55 research outputs found
MicroRNA-targeting in male infertility : Sperm microRNA-19a/b-3p and its spermatogenesis related transcripts content in men with oligoasthenozoospermia
Objective: To elucidate and validate the potential regulatory function of
miR-19a/b-3p and its spermatogenesis-related transcripts content in sperm
samples collected from men with oligoasthenozoospermia.
Methods: Men presenting at an infertility clinic were enrolled. MicroRNA
(miRNA) and target genes evaluation were carried out using in silico
prediction analysis, Reverse transcription-quantitative PCR (RT-qPCR)
validation, and Western blot confirmation.
Results: The expression levels of miRNA-19a/b-3p were significantly upregulated and 51 target genes were significantly down-regulated in
oligoasthenozoospermic men compared with age-matched
normozoospermic men as determined by RT-qPCR. Correlation analysis
highlighted that sperm count, motility, and morphology were
negatively correlated with miRNA-19a/b-3p and positively correlated with
the lower expression level of 51 significantly identified target genes.
Furthermore, an inverse correlation between higher expression levels of
miRNA-19a/b-3p and lower expression levels of 51 target genes was
observed. Consistent with the results of the RT-qPCR, reduced expression
levels of STK33 and DNAI1 protein levels were identified in an
independent cohort of sperm samples collected from men with
oligoasthenozoospermia.
Conclusion: Findings suggest that the higher expression of miRNA-19a/b3p or the lower expression of target genes are associated
with oligoasthenozoospermia and male infertility, probably
through influencing basic semen parameters. This study lay the
groundwork for future studies focused on investigating therapies for male
infertility
Expression of SPAG7 and its regulatory microRNAs in seminal plasma and seminal plasma-derived extracellular vesicles of patients with subfertility
Seminal plasma contains a variety of extracellular vesicles (EVs) that deliver RNAs including
microRNAs (miRNAs) molecules. However, the roles of these EVs along with their delivered RNAs
and their interactions with male infertility are not clear. Sperm-associated antigen 7 (SPAG 7) is
expressed in male germ cells and plays a crucial role in several biological functions associated with
sperm production and maturation. In this study, we aimed to identify the post-transcriptional
regulation of SPAG7 in seminal plasma (SF-Native) and seminal plasma-derived extracellular vesicles
(SF-EVs) collected from 87 men undergoing infertility treatment. Among the multiple binding sites
for miRNAs within its 3âUTR of SPAG7, we identifed the binding of four miRNAs (miR-15b-5p, miR195-5p, miR-424-5p, and miR-497-5p) to the 3âUTR of SPAG7 by the dual luciferase assays. Analyzing
sperm, we found reduced mRNA expression levels of SPAG7 in SF-EVs and SF-Native samples from
oligoasthenozoospermic men. By contrast, two miRNAs (miR-424-5p and miR-497-5p) form the
SF-Native samples, and four miRNAs (miR-195-5p, miR-424-5p, miR-497-5p, and miR-6838-5p) from
the SF-EVs samples showed signifcantly higher expression levels in oligoasthenozoospermic men. The
expression levels of miRNAs and SPAG7 were signifcantly correlated with basic semen parameters.
These fndings contribute signifcantly to our understanding of regulatory pathways in male fertility
by showing a direct link between upregulated miRNA, notably miR-424, and downregulated SPAG7
both in seminal plasma and in plasma-derived EVs likely contributing to oligoasthenozoospermia
Sperm Motility Annotated Genes: Are They Associated with Impaired Fecundity?
Sperm motility is a prerequisite for achieving pregnancy, and alterations in sperm motility,
along with sperm count and morphology, are commonly observed in subfertile men. The aim of the
study was to determine whether the expression level of genes annotated with the Gene Ontology
(GO) term âsperm motilityâ differed in sperm collected from healthy men and men diagnosed with
oligoasthenozoospermia. Reverse transcription quantitative real-time PCR (RT-qPCR), quantitative
mass spectrometry (LC-MS/MS), and enrichment analyses were used to validate a set of 132 genes
in 198 men present at an infertility clinic. Out of the 132 studied sperm-motility-associated genes,
114 showed differentially expressed levels in oligoasthenozoospermic men compared to those of
normozoospermic controls using an RT-qPCR analysis. Of these, 94 genes showed a significantly
lower expression level, and 20 genes showed a significantly higher expression level. An MS analysis
of sperm from an independent cohort of healthy and subfertile men identified 692 differentially
expressed proteins, of which 512 were significantly lower and 180 were significantly higher in
oligoasthenozoospermic men compared to those of the normozoospermic controls. Of the 58 gene
products quantified with both techniques, 48 (82.75%) showed concordant regulation. Besides the
sperm-motility-associated proteins, the unbiased proteomics approach uncovered several novel
proteins whose expression levels were specifically altered in abnormal sperm samples. Among
these deregulated proteins, there was a clear overrepresentation of annotation terms related to
sperm integrity, the cytoskeleton, and energy-related metabolism, as well as human phenotypes
related to spermatogenesis and sperm-related abnormalities. These findings suggest that many of
these proteins may serve as diagnostic markers of male infertility. Our study reveals an extended
number of sperm-motility-associated genes with altered expression levels in the sperm of men with
oligoasthenozoospermia. These genes and/or proteins can be used in the future for better assessments
of male factor infertility
Towards a More Comprehensive Picture of the MicroRNA-23a/b-3p Impact on Impaired Male Fertility
The expression levels of various genes involved in human spermatogenesis are influenced
by microRNAs (miRNAs), specifically microRNA-23a/b-3p. While certain genes are essential for
spermatogenesis and male germ cell function, the regulation of their expression remains unclear. This
study aimed to investigate whether microRNA-23a/b-3p targets genes involved in spermatogenesis
and the impact of this targeting on the expression levels of these genes in males with impaired fertility.
In-silico prediction and dual-luciferase assays were used to determine the potential connections
between microRNA-23a/b-3p overexpression and reduced expression levels of 16 target genes.
Reverse transcription-quantitative PCR (RT-qPCR) was conducted on 41 oligoasthenozoospermic
men receiving infertility treatment and 41 age-matched normozoospermic individuals to verify the
lower expression level of target genes. By employing dual-luciferase assays, microRNA-23a-3p was
found to directly target eight genes, namely NOL4, SOX6, GOLGA6C, PCDHA9, G2E3, ZNF695,
CEP41, and RGPD1, while microRNA-23b-3p directly targeted three genes, namely SOX6, GOLGA6C,
and ZNF695. The intentional alteration of the microRNA-23a/b binding site within the 30 untranslated
regions (30UTRs) of the eight genes resulted in the loss of responsiveness to microRNA-23a/b-3p.
This confirmed that NOL4, SOX6, GOLGA6C, PCDHA9, and CEP41 are direct targets for microRNA23a-3p, while NOL4, SOX6, and PCDHA9 are direct targets for microRNA-23b-3p. The sperm samples
of oligoasthenozoospermic men had lower expression levels of target genes than age-matched
normozoospermic men. Correlation analysis indicated a positive correlation between basic semen
parameters and lower expression levels of target genes. The study suggests that microRNA-23a/b-3p
plays a significant role in spermatogenesis by controlling the expression of target genes linked to
males with impaired fertility and has an impact on basic semen parameters
Integrated microRNA and mRNA Expression Profiling Identifies Novel Targets and Networks Associated with Ebsteinâs Anomaly
Little is known about abundance level changes of circulating microRNAs (miRNAs)
and messenger RNAs (mRNA) in patients with Ebsteinâs anomaly (EA). Here, we performed an
integrated analysis to identify the differentially abundant miRNAs and mRNA targets and to identify
the potential therapeutic targets that might be involved in the mechanisms underlying EA. A large
panel of human miRNA and mRNA microarrays were conducted to determine the genome-wide
expression profiles in the blood of 16 EA patients and 16 age and gender-matched healthy control
volunteers (HVs). Differential abundance level of single miRNA and mRNA was validated by RealTime quantitative PCR (RT-qPCR). Enrichment analyses of altered miRNA and mRNA abundance
levels were identified using bioinformatics tools. Altered miRNA and mRNA abundance levels
were observed between EA patients and HVs. Among the deregulated miRNAs and mRNAs,
76 miRNAs (49 lower abundance and 27 higher abundance, fold-change of â„2) and 29 mRNAs
(25 higher abundance and 4 lower abundance, fold-change of â„1.5) were identified in EA patients
compared to HVs. Bioinformatics analysis identified 37 pairs of putative miRNA-mRNA interactions.
The majority of the correlations were detected between the lower abundance level of miRNA and
higher abundance level of mRNA, except for let-7b-5p, which showed a higher abundance level
and their target gene, SCRN3, showed a lower abundance level. Pathway enrichment analysis
of the deregulated mRNAs identified 35 significant pathways that are mostly involved in signal
transduction and cellular interaction pathways. Our findings provide new insights into a potential
molecular biomarker(s) for the EA that may guide the development of novel targeting therapies
Characterization of micro-RNA in women with different ovarian reserve
Women undergoing infertility treatment are routinely subjected to one or more tests of ovarian reserve. Therefore, an adequate assessment of the ovarian reserve is necessary for the treatment. In this study, we aimed to characterize the potential role of microRNAs (miRNAs) as biomarkers for women with different ovarian reserves. A total of 159 women were recruited in the study and classified according to their anti-MĂŒllerian hormone (AMH) level into three groups: (1) low ovarian reserve (LAMH, nâ=â39), (2) normal ovarian reserve (NAMH, nâ=â80), and (3) high ovarian reserve (HAMH, nâ=â40). SurePrint Human miRNA array screening and reverse transcription-quantitative PCR (RT-qPCR) were respectively employed to screen and validate the miRNA abundance level in the three tested groups. Compared with NAMH, the abundance level of 34 and 98 miRNAs was found to be significantly altered in LAMH and HAMH, respectively. The abundance level of miRNAs was further validated by RT-qPCR in both, the screening samples as well as in an independent set of validation samples. The abundance levels of the validated miRNAs were significantly correlated with the AMH level. The best AUC value for the prediction of the increase and decrease in the AMH level was obtained for the miR-100-5p and miR-21-5p, respectively. The level of miRNAs abundance correlates with the level of AMH, which may serve as a tool for identifying women with a different ovarian reserve and may help to lay the ground for the development of novel diagnostic approaches
MicroRNA-targeting in spermatogenesis: Over-expressions of microRNA-23a/b-3p and its affected targeting of the genes ODF2 and UBQLN3 in spermatozoa of patients with oligoasthenozoospermia
Background
Male infertility is a multifactorial syndrome with diverse phenotypic representations. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Altered abundance levels of ODF2 and UBQLN3 have been reported in patients with different spermatogenic impairments. However, the transcriptional regulation of these two genes by miR-23a/b-3p is still unclear.
Objectives
To investigate experimentally whether miR-23a/b-3p targets the genes ODF2 and UBQLN3 and whether this targeting impacts abundance levels of ODF2 and UBQLN3 in patients with oligoasthenozoospermia.
Materials and methods
A total of 92 men attending a fertility clinic were included in the study, including 46 oligoasthenozoospermic men and 46 age-matched normozoospermic volunteers who served as controls. Reverse transcription-quantitative PCR (RT-qPCR), Western blot, and dual-luciferase (Firefly-Renilla) assays were used to validate the miRNAs and their target genes.
Results
RT-qPCR revealed that miR-23a/b-3p was more abundant and ODF2 and UBQLN3 targets were less abundant in men with impaired spermatogenesis. Besides, Western blot shows that ODF2 and UBQLN3 protein levels were reduced in men with impaired spermatogenesis. In silico prediction and dual-luciferase assays revealed that potential links exist between the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets. Mutations in the miR-23a/b-3p-binding site within the 3ËUTRs (3Ëuntranslated regions) of ODF2 and UBQLN3 genes resulted in abrogated responsiveness to miR-23a/b-3p. Correlation analysis showed that sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with the lower abundance level of UBQLN3, while ODF lower abundance level was positively correlated with sperm motility.
Conclusion
Findings indicate that the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets are associated with oligoasthenozoospermia and male subfertility
Dynamic and static circulating cancer microRNA biomarkers : a validation study
For cancers and other pathologies, early diagnosis remains the most promising path to survival. Profiling
of longitudinal cohorts facilitates insights into trajectories of biomarkers. We measured microRNA
expression in 240 serum samples from patients with colon, lung, and breast cancer and from cancerfree controls. Each patient provided at least two serum samples, one prior to diagnosis and one
following diagnosis. The median time interval between the samples was 11.6 years. Using computational
models, we evaluated the circulating profiles of 21 microRNAs. The analysis yielded two sets of
biomarkers, static ones that show an absolute difference between certain cancer types and controls
and dynamic ones where the level over time provided higher diagnostic information content. In the first
group, miR-99a-5p stands out for all three cancer types. In the second group, miR-155-5p allows to
predict lung cancers and colon cancers. Classification in samples from cancer and non-cancer patients
using gradient boosted trees reached an average accuracy of 79.9%. The results suggest that individual
change over time or an absolute value at one time point may predict a disease with high specificity and
sensitivity
Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy
Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
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