Evaluation of male infertility has been based on semen analysis for years. As this method can be subjective at times, there is a scientific tendency to discover stable and quantifiable biomarkers. This study included 28 couples who underwent an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle. The couples were assigned into two groups, according to sperm morphology. Couples where the males were normozoospermic were placed in the control group (15 participants), while couples where males had teratozoospermia were placed in the study group (13 participants). Thirteen candidate miRNAs were selected for qPCR analysis, based on our literature search. We determined significant under-expression of nine miRNAs (miR-10a-5p/-15b-5p/-26a-5p/-34b-3p/-122-5p/-125b-5p/-191-5p/-296-5p and let-7a-5p) in spermatozoa from patients with teratozoospermia compared to the controls, whereas expression levels of four miRNAs (miR-92a-3p/-93-3p/-99b-5p/-328-3p) did not significantly differ between the study and control groups. The expression levels of all 13 included miRNAs were significantly positively correlated with each other and significantly positively associated with spermatozoa morphology, excluding miR-99b-5p. There were no other significant associations between miRNA expression and sperm quality parameters. Only expression levels of miR-99b-5p were significantly positively correlated with good-quality day 3 embryo rate (ρ = 0.546; p = 0.003), while other variables of the IVF/ICSI cycle outcome showed no significant associations with miRNA expression profiles. This is one of the rare studies providing an insight directly into miRNA profiles in regard to sperm morphology. We identified nine miRNAs that could serve as biomarkers of spermatozoa quality in regard to teratozoospermia.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Objectives: To identify dysregulated microRNAs (miRNAs) and their gene targets in temporal arteries from GCA patients, and determine their association with GCA pathogenesis and related arterial wall remodelling. Methods: We included 93 formalin-fixed, paraffin-embedded temporal artery biopsies (TABs) from treatment-naïve patients: 54 positive and 17 negative TABs from clinically proven GCA patients, and 22 negative TABs from non-GCA patients. miRNA expression analysis was performed with miRCURY LNA miRNome Human PCR Panels and quantitative real-time PCR. miRNA target gene prediction and pathway enrichment analysis was performed using the miRDB and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases, respectively. Results: Dysregulation of 356 miRNAs was determined in TAB-positive GCA arteries, among which 78 were significantly under-expressed and 22 significantly overexpressed above 2-fold, when compared with non-GCA controls. Specifically, TAB-positive GCA arteries were characterized by a significant overexpression of ‘pro-synthetic’ (miR-21-3p/-21-5p/-146a-5p/-146b-5p/-424-5p) and under-expression of ‘pro-contractile’ (miR-23b-3p/-125a-5p/-143-3p/-143-5p/-145-3p/-145-5p/-195-5p/-365a-3p) vascular smooth muscle cell phenotype-associated regulatory miRNAs. These miRNAs targeted gene pathways involved in the arterial remodelling and regulation of the immune system, and their expression correlated with the extent of intimal hyperplasia in TABs from GCA patients. Notably, the expression of miR-21-3p/-21-5p/-146a-5p/-146b-5p/-365a-3p differentiated between TAB-negative GCA arteries and non-GCA temporal arteries, revealing these miRNAs as potential biomarkers of GCA. Conclusion: Identification of dysregulated miRNAs involved in the regulation of the vascular smooth muscle cell phenotype and intimal hyperplasia in GCA arterial lesions, and detection of their expression profiles, enables a novel insight into the complexity of GCA pathogenesis and implies their potential utilization as diagnostic and prognostic biomarkers of GCA.

Giant cell arteritis (GCA) is a primary systemic vasculitis affecting the elderly, characterized by a granulomatous vessel wall inflammation of large- and medium-sized arteries. The immunopathology of GCA is complex, involving both the innate and adaptive arms of the immune system, where a maladaptive inflammatory-driven vascular repair process ultimately results in vessel wall thickening, intramural vascular smooth muscle cell proliferation, neovascularization and vessel lumen occlusion, which can lead to serious ischemic complications such as visual loss and ischemic stroke. Over the past decade, microRNA (miRNA) dysregulation has been highlighted as an important contributing factor underlying the pathogenesis of GCA. Since current understanding of miRNA involvement in GCA remains largely based on extrapolation of previously determined miRNA functions in vitro or in loss- or gain-of-function studies, an overall insight into the role of miRNA alteration in GCA pathophysiology remains limited. In this narrative review, we summarize the current knowledge on aberrantly expressed miRNAs in GCA and thoroughly discuss the impact of their altered regulatory role in the context of GCA setting. Furthermore, we address challenges and future perspectives in utilization of miRNA-based diagnostic and prognostic biomarkers of GCA in clinical settings.

Objectives: To identify aberrantly expressed microRNAs (miRNAs) in sinonasal tissue biopsies of patients with granulomatosis with polyangiitis (GPA), associate their expression profiles to sinonasal histopathology, and assess their differential expression between subgroups of clinically proven GPA patients, healthy controls and patients exhibiting inflammation of other etiology.Methods: We included formalin-fixed, paraffin-embedded biopsy tissue samples of sinonasal mucosa from 37 patients with clinically proven GPA, 15 patients with inflammation of other etiology and 14 control patients with normal histology. Of the included GPA patients, 20 patients had characteristic GPA-related histological features, while 17 patients displayed non-specific GPA histopathology in their sinonasal biopsy. Assessment of histological parameters was performed using histopathological techniques, and analysis of miRNA expression with miRCURY LNA miRNA miRNome Human PCR Panels and quantitative real-time PCR.Results: We determined expression of 306 miRNAs in sinonasal biopsy samples, which displayed different extent of dysregulation between individual patient groups. Based on their potential to discriminate between the controls, non-GPA and GPA patient subgroups, dysregulation of 11 miRNAs was further assessed, of which miR-1-3p/-21-3p/-93-5p/-155-5p/-1248/-31-3p/-182-5p/-183-5p and let-7b-5p held the potential to stratify patients based on their sinonasal tissue miRNA profile. Notably, several of these miRNAs were associated with the presence of granulomas, vasculitis and necrosis in sinonasal biopsies of GPA patients.Conclusion: Our study identifies novel miRNAs putatively implicated in the pathogenesis of GPA, and highlights dysregulated miRNAs as supporting biomarkers in establishing GPA diagnosis, particularly in the early phases of the disease, or in patients with atypical GPA presentation.

Aims: The transcriptional activity of high-risk human papillomaviruses (HR-HPV) within oropharyngeal squamous cell carcinomas (OPSCC) has been linked to improved survival of patients. HR-HPV mRNA silver in situ hybridization (SISH) was evaluated on a cohort of OPSCC and compared with viral HPV DNA tests and p16 expression. Clinical outcomes of HPV-driven OPSCC and non-HPV related OPSCC were also studied. Methods: We evaluated 67 OPSCC and 3 papillomas, obtained from 62 patients, for detection of HR-HPV DNA by PCR tests. The positive samples were additionally studied by the SISH method using three probes of HPV16, HPV18, and HP33, and for p16 expression detected by immunohistochemistry. SISH assays were evaluated for the presence/number and intensity of signals in cancer cells. Prognostic significance of HPV status in our cohort was evaluated with univariate and multivariate statistics. Results: According to the HR-HPV PCR tests, 46 (69%) OPSCC cases were HPV positive, while three papillomas were negative. Of total 46 HPV-positive OPSCCs, 43 cases were also SISH-positive, while p16 overexpression was found in 45 of 46 HPV positive OPSCC cases. In OPSCC specimens, the sensitivity and specificity of the combined SISH probes (HPV16 and 33) were both 100.00%, when compared to HPV PCR. HPV positivity of the tumors appeared significant for predicting progression-free survival, cause specific survival and overall survival in a multivariate setting. Conclusions: The recently developed mRNA SISH methodology can detect HPV-driven OPSCCs without any additional test in 79% of cases. Positive SISH signals enable the visualization of viral transcripts required to recognize clinically relevant HPV infection. However, rare and tiny signals require an experienced pathologist to establish a consensus interpretation of results. The currently applied HR-HPV mRNA SISH analysis may serve as a groundwork for additional studies.