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 3ˊUTRs (3ˊuntranslated 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

Elevated seminal protein carbonyl concentration is correlated with asthenozoospermia and affects adversely the laboratory intracytoplasmic sperm injection (ICSI) outcomes

Elevated concentrations of reactive oxygen species (ROS) in the semen can lead to oxidative protein damage as they react with the amino acids' side chains in the protein, leading to the generation of carbonyl groups. This study aimed to investigate the effect of protein carbonyl (PC) concentration on sperm motility and the laboratory intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study and were divided into three groups (G) according to the PC concentration as following, G1 included samples with PC concentrations <0.65 nmol/mg, G2 included samples with 0.65≤PC≤2.23 nmol/mg and G3 included samples with PC>2.23 (nmol/mg). PC concentrations were measured in all semen samples, and the laboratory ICSI outcomes were evaluated for all injected oocytes. The Kruskal–Wallis p-values for the differences in the medians of sperm motility, fertilisation rate, embryo cleavage score and embryo quality score were <0.05. Furthermore, Dunn's post hoc test showed a significant difference between all groups, p-values <0.05, except for the medians of embryo quality score between G2 and G3. In conclusion, our results showed that sperm motility and laboratory ICSI outcomes are affected negatively by higher concentrations of PC in the semen

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-Müllerian hormone (AMH) level into three groups: (1) low ovarian reserve (LAMH, n = 39), (2) normal ovarian reserve (NAMH, n = 80), and (3) high ovarian reserve (HAMH, n = 40). 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

Impact of Mitochondrial Genetic Variants in ND1, ND2, ND5, and ND6 Genes on Sperm Motility and Intracytoplasmic Sperm Injection (ICSI) Outcomes

Sperm mitochondrial dysfunction causes the generation of an insufficient amount of energy needed for sperm motility. This will affect sperm fertilization capacity, and thus, most asthenozoospermic men usually require assisted reproductive techniques. The etiology of asthenozoospermia remains largely unknown. The current study aimed to investigate the effect of mitochondrial genetic variants on sperm motility and intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study. One hundred five of the male partners were asthenozoospermic patients, and they were subdivided into three groups according to their percentage of sperm motility, while forty-five of the male partners were normozoospermic. Genetic variants were screened using direct Sanger’s sequencing in four mitochondrial genes (nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase 1 (ND1), NADH dehydrogenase 2 (ND2), NADH dehydrogenase 5 (ND5), and NADH dehydrogenase 6 (ND6)). We identified three significant variants: 13708G>A (rs28359178) in ND5, 4216T>C (rs1599988) in ND1, and a novel 12506T>A in ND5 with P values 0.006, 0.036, and 0.013, respectively. The medians of sperm motility, fertilization rate, embryo cleavage score, and embryo quality score were significantly different between men showing 4216T>C, 12506T>A, 13708G>A and wild type, Mann-Whitney P values for the differences in the medians were A, 12506T>A, and 4216 T>C variants in sperm mitochondrial DNA negatively affect sperm motility and ICSI outcomes

RNA-Containing Cytoplasmic Inclusion Bodies in Ciliated Bronchial Epithelium Months to Years after Acute Kawasaki Disease

Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common.Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9-84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI.These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD

Effects of the Insemination of Hydrogen Peroxide-Treated Epididymal Mouse Spermatozoa on γH2AX Repair and Embryo Development

BACKGROUND: Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation. METHODS AND FINDINGS: We used exogenous ROS (H(2)O(2)) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H(2)O(2)-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium (containing 0, 0.1, 0.5 and 1 mM H(2)O(2)) or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H(2)O(2) treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos. CONCLUSIONS: γH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa

MRI of the kidney—state of the art

Ultrasound and computed tomography (CT) are modalities of first choice in renal imaging. Until now, magnetic resonance imaging (MRI) has mainly been used as a problem-solving technique. MRI has the advantage of superior soft-tissue contrast, which provides a powerful tool in the detection and characterization of renal lesions. The MRI features of common and less common renal lesions are discussed as well as the evaluation of the spread of malignant lesions and preoperative assessment. MR urography technique and applications are discussed as well as the role of MRI in the evaluation of potential kidney donors. Furthermore the advances in functional MRI of the kidney are highlighted

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation

Abstract BACKGROUND: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation. METHODS: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis. 2-D gels were stained with Sypro Ruby. Map images of DC and SU spermatozoa were compared using dedicated software. Intensities of a given spot were considered different between DC and SU when their group mean differed by >1.5-fold (p<0.05, Anova). RESULTS: No differences were observed for 853 spots, indicating a 98.7% similarity between DC and SU. Five spots were DC>SU and 1 was SU>DC. Proteins present in 3 of the differential spots could be identified. One DC>SU spot contained lactate dehydrogenase C and gamma-glutamylhydrolase, a second DC>SU spot contained fumarate hydratase and glyceraldehyde-3-phosphate dehydrogenase-2, and a SU>DC spot contained pyruvate kinase M1/M2. CONCLUSIONS: The differences in protein levels found on comparison of DC with SU spermatozoa indicate possible dissimilarities in their glycolytic metabolism and DNA methylation and suggest that DC cells may have a better capacitation potential

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation