkers for the identification of prostate and bladder cancer.

BACKGROUND: Early detection of cancers improves patients' survival and decreases the treatment cost. Unfortunately, the current methods for diagnosis of bladder and prostate cancers, two most common urothelial malignancies, suffer from a low sensitivity and specificity. MicroRNAs, as a group of endogenously produced non-coding RNAs, regulate gene expression and their expression is observed to be altered in many cancers and cancer progression phenomena. The remarkable stability of microRNAs in biofluids and their unique expression pattern in different pathological conditions make them an appealing, noninvasive diagnostic method in cancer diagnosis. Our objective is to identify microRNAs as biomarkers in urine samples of bladder and prostate cancers to improve the existing diagnostic methods in this field. MATERIALS AND METHODS: In this study, urine samples from 110 men with either bladder (n = 45) or prostate (n = 23) cancer, benign prostatic hyperplasia (n = 22) and healthy controls (n = 20) were collected. qPCR was used to evaluate the expression level of miR-21-5p, miR-141-3p, and miR-205-5p in these samples. The sensitivity and specificity of these microRNAs were determined using ROC curve analysis. RESULTS: The analysis of the data revealed that miR-21-5p, miR-141-3p, and miR-205-5p are differentially expressed in urine of bladder and prostate cancer patients. All these three microRNAs were upregulated in these samples and they were also able to differentiate benign prostatic hyperplasia from malignant cases. The statistical analyses revealed a good specificity for each individual microRNA. CONCLUSION: The results show that these three urine-based microRNAs might be a good choice to implement a specific and non-invasive diagnostic tool for bladder and prostate cancer. The expression pattern of all three microRNAs was particularly useful to distinguish benign and invasive tumors in prostate cases. From the patients' perspective the improvement of the diagnostic situation is awaited eagerly.

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.

The mitochondrial DNA copy number, cytochrome c oxidase activity and reactive oxygen species level in metaphase II oocytes obtained from in vitro culture of cryopreserved ovarian tissue in comparison with in vivo-obtained oocyte.

AIM: To evaluate the mitochondrial DNA (mtDNA) copy number, reactive oxygen species (ROS) level and intensity of mitochondrial enzyme activity in metaphase II oocytes derived from vitrified cultured immature mouse ovarian tissue in comparison with nonvitrified group and in vivo-obtained oocytes. METHODS: Vitrified and nonvitrified ovaries from neonate female mice were cultured for 7 days. Then, preantral follicles were isolated and cultured in a three-dimensional culture system. Follicular development and oocyte maturation were evaluated and compared in both groups. Some of the collected metaphase II oocytes derived from in vitro and in vivo conditions were inseminated with capacitated spermatozoa, and then, the fertilization and embryo developmental rates were assessed. In the other series of oocytes, mtDNA copy number, distribution and enzyme activity and ROS level were analyzed. RESULTS: The embryo development, mtDNA copy number and mitochondrial enzyme activity in collected metaphase II oocytes from two in vitro-cultured groups were significantly lower, and the ROS level was higher than those of the in vivo group (P < 0.05), but there was no significant difference between vitrified and nonvitrified groups. CONCLUSION: This study showed that a two-step in vitro culture of mouse ovarian tissue decreased the mtDNA copy number and cytochrome c oxidase activity of metaphase II oocytes through an increase in their ROS level in comparison with in vivo-obtained oocytes. Thus, the in vitro culture methods should be improved.

The 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 MitoTracker 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.

The 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&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). 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.

Effect of lysophosphatidic acid on the follicular development and the expression of lysophosphatidic acid receptor genes during in vitro culture of mouse ovary.

Lysophosphatidic acid (LPA) known as a serum-derived growth factor, is involved in several cell physiological functions in the female reproductive system including: oocyte maturation, in vitro fertilization and embryo implantation by its transmembrane G protein-coupled receptors. The aim of the present study was to examine the effect of LPA on in vitro follicular development of mouse ovarian tissue. Neonatal mouse ovarian tissues were cultured in five different concentrations of LPA (0, 5, 10, 20 and 40 µM). The developmental competence and the function of cultured ovarian tissue were assessed by morphological study using hematoxylin and eosin staining and hormonal analysis. The expression of LPA receptor (LPAR 1-4) genes were analyzed by real-time RT-PCR. The proportion of preantral follicles and the level of E2 hormone were significantly higher in the 20 µM LPA-treated group than those in the other treatment groups. There was a significant difference in the expression of LPAR 1-4 genes in 20 µM LPA treated group in comparison with 0 µM LPA (control group) treated and non-cultured groups. In addition, the expression of LPAR1 gene was higher than other receptor genes in all studied groups. In conclusion supplementation of the media with 20 µM LPA, could improve the survival and developmental potential of follicles and it had positive effects on cell function and stimulation of E2 synthesis in mouse whole ovarian tissues.

Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

OBJECTIVE: This study aimed to evaluate the expression of the genes related to folliculogenesis 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 (α-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 α-MEM medium for 2 weeks) subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin (H&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.

Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics.

AIM: To evaluate the role of biofilm formation on the resistance of Helicobacter pylori (H. pylori) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared. METHODS: A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA. RESULTS: The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones. CONCLUSION: The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA.

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-ß 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 stabil- ity 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.