Oxytocin preprotein and oxytocin receptor mRNA expression is altered in semen samples with abnormal semen parameters.

RESEARCH QUESTION: Are oxytocin preprotein and the oxytocin receptor expressed in human spermatozoa and is their mRNA expression different between normal semen samples and samples with at least one abnormal parameter? DESIGN: An in-vitro prospective study of 175 semen samples from Greek men, according to World Health Organization criteria, 2010. mRNA expression levels were compared between different categories of semen samples, classified according to their concentration, total number, motility and morphology. Immunohistochemistry was used to detect oxytocin preprotein and its receptor on spermatozoa smears. RESULTS: Compared with normal samples (normal motility and normal concentration), samples with at least one abnormal sperm parameter had statistically significantly lower oxytocin preprotein mRNA expression (P = 0.019) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oligozoospermic samples had statistically significantly higher oxytocin preprotein mRNA expression levels (P = 0.002) and lower oxytocin receptor mRNA expression levels (P = 0.047). Asthenozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P < 0.001). Teratozoospermic samples had statistically significantly lower oxytocin preprotein mRNA expression levels (P = 0.049) and higher oxytocin receptor mRNA expression levels (P < 0.001). Oxytocin preprotein mRNA expression was positively associated with total progressive motility (P < 0.001) and negatively associated with the percentage of immotile spermatozoa (P = 0.001). Oxytocin receptor mRNA expression was negatively associated with the percentage of normal forms (P < 0.001). CONCLUSION: Oxytocin preprotein and oxytocin receptor mRNA expression in spermatozoa could be used as a novel and unbiased diagnostic tool for male infertility.

Improvement in sperm quality by oral antioxidant supplementation in infertile men with varicocele who have not undergone surgical repair: Systematic review and meta-analysis.

The aim of this systematic review and meta-analysis was to assess whether oral antioxidant supplementation improves sperm quality in men with infertility and varicocele (VCL) who have not undergone surgical repair. In men with infertility and VCL who had not undergone surgical repair oral antioxidant supplementation significantly increased sperm concentration (WMD +5.86 × 106 /ml 95% CI: +1.47 to +10.24, p < 0.01; random effects model, six studies, 213 patients), total motility (WMD + 3.76%, 95% CI: +0.18 to +7.34, p = 0.04; random effects model, three studies, 93 patients), progressive motility (WMD + 6.38%, 95% CI: +3.04 to +9.71, p < 0.01; random effects model, three studies, 84 patients) and seminal volume (WMD +0.55 ml, 95%CI: +0.06 to +1.04, p = 0.03; random effects model, four studies, 120 patients). On the other hand, no significance difference was observed in sperm morphology (WMD +3.89%, 95% CI: -0.14 to +7.92, p = 0.06; random effects model, five studies, 187 patients). In conclusion, limited evidence suggests that the use of oral antioxidants in men with infertility and VCL, who have not undergone surgical repair improves their seminal volume, sperm concentration, total and progressive motility.

Effect of Bladder Injection of OnabotulinumtoxinA on the Central Expression of Genes Associated with the Control of the Lower Urinary Tract: A Study in Normal Rats.

To investigate a possible central mechanism of action of Botulinum toxin A (BoNT/A) following injection in the bladder, complementary to the acknowledged peripheral bladder effect, we studied changes in the expression of neuropeptides and receptors involved in lower urinary tract function in the spinal cord (SC) and dorsal root ganglia (DRG) of normal rats following BoNT/A bladder injection. Thirty-six Sprague-Dawley rats, divided into three groups of n = 12, received bladder injections of 2U or 5U OnabotulinumtoxinA (BOTOX®), or saline. Six animals from each group were sacrificed on days 7 and 14. Expression of Tachykinin 1 (Tac1), capsaicin receptor (TRPV1), neuropeptide Y (NPY), proenkephalin (PENK) and muscarinic receptors M1, M2, M3, was evaluated in the bladder, L6-S1 DRG, and SC segments using real-time PCR and Western blotting. Real-time PCR revealed increased expression of NPY in all tissues except for SC, and increased TRPV1 and PENK expression in DRG and SC, whereas expression of Tac1, M1 and M2 was decreased. Less significant changes were noted in protein levels. These findings suggest that bladder injections of OnabotulinumtoxinA may be followed by changes in the expression of sensory, sympathetic and cholinergic bladder function regulators at the DRG/SC level.

The protective variant rs7173049 at LOXL1 locus impacts on retinoic acid signaling pathway in pseudoexfoliation syndrome.

LOXL1 (lysyl oxidase-like 1) has been identified as the major effect locus in pseudoexfoliation (PEX) syndrome, a fibrotic disorder of the extracellular matrix and frequent cause of chronic open-angle glaucoma. However, all known PEX-associated common variants show allele effect reversal in populations of different ancestry, casting doubt on their biological significance. Based on extensive LOXL1 deep sequencing, we report here the identification of a common non-coding sequence variant, rs7173049A>G, located downstream of LOXL1, consistently associated with a decrease in PEX risk (odds ratio, OR = 0.63; P = 6.33 × 10-31) in nine different ethnic populations. We provide experimental evidence for a functional enhancer-like regulatory activity of the genomic region surrounding rs7173049 influencing expression levels of ISLR2 (immunoglobulin superfamily containing leucine-rich repeat protein 2) and STRA6 [stimulated by retinoic acid (RA) receptor 6], apparently mediated by allele-specific binding of the transcription factor thyroid hormone receptor beta. We further show that the protective rs7173049-G allele correlates with increased tissue expression levels of ISLR2 and STRA6 and that both genes are significantly downregulated in tissues of PEX patients together with other key components of the STRA6 receptor-driven RA signaling pathway. siRNA-mediated downregulation of RA signaling induces upregulation of LOXL1 and PEX-associated matrix genes in PEX-relevant cell types. These data indicate that dysregulation of STRA6 and impaired retinoid metabolism are involved in the pathophysiology of PEX syndrome and that the variant rs7173049-G, which represents the first common variant at the broad LOXL1 locus without allele effect reversal, mediates a protective effect through upregulation of STRA6 in ocular tissues.

Expression of genes that regulate follicle development and maturation during ovarian stimulation in poor responders.

RESEARCH QUESTION: Sex hormone-binding globulin (SHBG), androgen receptor (AR), LH beta polypeptide (LHB), progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) regulate follicle development and maturation. Their mRNA expression was assessed in peripheral blood mononuclear cells (PBMC) of normal and poor responders, during ovarian stimulation. DESIGN: Fifty-two normal responders and 15 poor responders according to the Bologna criteria were enrolled for IVF and intracytoplasmic sperm injection and stimulated with 200 IU of follitrophin alpha and gonadotrophin-releasing hormone antagonist. HCG was administered for final oocyte maturation. On days 1, 6 and 10 of stimulation, blood samples were obtained, serum hormone levels were measured, RNA was extracted from PBMC and real-time polymerase chain reaction was carried out to identify the mRNA levels. Relative mRNA expression of each gene was calculated by the comparative 2-DDCt method. RESULTS: Differences between mRNA levels of each gene on the same time point between the two groups were not significant. PGRMC1 and PGRMC2 mRNA levels were downregulated, adjusted for ovarian response and age. Positive correlations between PGRMC1 and AR (standardized beta = 0.890, P < 0.001) from day 1 to 6 and PGRMC1 and LHB (standardized beta = 0.806, P < 0.001) from day 1 to 10 were found in poor responders. PGRMC1 and PGRMC2 were positively correlated on days 6 and 10 in normal responders. CONCLUSIONS: PGRMC1 and PGRMC2 mRNA are significantly decreased during ovarian stimulation, with some potential differences between normal and poor responders.

Association of NFKB1 -94ATTG ins/del polymorphism (rs28362491) with pemphigus vulgaris.

Pemphigus vulgaris is a rare chronic blistering skin disease resulting from IgG autoantibodies directed against transmembrane desmosomal glycoprotein desmoglein 3 and is the most common form of pemphigus. Since interleukin-1 receptor-associated kinase (IRAK-1)/nuclear factor-kappa B (NF-kappa B) pathway plays an essential role in the pathogenesis of autoimmune diseases, the aim of the present study was to explore the role of polymorphisms in three genes, named IRAK1 (rs3027898), NFKBIA (rs696) and NFKB1 (-94ATTG insertion/deletion variant, - rs28362491), in PV susceptibility. Forty-four unrelated patients with PV (23 males) were enrolled in the study. Additionally, 77 ethnic matching healthy volunteers (45 males) with no personal or family history of chronic autoimmune or infectious diseases were studied. Strong statistical significant difference was observed between PV patients and controls for polymorphism -94 insertion/deletion ATTG in the promoter region of NFKB1 gene (P = 0.00005). Additional dedicated studies in larger groups of patients of various ethnicities are needed to replicate and confirm the preliminary findings.

Ethnicity-Based Differences in the Association of LOXL1 Polymorphisms with Pseudoexfoliation/Pseudoexfoliative Glaucoma: A Meta-Analysis.

Pseudoexfoliation (PEX) is an age-related disorder of the extracellular matrix; it is strongly associated with glaucoma, the leading cause of irreversible blindness worldwide. We conducted an ethnic-based meta-analysis of the association of LOXL1 polymorphisms with PEX/pseudoexfoliative glaucoma (PEXG). Association studies were retrieved systematically from PubMed, EMBASE, and Web of Knowledge. Allelic and genotype frequencies of rs3825942, rs1048661, and rs2165241 were compared between PEX/PEXG and controls. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using a random effects model. Overall, 39 independent cohorts were included. Rs3825942 (G) was an at risk allele for PEX/PEXG in Caucasians, Japanese, Koreans, Chinese, South Asians, and Middle Easterners, but protective in Black South Africans (OR = 0.10, 95%CI:0.06-0.16). Rs1048661 (G) was an at risk allele for PEX/PEXG in Caucasians, South Asians, Middle Easterners and Black South Africans, but was protective in Japanese (OR = 0.03, 95%CI:0.02-0.06) and Koreans (OR = 0.10, 95%CI:0.05-0.22). These associations we-re confirmed for the genotypic recessive models. Rs2165241 (C) was a protective allele for PEX/PEXG in Caucasians, but was an at risk allele in Japanese (OR = 7.49, 95%CI:3.22-17.41) and Koreans (OR = 6.63, 95%CI:2.60-16.90). This was confirmed for the genotypic dominant model. Other genetic and/or environmental factors may modify the effect of LOXL1 polymorphisms in certain ethnic groups.

Serous ovarian cancer signaling pathways.

Ovarian cancer is the most lethal malignancy of the female genital tract, mainly due to the failure of early diagnosis and the limitations posed by the conventional chemotherapies. Current research has focused in the study of cascades of various cellular molecular reactions, known as signaling pathways. In this review article, authors try to describe the current knowledge regarding the signaling pathways that influence multiple cellular processes in serous ovarian cancer and especially the pathogenesis. Thorough understanding of the precise role of these pathways can lead to the development of new and more effective targeted therapies as well as novel biomarkers in ovarian cancer.

Clinical significance of anti-desmoglein-1 and -3 circulating autoantibodies in Pemphigus Patients Measured by Area Index and Intensity Score.

Detection of anti-desmoglein-1 (anti-DSG-1) and anti-DSG-3 autoantibodies is widely used in the diagnosis of pemphigus. Two validated scoring systems, Pemphigus Disease Area Index (PDAI) and Autoimmune Bullous Skin Disorder Intensity Score (ABSIS), are used for the evaluation of clinical severity. The aim of this cross-sectional study was to interpret the titres of pemphigus autoantibodies in correlation with either total or location-dependent PDAI scores and ABSIS. A total of 35 pemphigus patients were selected and evaluated at 3 time points. Total PDAI and ABSIS seemed useful in pemphigus with cutaneous lesions or in the mucocutaneous form, while location-dependent PDAI and ABSIS scores were useful in the mucosal form. Anti-DSG-1 autoantibodies titres better showed the disease extent in pemphigus with cutaneous only or with mucocutaneous lesions. Anti-DSG-3 autoantibodies titres did not correlate to disease activity.

The Association of Vitamin D Receptor Polymorphisms with COVID-19 Severity.

BACKGROUND: Association studies of vitamin D receptor (VDR) polymorphisms with COVID-19 severity have produced inconsistent results in different populations. Herein we examined VDR gene polymorphisms in a Caucasian Greek cohort of COVID-19 patients. METHODS: This was a case-control study in a tertiary university hospital in Greece including 137 COVID-19 patients with varying disease severities and 72 healthy individuals. In total 209 individuals were genotyped for the FokI (rs10735810), ApaI (rs7975232), TaqI (rs731236) and BsmI (rs1544410) single-nucleotide polymorphisms (SNP) of the VDR gene by polymerase chain reaction and restriction fragment length polymorphism analysis (PCR-RFLPs). Statistical analyses were performed to determine the association between genotype and disease severity, adjusting for various confounding factors. RESULTS: Genotype distribution of the studied VDR SNPs in the control group was in Hardy-Weinberg equilibrium. The TaqI variant was differentially distributed between controls and COVID-19 patients according to the additive model (p = 0.009), and the CC genotype was significantly associated with an increased risk for severe COVID-19 according to the recessive model [OR: 2.52, 95%CI:1.2-5.29, p = 0.01]. Multivariate analysis demonstrated a robust association of COVID-19 severity and TaqI polymorphism in the recessive model even after adjusting for multiple confounders, including age, sex and CRP levels [Adj.OR:3.23, 95%CI:1.17-8.86, p = 0.023]. The distribution of FokI, ApaI and BsmI genotypes was similar between COVID-19 patients and controls. CONCLUSIONS: The CC genotype of TaqI polymorphism is significantly associated with an increased risk for severe COVID-19 independently of age, sex or degree of inflammation.

Haloperidol's Cytogenetic Effect on T Lymphocytes of Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients: An In Vitro Study.

OBJECTIVES: Investigating haloperidol's cytogenetic behavior in cultured human T lymphocytes of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). METHODS: Four haloperidol solutions were added in cultures of peripheral blood lymphocytes of healthy individuals, SLE, and RA patients. After 72 hours of incubation, the cultured lymphocytes were plated on glass slides, and stained with the fluorescence plus Giemsa method, and sister chromatid exchanges (SCEs), proliferation rate index (PRI), and mitotic index (MI) were measured with the optical microscope. RESULTS: Result analysis revealed: (a) a statistically significant (p=0.001) dose-dependent increase of SCEs in SLE patients compared to healthy individuals; (b) a statistically significant (p=0.001) dose-dependent decrease of SCEs in RA patients for haloperidol concentrations 5, 10µg/mL; (c) a statistically significant (p=0.001) dose-dependent increase of SCEs in RA patients for haloperidol concentrations 20, 100µg/mL; and (d) a statistically significant (p=0.001) dose-dependent reduction of PRI and MI in both patient groups compared to healthy individuals. Furthermore, a correlation was observed between (a) SCE and PRI index variations, (b) MI and SCE index variations, and (c) PRI and MI index variations. CONCLUSIONS: Haloperidol affects T lymphocytes from SLE and RA patients by modifying DNA replication procedures, DNA damage response, and ferroptosis. Considering the wide use of haloperidol in neuropsychiatric symptoms of SLE and RA patients, further studies with more immune cell subsets are needed to evaluate its effects on human genetic material.

Olanzapine's Cytogenetic Effect on T Lymphocytes in Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients: In Vitro Study.

OBJECTIVES: This study will investigate olanzapine's cytogenetic behavior in cultured human T lymphocytes in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). METHODS: Three olanzapine solutions were added in cultures of peripheral blood lymphocytes of healthy individuals, SLE, and RA patients. After 72 hours of incubation, the cultured lymphocytes were plated on glass slides and stained with the fluorescence plus Giemsa method. Sister chromatid exchanges (SCEs), proliferation rate index (PRI), and mitotic index (MI) were measured with the optical microscope. RESULTS: There was a statistically significant (p=0.001) dose-dependent increase of SCEs in SLE and RA patients compared to healthy individuals and a statistically significant (p=0.001) reduction of PRI and MI in the highest concentration in the SLE group. Moreover, Spearman's rank correlation coefficient was applied to calculate the correlation between SCEs, PRI, and MI. Negative significant correlations were noticed for both patient groups concerning SCEs-PRI alterations and SCEs-MI alterations. Conversely, positive correlations were noticed for both patient groups for PRI-MI alterations.  Conclusions: Olanzapine affects T lymphocytes from SLE and RA patients by modifying DNA replication procedures and DNA damage response. Considering the use of olanzapine in neuropsychiatric symptoms of SLE, further in vivo studies are necessary to evaluate its effect on human DNA.

Association between rs738409 and rs2896019 single-nucleotide polymorphisms of phospholipase domain-containing protein 3 and susceptibility to nonalcoholic fatty liver disease in Greek children and adolescents.

Background: Several studies have detected a strong association linking rs738409 and rs2896019 polymorphisms in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene with hepatic steatosis and steatohepatitis. In the present study we aimed to determine the association of those PNPLA3 variants with nonalcoholic fatty liver disease (NAFLD) susceptibility in obese and nonobese Greek children and adolescents. Methods: The study recruited 91 children and adolescents of Greek descent with NAFLD or biopsy-proven nonalcoholic steatohepatitis, and 91 healthy subjects of normal weight (control group) with sex distribution similar to the patient group. DNA samples were amplified using polymerase chain reaction with specifically designed primers. Data were analyzed using the statistical software SPSS version 24.0. Results: A significant correlation was shown between the rs738409 polymorphism (CG and GG genotypes) and the rs2896019 polymorphism (TG genotype) with the development of hepatic steatosis (P<0.001). The incidences of rs738409 GG, rs738409 CG and rs2896019 TG genotypes were found to be increased in patients with hepatic steatosis (obese and nonobese), but not in obese patients without liver disease. The combined expression of the 2 polymorphisms was associated with a lower age of diagnosis of hepatic steatosis in nonobese patients. Conclusions: We confirmed a strong association between the 2 polymorphisms and hepatic steatosis. The association of the rs2896019 single-nucleotide polymorphism with hepatic steatosis in obese and nonobese pediatric patients, and the combined study of both polymorphisms in a pediatric population of Greek origin are described for the first time.

Association of rs738409 Polymorphism in Adiponutrin Gene with Liver Steatosis and Atherosclerosis Risk Factors in Greek Children and Adolescents.

Non-alcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis, as it is associated with components of the metabolic syndrome. However, genetic variations have also been linked to the risk of NAFLD, such as adiponutrin/patatin-like phospholipase domain-containing the protein 3 (PNPLA3) rs738409 polymorphism. The aim of the study was to determine the associations of thePNPLA3 rs738409 polymorphism with NAFLD and atherosclerosis risk factors in children and adolescents from northern Greece. A total of 91 children/adolescents who followed a Mediterranean eating pattern with no particular restrictions were studied. They were divided into three subgroups, according to their body mass index (BMI) and the presence or absence of liver disease. Diagnosis of NAFLD was based on a liver ultrasound, while the distribution of the PNPLA3 rs738409 polymorphism was investigated in all the participants. From the components of metabolic syndrome, only BMI, waist circumference, blood pressure, and the homeostasis model of insulin resistance (HOMA-IR) differed significantly between groups. The rs738409 polymorphism was significantly associated with BMI and NAFLD, while lipid values had no significant association with either NAFLD or gene polymorphism. This study shows that in Greekchildren, there is a significant association between the rs738409polymorphism in the PNPLA3 gene and hepatic steatosis, regardless of bodyweight.

Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study.

BACKGROUND: Recent scientific data support that the mode of conception and delivery may influence epigenetic regulation and therefore embryo development. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel variant of OCT4 with yet unknown biological function, is suggested to have a potential role in mediating cellular stress response. Furthermore, Insulinlike Growth Factor 2 (IGF2), Mesoderm-specific Transcript (MEST) and paternally expressed gene 10 (PEG10) are genes known as imprinted and are regulated via means of epigenetic regulation. The influence of delivery mode and conception on epigenetic regulation is an active research field. OBJECTIVE: Our aim was to correlate the expression level of Oct4B1 and the expression and methylation level of IGF2, MEST, and PEG10 imprinted genes with the mode of delivery and conception in the umbilical cord blood of newborns. MATERIALS AND METHODS: Samples of umbilical cord blood from infants born after vaginal delivery, caesarean section (CS) with the infant in cephalic position and CS due to breech position were examined. Furthermore, the investigation included infants conceived through means of assisted reproductive technology. RESULTS: No statistically significant differences were found in mRNA expression levels between different modes of conception and delivery (p = 0.96). Oct4B1, IGF2, MEST, and PEG10 expression levels do not seem to be significantly affected by different modes of conception and delivery. CONCLUSION: These results indicate that the expression and methylation patterns of Oct4B1, IGF2, MEST and PEG10 in umbilical cord blood are not affected by the conception and delivery mode.

OCT4B1 isoform: the novel OCT4 alternative spliced variant as a putative marker of stemness.

A novel OCT4 alternative spliced variant (OCT 4B1) is deduced to be the isoform present in 59 hESC lines characterised by the International Stem Cell Initiative (ISCI) rather than OCT4A as previously assumed. The new variant may be a more reliable marker of stemness than OCT4A and studies are needed to test this.

Revisiting OCT4 expression in peripheral blood mononuclear cells.

The transcription factor OCT4 (officially POU5F1; alternatively OCT3, OCT3/4, OTF3, and OTF4) is currently considered a main regulator of human embryonic stem cell pluripotency and self-renewal capacities. Importantly, these stemness properties are attributed to OCT4A, which is one of the two isoforms produced by the OCT4 gene. The second OCT4 isoform, OCT4B, does not share the stemness factor characteristics of OCT4A and is currently considered of unknown function. Hence, when investigating OCT4 expression at the mRNA and protein level, it is important to specify which OCT4 isoform is detected by the applied methods, such as polymerase chain reaction assays and immunocytochemistry antibodies. Here, we discuss the need to distinguish between OCT4A and OCT4B when interpreting OCT4 expression in differentiated cells, such as peripheral blood mononuclear cells.

Detection of IgG Autoantibodies against Desmocollin-3 in Greek Patients with Pemphigus.

Pemphigus is an autoimmune bullous disorder caused by autoantibodies against desmosomal cadherins. The most common clinical forms are pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Among the numerous proteins that are considered responsible for the cohesion of keratinocytes in epidermis, desmocollin-3 (Dsc-3) has been initially reported to participate in epidermal blistering in mice. There have been reports in which autoantibodies against Dsc-3 have been detected. In PV, a limited number of studies found no presence of IgG or IgA autoantibodies against Dsc-3. In this study we examined sera from Greek patients with PV and PF for the presence of IgG autoantibodies against Dsc-3. Immunoblotting for the detection of autoantibodies against Dsc-3 was performed in sera from all cases. Dsc-3 autoantibodies were not detected in either group (PV and PF). Our results confirm the hypothesis that the pathogenic role of Dsc-3 in epidermal blistering in PV and PF remains controversial.