miRNAs In Follicular Fluid Research Articles

MiRNAs in Follicular and Oviductal Fluids Support Global DNA Demethylation in Early-Stage Embryos.

Global methylation levels differ in in vitro- and in vivo-developed embryos. Follicular fluid (FF) contains extracellular vesicles (EVs) containing miRNAs that affect embryonic development. Here, we examined our hypothesis that components in FF affect global DNA methylation and embryonic development. Oocytes and FF were collected from bovine ovaries. Treatment of zygotes with a low concentration of FF induced global DNA demethylation, improved embryonic development, and reduced DNMT1/3A levels. We show that embryos take up EVs containing labeled miRNA secreted from granulosa cells and the treatment of zygotes with EVs derived from FF reduces global DNA methylation in embryos. Furthermore, the methylation levels of in vitro-developed blastocysts were higher than those of in their vivo counterparts. Based on small RNA-sequencing and in silico analysis, we predicted miR-29b, -199a-3p, and -148a to target DNMTs and to induce DNA demethylation, thereby improving embryonic development. Moreover, among FF from 30 cows, FF with a high content of these miRNAs demethylated more DNA in the embryos than FF with a lower miRNA content. Thus, miRNAs in FF play a role in early embryonic development.

Extracellular microRNA profiling in human follicular fluid: new biomarkers in female reproductive potential.

MicroRNAs (miRNAs) are small, about 22 nucleotides, non-coding RNAs which regulate a wide range of gene expression during post-transcriptional stage. They are released into intra- and extracellular microenvironments and play vital roles in different physiological and pathological pathways. Due to easy accessibility, detection of extracellular miRNAs in body fluids, e.g. serum, plasma, cerebrospinal fluid, and follicular fluid, has been explored in recent years. Since miRNAs are stable at unsuitable conditions, scientists have been investigating to use them as biomarkers in different fields of medicines. It goes without saying that experienced biomarkers would be required in reproductive medicine as well. Biomarkers can help clinicians and embryologists to diagnose disorders and assess the embryo quality via molecular pattern which is more reliable than nowadays routine methods. Follicular fluid as a noninvasive fluid in assisted reproductive techniques (ART) has attracted researchers as a rich pool for biomarkers, and miRNAs are not exception. Although miRNA biomarkers in reproduction field are located on their initial stage and there is a long path to move forward, several meticulous studies have been performed and discovered their associations with various conditions. In this regard, we summarize the reported miRNAs in follicular fluid and their correlations with female infertility and ART success rate, while subsequent investigations are required.

Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency.

Analysis of the MicroRNA Profile by Sequencing in Ovarian Granular Cells from Women Suffering Fluorosis with Reproductive Dysfunction

Excessive intake of fluoride may cause female reproductive dysfunction but pathological mechanism is unclear. The miRNAs in follicular fluid are a class of small non-coding RNAs from granulosa cells. The aim of this study is to examine the differential expressions of miRNAs in ovarian granulosa cells from women suffering from fluorosis and infertility. Five fluorosis women suffering infertility and 5 non-fluorosis (normal ovarian secretory function control) women were included as two groups. These two groups were indexed by serum and urine fluoride (F) levels as high F group and control group. The concentrations of estradiol (E2), progesterone (P), human chorionic gonadotropin (HCG), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured by ELISA kits. The characteristics of menstruation from each woman were collected. The total RNA was isolated from granulosa cells for sequencing. The validation was completed by RT-qPCR. There was no significant difference between the two groups on age, the levels of E2, FSH, P, HCG, and LH in serum, and the characteristics of menstruation. A total of 37 miRNAs were upregulated and 40 miRNAs downregulated in follicular fluid from granulosa cells in the high F group compared with that in the control group. The expression of hsa-miR-29b increased in fluorosis patients identified by the miRNA sequence analysis. The distinct expression patterns of miRNAs in women requiring IVF from fluorosis area provide a new direction for molecular mechanism of fluorosis caused reproductive dysfunction.

The profile of free and exosomal mirnas in follicular fluid of OHSS women

The profile of free and exosomal mirnas in follicular fluid of OHSS women

Identification of microRNAs and genes associated with hyperandrogenism in the follicular fluid of women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease, which is characterized by hyperandrogenism (HA), chronic anovulation, polycystic ovaries, insulin resistance, and obesity. At present, the mechanism by which PCOS/HA occurs has not been fully elucidated, thus, the mechanisms behind and interventions for HA in PCOS are current hot topics in research. MiRNAs have recently been shown to serve as diagnostic or prognostic biomarkers in patients with cancer. Thus, we are currently focused on studying the altered expression of miRNAs in follicular fluid and their correlation with HA in PCOS. Illumina deep sequencing technology was used to explore different miRNAs in the follicular fluid of women with PCOS/HA and in the follicular fluid of women in a control group. Target prediction databases were then used to analyse the target genes of different expressed miRNAs, and GO analysis and the KEGG pathway database were used to identify the functions and the main biochemical and signalling pathways of differentially expressed target genes. The expression levels of 263 miRNAs were significantly different (>2-fold up-regulated or <0.5-fold down-regulated, P < 0.05) between the two groups of women. For example, the expression levels of miRNA (200a-3p, 10b-3p, 200b-3p, 29c-3p, 99a-3p, and 125a-5p) were significantly increased, while there was a decreased expression of miR-105-3p in PCOS patients with respect to the control. Literature has shown that the above seven miRNAs were associated with HA in PCOS. Furthermore, 31 770 genes were predicted to be targets of the 263 differentially expressed microRNAs. GO analysis and the KEGG pathway database showed involvement of these target genes in HA in PCOS. These results suggest the presence of differentially expressed miRNAs in the follicular fluid of women with PCOS/HA versus women in the control group. The potential role of these microRNAs was elucidated using bioinformatics tools and was found to be involved in the regulation of different pathways, biological functions, and cellular components underlying PCOS. The results of this research may reveal new mechanisms of PCOS/HA and suggest potential treatment targets.

Expression of miR-15a, miR-145, and miR-182 in granulosa-lutein cells, follicular fluid, and serum of women with polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies that affects women in reproductive age. MicroRNAs (miRNAs) play crucial roles in normal function of female reproductive system and folliculogenesis. Deregulated expression of miRNAs in PCOS condition may be significantly implicated in the pathogenesis of PCOS. We determined relative expression of miR-15a, miR-145, and miR-182 in granulosa-lutein cells (GLCs), follicular fluid (FF), and serum of PCOS patients. Human subjects were divided into PCOS (n=20) and control (n=21) groups. GLCs, FF, and serum were isolated and stored. RNA isolation was performed and cDNA was reversely transcribed using specific stem-loop RT primers. Relative expression of miRNAs was calculated after normalization against U6 expression. Correlation of miRNAs' expression level with basic clinical features and predictive value of miRNAs in FF and serum were appraised. Relative expression of miR-145 and miR-182 in GLCs was significantly decreased in PCOS, but miR-182 in FF of PCOS patients revealed up-regulated levels. Significant correlations between level of miRNAs in FF and serum and hormonal profile of subjects were observed. MiR-182 in FF showed a significant predictive value with AUC of 0.73, 76.4% sensitivity, and 70.5% specificity which was improved after combination of miR-182 and miR-145. A significant dysregulation of miR-145 and miR-182 in GLCs of PCOS may indicate their involvement in pathogenesis of PCOS. Differential up-regulation of miR-182 in FF of PCOS patients with its promising predictive values for discrimination of PCOS reinforced the importance of studying miRNAs' profile in FF.

Dynamics and role of MicroRNAs during mammalian follicular development

Ovarian functions, which involve dynamically regulated processes of selection, recruitment and dominance, are known to be regulated by an array of genes, which are expressed in spatiotemporal manner in follicular somatic cells and gametes. This differential expression of genes in mammalian follicular cells is partly regulated by posttranscriptional gene regulators named microRNAs (miRNAs). In addition to the cellular miRNAs, growing number of evidences are available on the potential role of extracellular vesicles mediated transfer of miRNAs in follicular fluid. These extracellular vesicles are shown to be involved in cellto-cell communication within the follicular environment. The molecular messages carried by the extracellular vesicles released into extracellular space are thought to be reflections of the physiological status of the cells from where they are released. Therefore due to their structural nature and potential to cargo several physiologically relevant molecules, exosomes have a great potential to be used as markers of oocyte developmental competence in follicular environment. Here, we review large sets of literatures to show the dynamic nature of miRNAs during various stages of mammalian follicular development, oocyte growth and the role of some of the miRNAs in ovarian cell functions. Moreover, the presence of microvesicle and exosome-coupled extracellular miRNAs in mammalian follicular fluid and their potential involvement in cellto-cell communication are briefly discussed.

Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma.

BackgroundDespite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract.MethodTwelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs.ResultsA total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis.Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma.ConclusionOur data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.

Follicular fluid and mural granulosa cells microRNA profiles vary in in vitro fertilization patients depending on their age and oocyte maturation stage

To determine whether there is any difference in the follicular fluid (FF) microRNA (miRNA) profiles from in vitro fertilization (IVF) patients according to their age and oocyte maturation stage. Observational prospective study. IVF clinic/hospital facilities. We included 30 women with primary infertility undergoing intracytoplasmic sperm injection treatment and excluded patients with polycystic ovarian syndrome, endometriosis, severe male factor, and low ovarian reserve. After the collection of FF and granulosa cells from each patient, the samples were processed for total RNA extraction. RNA was pooled into different groups (three samples per pool) for microarray analysis to evaluate the expression of a total of 866 human miRNAs. Individual samples were analyzed to validate the pooled microarray results using real-time polymerase chain reaction. Evaluation of the expression of a total of 866 human miRNAs in FF and granulosa cells. We identified only one differentially expressed miRNA, hsa-miR-424, which is present in higher proportions in FF from patients with advanced age. When we compared the FF from metaphase II (MII) versus GV (germinal vesicle) oocytes, we found 13 differentially expressed miRNAs (two up- and 11 downregulated). When we compared FF from MII versus MI, we found seven differentially expressed miRNAs in MII (three up- and four downregulated). We have described the FF miRNA profiles according to IVF patients' age and the maturation stage of their oocytes. This descriptive study may aid our understanding of the physiology and regulation of oocyte maturation and could identify some potential miRNA biomarkers for this process. Not applicable.

Identification of MicroRNAs in Human Follicular Fluid: Characterization of MicroRNAs That Govern Steroidogenesis in Vitro and Are Associated With Polycystic Ovary Syndrome in Vivo

Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids. We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS). Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays. We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively). These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.