Investigation of sperm hsa-mir-145-5p and MLH1 expressions, seminal oxidative stress and sperm DNA fragmentation in varicocele.

BACKGROUND: Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. METHODS: Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. RESULTS: Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. CONCLUSION: This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.

Evaluation of Transforming Growth Factor Beta-1 in the vein wall of males with varicocele.

OBJECTIVE: This study aims to investigate potential differences in the presence of Transforming Growth Factor-Beta 1 (TGF-ß1) between the vein walls of patients with varicocele and those of healthy individuals. PATIENTS AND METHODS: The study comprised a total of 40 participants, divided into two groups. The control group (Group 1) consisted of 20 patients who underwent coronary bypass surgery, while the varicocele group (Group 2) included 20 patients scheduled for varicocelectomy. The cytoplasmic and nuclear staining patterns of TGF-ß1 immunohistochemistry were assessed in tissue samples under light microscopy, identifying any differences in TGF-ß1 presence between varicocele patient vein walls and normal (saphenous) veins. RESULTS: The varicocele group demonstrated lower nuclear and cytoplasmic TGF-ß1 staining rates compared to the control group. After controlling for the independent factor of age, significantly lower nuclear and cytoplasmic staining was still observed in the varicocele group. CONCLUSIONS: This study is the first of its kind to compare TGF-ß1 staining in the vein walls of varicocele patients and healthy individuals. Previous studies focusing on varicose veins reported elevated TGF-ß1 expression. Contrarily, our study observed lower TGF-ß1 expression in varicocele patient veins, marking a unique contribution to the field.

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms.

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR.

Oxidative Stress and Cell Cycle Arrest in Seminiferous Tubules Nearby Varicose Vessels: New Perspectives from Experimental Varicocele.

Varicocele (VCL) has been shown to induce severe oxidative stress in the testicular tissue resulting in 35% of males with primary infertility. To compare the exacerbating impacts of varicose on oxidative DNA damage and homeostatic antioxidant reactions in the seminiferous tubules (ST), enclosed and far from varicose vessels. Thirty mature Wistar rats were divided into control and VCL-induced groups. To approve VCL, the testicular diameters, volume, and blood circulation were measured using B-mode and Doppler ultrasonography. Next, to confirm oxidative stress (OS), the global homeostatic antioxidant biomarkers were evaluated. Moreover, the OS-induced oxidative DNA damage and homeostatic antioxidant reactions were compared between STs nearby and far from varicose vessels. Finally, to clarify the DNA damage-induced impact on the cell cycle progression, the global and local expressions of Cyclin D1, Cdk4, and p21 were examined. The VCL-induced group exhibited diminished global antioxidant status (marked with TAC, GPX, SOD, and CAT) and UNG and MPG expression levels. Moreover, the cross-sections of the VCL group represented a prominent reduction in the UNG, MPG, Cyclin D1, and cdk4, and upregulation in the p21 expression levels, more prominently in the STs nearby varicose vessels. Concerning severe oxidative DNA damage and intensive molecular changes in the STs nearby the varicose vessels, they can be considered the main cause of oxidative DNA damage in enclosed tubules. Thus, the varicose-mediated oxidative DNA damage negatively impacts the cell cycle progression in the tubules more intensively in the subcapsular area.

Ergothioneine attenuates varicocele-induced testicular damage by upregulating HSP90AA1 in rats.

This study investigates the therapeutic effect and the underlying mechanisms of ergothioneine (EGT) on the testicular damage caused by varicocele (VC) in vivo, in vitro, and in silico. This preclinical study combines a series of biological experiments and network pharmacology analyses. A total of 18 Sprague Dawley (SD) male rats were randomly and averagely divided into three groups: the sham-operated, VC model, and VC model with EGT treatment (VC + EGT) groups. The left renal vein of the VC model and the VC + EGT groups were half-ligated for 4 weeks. Meanwhile, the VC + EGT group was intragastrically administrated with EGT (10 mg/kg). GC1 and GC2 cells were exposed to H2 O2 with or without EGT treatment to re-verify the conclusion. The structure disorder of seminiferous tubules ameliorated the apoptosis decrease in the VC rats receiving EGT. EGT can also increase the sperm quality of the VC model rats (p < 0.05). The exposure to H2 O2 decreased proliferation and increased apoptosis of GC1 and GC2 cells, which was revisable by adding EGT to the plates (p < 0.05). The network pharmacology and molecular docking were conducted to explore the potential targets of EGT in VC, and HSP90AA1 was identified as the pivotal gene, which was validated by western blot, immunohistochemistry, and RT-qPCR both in vivo and in vitro (p < 0.05). Overall, EGT attenuates the testicular injury in the VC model both in vivo and in vitro by potentially potentiating the expression of HSP90AA1.

Is Parthanatos Involved in Varicocele?

Varicoceles (VCs) have received widespread attention as a primary factor affecting male fertility and a pathological condition that may lead to decreased sperm count and motility in patients. Many studies have shown that an imbalance of local antioxidant balance exists in patients with VC, leading to an obvious increase in the content of reactive oxygen species (ROS) and may cause reductive stress. Excessive ROS may aggravate spermatogenesis dysfunction and affect male fertility. Poly(ADP-ribose) polymerase (PARP) is an enzyme associated with DNA repair in eukaryotic cells, can be activated by DNA fragments with structural damage, and has been considered a DNA damage receptor in DNA damage repair and apoptosis. We built a rat model of VC and an oxidative damage model of a spermatocyte-derived cell line (GC-2 cells) induced by hydrogen peroxide to study the role of PARP1 in VC. Differentially expressed genes (DEGs) were obtained by RNA sequencing in the testes of VC rats. Analysis of DEGs revealed some genes with significantly altered expression, which were validated in rat and cell models. Immunofluorescence, real-time quantitative PCR analysis, Western blot, and flow cytometry were used to analyze the changes between the control group and the VC or hydrogen peroxide group. Overall, we found that PARP1 protein expression increased in VC rats and in the hydrogen peroxide-induced oxidative stress model of GC-2 cells. Parthanatos may be one of the factors leading to reduced reproductive capacity in VC patients. Our study provides novel insights into the mechanisms of male infertility induced by oxidative stress and provides a new therapeutic target for VC.

Varicocoele-associated male infertility: Cellular and molecular perspectives of pathophysiology.

BACKGROUND: Varicocoele is a common risk factor associated with reduced male fertility potential. The current understanding of varicocoele pathophysiology does not completely explain the clinical manifestation of infertility. The present treatment options such as antioxidant supplementation and varicocoelectomy only help ≈35% of men to achieve spontaneous pregnancy. OBJECTIVE: This review aims to summarize the available knowledge on cellular and molecular alterations implicated to varicocoele-associated male infertility and also highlights the new knowledge generated by "omics" technologies. MATERIALS AND METHODS: PubMed, MEDLINE, Cochrane and Google Scholar databases are searched using different combinations of keywords (varicocoele, infertile/fertile men with varicocoele, cellular changes, molecular mechanisms, proteome, epigenome, transcriptome and metabolome). A total of 229 relevant human and animal studies published till 2021 were included in this review. RESULTS: Current understanding advocates oxidative stress (OS) as a major contributory factor to varicocoele-associated male infertility. Excessive OS causes alteration in testicular microenvironment and sperm DNA fragmentation, which further contributes to infertility. Molecular and omics studies have identified several promising biomarkers such as AAMP, SPINT1, MKI67 (genetic markers), sperm quality and function related protein markers, global sperm DNA methylation level (epigenetic marker), Hspa2, Protamine, Gadd7, Dynlt1 and Beclin1 (mRNA markers), PRDX2, HSPA, APOA2, YKL40 (seminal protein markers), total choline and PHGDH (metabolic markers). DISCUSSION: Mature spermatozoa harbours a plethora of molecular information in form of proteome, epigenome and transcriptome, which could provide very important clues regarding pathophysiology of varicocoele-associated infertility. Recent molecular and omics studies in infertile men with varicocoele have identified several promising biomarkers. Upon further validation with larger and well-defined studies, some of these biomarkers could aid in varicocoele management. CONCLUSION: The present evidences suggest that inclusion of OS and sperm DNA fragmentation tests could be useful to the diagnostic workup for men with varicocoele. Furthermore, including precise molecular markers may assist in diagnostics and prognostics of varicocoele-associated male infertility.

The effect of Fumaria parviflora on the expression of sexual hormones along with their receptors in testicles of adult rats induced by varicocele.

Varicocele (VCL) is a pathological dilation of the venous pampiniform plexus of the spermatic cord and is also classified as male factor infertility. The current experiment aimed to examine the protective effect of Fumaria parviflora (FP), as a powerful antioxidant, against reproductive damage induced by VCL. In this experimental study, 32 male rats were randomly allocated into four groups, namely sham (simple laparotomy without additional intervention), FP (healthy rats administered 250 mg/kg FP), VCL + FP (underwent VCL and received 250 mg/kg FP), VCL (underwent VCL without receiving any treatment). The results showed that the number of Sertoli and germ cells were markedly reduced in the VCL group in comparison to the FP-treated and sham groups. The VCl + FP group had significantly higher serum levels of testosterone (T), FSH, and LH hormones than the VCL group. The quality and motility of spermatozoa were reduced in the VCL group compared with other groups (p ≤ 0.05). Moreover, our findings demonstrated that the administration of FP considerably enhanced the mRNA levels of CatSper-1 and -2, SF-1, 3ß-HSD, 17ß-HSD3, LHCGR, and FSHR (p ≤ 0.05). Based on the obtained results, treatment with FP is capable of preventing testicular dysfunction and elevating the concentration of hormones and some crucial genes, such as CatSper1 and 2, SF-1, 3ß-HSD, 17ß-HSD3, LHCGR, and FSHR that contribute to the spermatogenesis process.

Minocycline can reduce testicular apoptosis related to varicocele in male rats.

The current research aimed to assess the impacts of Minocycline on varicocele-induced regulation of apoptotic-related genes and oxidative stress in the testis of adult Wistar rats. Thirty-two rats were divided into 4 groups: sham, varicocele (VcI), varicocele treated with Minocycline (VcI + Mno) for 56 days and healthy rats treated with minocycline (Mno). After 8 weeks, the oxidative stress markers levels in serum were investigated, afterwards, the level of Bax and Bcl-2 expression were assessed through 'immunocytochemistry' and RT-qPCR assays. Also, the rate of apoptosis was evaluated through the TUNEL method. Johnson's score, 'the width of epithelium' and 'seminiferous tubules diameter' were ameliorated in the VcI + Mno group in comparison with the Vcl group. Administration of Minocycline raised the 'Glutathione peroxidase' and 'Superoxide dismutase' levels in serum and declined the Malondialdehyde level in serum (p = 0.001). Furthermore, current study represented that minocycline reduced Bax and enhanced the expression of Bcl-2 gene and protein in comparison with the Vcl group (p < 0.05). In addition, Minocycline administration significantly declined the rate of apoptosis in germ cells (p < 0.05). Our study demonstrated that the administration of Minocycline could improve testicular injury in varicocele-induced rats by its antioxidant activity.

Long noncoding RNAs regulated spermatogenesis in varicocele-induced spermatogenic dysfunction.

OBJECTIVES: To evaluate the expression, potential functions and mechanisms of long noncoding RNAs (lncRNAs) in the pathogenesis of varicocele (VC)-induced spermatogenic dysfunction. MATERIALS AND METHODS: We established a rat model with left experimental VC and divided rats into the sham group, the VC group, and the surgical treatment group (each group, n = 10). Haematoxylin and eosin (HE) staining and sperm quality were analysed to evaluate spermatogenesis function. LncRNA expression profiles were analysed using lncRNA-Seq (each group n = 3) and validated using quantitative real-time polymerase chain reaction (each group n = 10). Correlation analysis and gene target miRNA prediction were used to construct competing endogenous RNA network. The regulated signalling pathway and spermatogenic dysfunction of differentially expressed lncRNAs (DE lncRNAs) were validated by Western blot. RESULTS: HE detection and sperm quality analysis showed that VC could induce spermatogenic dysfunction. Eight lncRNAs were upregulated and three lncRNAs were downregulated in the VC group compared with the sham group and surgical treatment group. The lncRNA of NONRATG002949.2, NONRATG001060.2, NONRATG013271.2, NONRATG022879.2, NONRATG023424.2, NONRATG005667.2 and NONRATG010686.2 were significantly negatively related to sperm quality, while NONRATG027523.1, NONRATG017183.2 and NONRATG023747.2 were positively related to sperm quality. The lncRNAs promote spermatogenic cell apoptosis and inhibit spermatogonia and spermatocyte proliferation and meiotic spermatocytes by regulating the PI3K-Akt signalling pathway. CONCLUSION: DE lncRNAs may be potential biomarkers for predicting the risk of spermatogenic dysfunction in VC and the effect of surgical repair. These DE lncRNAs promote spermatogenic dysfunction by regulating the PI3K-Akt signalling pathway.

MIR210HG is aberrantly expressed in the seminal plasma of varicocele patients and associated with varicocele-related dyszoospermia.

This study aimed to confirm the expression of the seminal plasma long noncoding RNAs (lncRNAs) microRNA210 host gene (MIR210HG) in varicocele (VC) patients, to further explore the association between MIR210HG and VC severity and to evaluate whether MIR210HG can predict VC-related dyszoospermia. Semen samples from 188 VC patients and 92 healthy men were collected. Quantitative reverse transcriptase PCR detected seminal plasma MIR210HG levels. Receiver operating characteristic analysis assessed the ability of MIR210HG to screen patients with VC, or to screen VC patients with abnormal semen quality. Logistic analysis assessed the value of MIR210HG in predicting dyszoospermia in VC patients. The levels of MIR210HG in seminal plasma of VC patients were upregulated, which could screen VC patients. In addition, the levels of seminal plasma MIR210HG were upregulated with VC severity and were downregulated at 6 months after surgery in VC patients. Moreover, elevated MIR210HG levels in VC patients with abnormal semen quality could screen patients with abnormal semen quality and could independently predict the occurrence of dyszoospermia in VC patients. Seminal plasma MIR210HG expression is upregulated in VC patients, is associated with the severity of VC and may function as an independent predictor of VC-related dyszoospermia.

Melatonin epigenetic potential on testicular functions and fertility profile in varicocele rat model is mediated by silent information regulator 1.

BACKGROUND AND PURPOSE: Varicocele is a leading cause of male infertility. Melatonin is a highly pleiotropic neurohormone. We aimed to characterize the melatonin epigenetic potential in varicocele and the involved molecular mechanisms. EXPERIMENTAL APPROACH: Fifty-two male albino rats were randomly divided into four groups (13 rats each): control (I), melatonin (II), varicocele (III) and melatonin treated varicocele (IV) groups. Left varicocele was induced by partial left renal vein ligation. Reproductive hormones, epididymal sperm functional parameters, testicular 3/17 ß-hydroxysteroid dehydrogenases, antioxidant enzymes, malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase, 8-hydroxy-2'-deoxyguanosine and histopathological/Johnsen's score were evaluated. Flow cytometry and Comet were carried out to explore extent of sperm and testicular DNA damage. Testicular expression of silent information regulator 1 (SIRT1), forkhead transcription factors-class O (type1) (FOXO1), tumour suppressor gene, P53, cation channels of sperm (CatSper) and steroidogenic acute regulatory protein was evaluated by western blot technique. Testicular expression of Bcl-2 and its associated X protein and nuclear factor kappa-light-chain-enhancer of activated B cells were assayed by immunohistochemical staining. Testicular miR-34a expression was quantified by quantitative reverse transcription-polymerase chain reaction. KEY RESULTS: The varicocele induced testicular histological injury, enhanced oxidative stress, P53-mediated apoptosis, DNA damage and increased testicular miR-34a expression paralleled with down-regulated SIRT1/FOXO axis. Melatonin treatment of varicocele rats displayed antioxidant/anti-apoptotic efficacy and improved reproductive hormones axis, CatSper expression and fertility parameters. MiR-34a/SIRT1/FOXO1 epigenetic axis integrates testicular melatonin mediated intracellular transduction cascades in varicocele. CONCLUSION AND IMPLICATIONS: Melatonin can be used as an adjuvant therapy to improve varicocele and its complication.

Exosomes as Potential Biomarkers for Erectile Dysfunction, Varicocele, and Testicular Injury.

INTRODUCTION: Optimal male reproductive health is dependent upon critical mediators of cell-cell communication: exosomes or extracellular vesicles. These vesicles are nano-sized particles released into a variety of bodily fluids, such as blood and semen. Exosomes are highly stable and can carry genetic and other molecules, including DNA, RNA, and proteins, which provide information about their origin cells. OBJECTIVE: To identify exosomes as potential biomarkers or therapeutic mediators in male sexual and reproductive disorders like erectile dysfunction (ED), varicocele, and testicular injury. METHODS: A PubMed search was performed to highlight all articles available relating to exosomes and extracellular vesicles in the pathogenesis of different male sexual and reproductive disorders, and their importance in clinical use as both diagnostic markers and potential therapeutic mediators. RESULTS: Various male reproductive system disorders, such as ED, varicocele, and testicular injury, are linked to increased or decreased levels of exosomes. Exosomes have a higher number of molecules such as DNA, RNA, and proteins, which can give a more precise and comprehensive result when compared to other biomarkers. Exosomes can be considered as plausible diagnostic biomarkers for male sexual and reproductive diseases, with considerable advantages over other diagnostic procedures such as invasive tissue biopsy. Exosomes can carry cargo such certain drugs and therapeutic molecules making them a promising therapeutic approach. Several studies have begun to test treating various male sexual reproductive disorders with exosomes. CONCLUSION: Exosomes deliver many components that can regulate gene expression and target signaling pathways. Understanding how extracellular vesicles can be utilized as biomarkers in diagnosing men, particularly those with idiopathic erectile dysfunction, will not only aid in diagnosis but also help with making therapeutic targets. Khodamoradi K, Golan R, Dullea A, et al. Exosomes as Potential Biomarkers for Erectile Dysfunction, Varicocele, and Testicular Injury. Sex Med Rev 2022;10:311-322.

[Pricking-reinforcing -reducing therapy improves semen quality and seminal plasma biochemical indexes in patients with varicocele infertility].

OBJECTIVE: To investigate the effect of pricking-reinforcing -reducing therapy (PRRT) on the semen quality and seminal plasma biochemical indexes of varicocele (VC) infertility patients. METHODS: We randomly and equally assigned 160 patients with VC infertility into a PRRT and a control group, the former treated by PRRT and the latter with oral ShengjingCapsules. Before and after treatment, we obtained the semen parameters, sperm morphology, sperm survival rate, sperm acrosin activity, seminal plasma neutral α glucosidase and seminal plasma zinc in the patients and compared them between the two groups. RESULTS: Before treatment, there were no statistically significant differences between the PRRT and control groups in sperm concentration (ï¼»16.81 ± 7.83ï¼½ vs ï¼»16.80 ± 7.54ï¼½ ×106 /ml, P > 0.05), total sperm count (ï¼»42.01 ± 19.57ï¼½ vs ï¼»41.99 ± 18.84ï¼½ ×106, P > 0.05), percentages of progressively motile sperm (PMS) (ï¼»15.37 ± 11.03ï¼½% vs ï¼»14.68 ± 10.27ï¼½%, P > 0.05) and morphologically normal sperm ( MNS) (1.62 ± 1.51ï¼½% vs ï¼»1.62 ± 1.13ï¼½%, P > 0.05), sperm survival rate (ï¼»28.11 ± 18.95ï¼½% vs ï¼»28.23±18.38ï¼½%, P > 0.05) and sperm acrosin activity (ï¼»28.11 ± 14.64ï¼½ vs ï¼»27.19 ± 14.07ï¼½ U/L, P > 0.05). After three months of treatment, all the patients showed evident increases in the above parameters (P < 0.05), even higher in the PRRT than in the control group, more significantly in sperm concentration (ï¼»38.88 ± 30.54ï¼½ vs ï¼»25.60 ± 14.71ï¼½ ×106 /ml, P < 0.05), PMS (ï¼»32.60 ± 12.46ï¼½% vs ï¼»27.67 ± 12.27ï¼½%, P < 0.05) and sperm acrosin activity (ï¼»65.74±31.81ï¼½ vs ï¼»67.94±17.95ï¼½ U/L, P < 0.05), though not significantly in total sperm count (97.20 ± 76.35ï¼½ vs ï¼»88.19 ± 39.56ï¼½ ×106, P > 0.05), MNS (ï¼»2.35 ± 1.83ï¼½% vs ï¼»1.87 ± 1.20ï¼½%, P > 0.05) and sperm survival rate (ï¼»61.44 ± 20.02ï¼½% vs ï¼»59.12 ± 22.48ï¼½%, P > 0.05). Compared with the baseline, after treatment, the patients in the PRRT group also exhibited elevated levels of neutral α-glucosidase (ï¼»14.42 ± 5.90ï¼½ vs ï¼»28.43 ± 19.76ï¼½ U/L, P < 0.05) and seminal plasma zinc (ï¼»2.11 ± 1.22ï¼½ vs ï¼»2.89 ± 1.23ï¼½ mmol/L, P < 0.05), and so did the controls (ï¼»14.44 ± 5.61ï¼½ vs ï¼»26.66 ± 17.69ï¼½ U/L , P < 0.05) and (ï¼»2.09 ± 1.10ï¼½ vs ï¼»2.82±1.08ï¼½ mmol/L, P < 0.05). No statistically significant difference, however, was observed between the two groups after treatment (P > 0.05). CONCLUSION: PRRT can significantly improve semen quality in patients with VC infertility, even more effective than ShengjingCapsules in improving sperm concentration, PMS, sperm survival rate, and sperm acrosin activity, which may be related to its effect of elevating the levels of seminal plasma neutral-α glucosidase and zinc providing sufficient energy for basic sperm metabolism, maturation, energy acquisition and motility.

Expression of Hypoxia-Inducible Factor1-α in Varicocele Disease: a Comprehensive Systematic Review.

Hypoxia has been suggested as an important pathophysiological feature in varicocele disease. On the other hand, the expression of hypoxia-inducible factor 1-alpha (HIF1-α) is associated with the incidence of hypoxia. In this study, we investigated the expression of HIF1-α in varicocele disease through a comprehensive systematic review. We searched PubMed, Scopus, Web of Science, and Embase databases to identify the related studies published up to February 2021. Human studies have demonstrated an increase in the HIF-1α protein expression in the internal spermatic vein (ISV) of the varicocele testicle. HIF-1α mRNA expression in the seminal plasma was significantly higher in infertile varicocele patient compared with fertile ones. Similarly, most animal studies demonstrated a significant increase in HIF-1α gene and protein expression in varicocele testicular tissue compared with control groups. The studies illustrated that hypoxia followed by increased expression of hypoxia-inducible factor 1-alpha (HIF1-α) mRNA and protein occurs in varicocele disease. Expression of HIF-1α regulates the expression of many genes, including VEGF, p53, GLUT, Bax, and Caspase-3, that could be involved in many of the varicocele pathophysiological effects such as DNA fragmentation and apoptosis of sperm cells. Further studies with a large number of patients are necessary and can provide more definitive evidence.

Expression of proapoptotic receptors Fas/Fas-L on sperm cells and levels of their soluble forms in seminal fluid from patients with varicocele.

Varicocele is a condition in which the pampiniform venous plexus in the scrotum dilates abnormally during puberty, affecting testicular growth and semen parameters, and is thought to be a major cause of male infertility. The findings of researches on the presence of the Fas system in sperm are controversial. As the main triggers of apoptosis in the semen of patients with varicocele, in this study, we examined the expression of Fas/Fas-L on sperm cells and also the levels of their soluble forms in seminal plasma. Semen samples were taken from 45 patients with varicocele (study group) and 45 healthy subjects without varicocele (control group) after 3-5 days of ejaculatory abstinence. Flow cytometry was used to examine the expression of Fas and Fas ligand (Fas-L) on Sperm cells. Furthermore, soluble Fas (sFas) and soluble Fas-Ligand (sFas-L) levels in the seminal fluid were determined using an ELISA (enzyme-linked immunosorbent assay). The presence of Fas and Fas-L proteins on the sperm ejaculation surface was not found in the patients with varicocele or the control groups. However, in the case group (3.32 ± 0.31 ng/mL), sFas seminal concentrations were slightly lower than in the control group (5.50 ± 0.36 ng/mL) (p < 0.001). In varicocele patients, there was a strong negative association between seminal sFas and sperm motility. Apoptosis effects through this system on key sperm parameters were not found based on our findings. In varicocele, a reduction in sperm count and motility does not seem to be due to Fas-related mechanisms, but rather to other mechanisms.

Varicocele-Mediated Male Infertility: From the Perspective of Testicular Immunity and Inflammation.

Background: Varicocele (VC) is present in 35 - 40% of men with infertility. However, current surgical and antioxidant treatments are not completely effective. In addition to oxidative stress, it is likely that other factors such as testicular immune microenvironment disorder contribute to irreversible testicular. Evidence suggests that VC is associated with anti-sperm antibodies (ASAs), spermatogenesis and testosterone secretion abnormalities, and testicular cytokine production. Moreover, inhibition of inflammation can alleviate VC-mediated pathogenesis. The normal function of the testis depends on its immune tolerance mechanism. Testicular immune regulation is complex, and many infectious or non-infectious diseases may damage this precision system. Results: The testicular immune microenvironment is composed of common immune cells and other cells involved in testicular immunity. The former includes testicular macrophages, T cells, dendritic cells (DCs), and mast cells, whereas the latter include Leydig cells and Sertoli cells (SCs). In animal models and in patients with VC, most studies have revealed an abnormal increase in the levels of ASAs and pro-inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha in the seminal plasma, testicular tissue, and even peripheral blood. It is also involved in the activation of potential inflammatory pathways, such as the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing (NLRP)-3 pathway. Finally, the development of VC-mediated infertility (VMI) may be facilitated by abnormal permeability of proteins, such as claudin-11, that constitute the blood-testis barrier (BTB). Conclusions: The testicular immune response, including the production of ASAs and inflammatory factors, activation of inflammatory pathways, and destruction of the BTB may be involved in the pathogenesis of VMI it is necessary to further explore how patient outcomes can be improved through immunotherapy.

The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation-Reduction Potential in Semen.

Since varicocele is so common in infertile men, this study intends to analyse the relationships between varicocele and conventional semen characteristics, sperm nuclear DNA dispersion and oxidation-reduction potential (ORP) in semen. Varicocele-positive and varicocele-negative infertile men (study groups) showed significantly lower standard sperm parameters and higher sperm DNA fragmentation (SDF) and ORP in semen than healthy volunteers and subjects with proven fertility (control groups). A lower proportion of low SDF levels (0-15% SDF) and higher incidence of high SDF levels (>30% SDF), as well as a higher prevalence of high ORP values (>1.37 mV/106 sperm/mL), were found in the study groups vs. the control groups. Moreover, infertile men had significantly lower odds ratios (ORs) for low SDF levels and significantly higher ORs for high SDF levels and high ORP. SDF and ORP were negatively correlated with sperm number, morphology, motility and vitality. Furthermore, a significant positive correlation was found between SDF and ORP. The obtained results suggest that disorders of spermatogenesis may occur in varicocele-related infertility. These abnormalities are manifested not only by reduced standard semen parameters but also by decreased sperm DNA integrity and simultaneously increased oxidative stress in semen.

Verbascoside attenuates experimental varicocele-induced damage to testes and sperm levels through up-regulation of the hypothalamus-pituitary-gonadal (HPG) axis.

CONTEXT: Verbascoside (VB), which is found in many medicinal plant families, exhibits biological activities in various diseases. However, its effects on varicocele (VCL)-induced damage remain unknown. OBJECTIVE: To investigate the effects and mechanism of VB on experimental rats with varicocele (VCL)-induced damage. MATERIALS AND METHODS: Sixty sexually mature male Sprague-Dawley (SD) rats were divided into six groups (n = 10): control, control-sham, VCL-vehicle (normal saline), and VCL + VB groups (50, 100, and 200 mg/kg/day, intraperitoneally). After 4 weeks of VB treatment, all animals were sacrificed, and the body and testicular weight, sperm quality parameters, histopathology, antioxidant status, and hormone levels were tested. The levels of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone in the hypothalamus were detected by western blot. RESULTS: Compared with the VCL-vehicle group (41.14%), administration of VB significantly increased the sperm viability (59.29, 65.45, 84.93%). VB groups showed higher Johnson's score (3.57 ± 0.15, 4.71 ± 0.26, 7.93 ± 0.37) than VCL-vehicle group (2.72 ± 0.24). Antioxidant status and hormone levels alterations were also observed. Meanwhile, the mean number of apoptotic tubules (8.15 ± 0.96, 6.61 ± 1.05, 2.17 ± 0.08) and apoptotic index showed a marked decrease. Compared with the VCL-vehicle group (0.21 ± 0.09), the VB groups (0.36 ± 0.07, 0.42 ± 0.06, 0.88 ± 0.10) showed considerable increases in GnRH. DISCUSSION AND CONCLUSIONS: VB has protective effects on reproductive organs and VB may be therapeutically useful in the treatment of varicocele through up-regulation of the HPG axis.

Assessment of seminal calcium and magnesium levels in infertile men with varicocele before and after varicocelectomy.

BACKGROUND: Trace elements perform a vital role in all stages of human physiology, as well as reproduction. OBJECTIVE: This study aimed to assess seminal calcium (Ca) and magnesium (Mg) in infertile men associated with varicocele (Vx). MATERIALS AND METHODS: Overall, 50 men were divided into two groups: fertile men (n = 20) and infertile men who were scheduled for Vx surgical repair (n = 30). Exclusion criteria were as follows: azoospermia, smoking, leukocytospermia, and consumption of Ca and/or Mg supplements. All cases were subjected to history taking and clinical examination. Semen analysis and assessment of seminal Ca and Mg by the colorimetric method were carried out for all cases at the base point and 3 months postvaricocelectomy. RESULTS: Generally, the mean seminal Ca and Mg levels demonstrated significant decreases in infertile men with Vx compared with the healthy fertile men linked to higher Vx grade as well as Vx bilaterality. These seminal decreases demonstrated significant increases after Vx surgical repair. Collectively, seminal Ca and Mg levels showed a significant positive correlation (r = 0.665, p= 0.001). Besides, seminal Ca, Mg levels, and Ca/Mg ratio showed significant positive correlation with sperm concentration (r = 0.479, p = 0.001; r = 0.541, p = 0.001; r = 0.282, p = 0.001, respectively), sperm motility percentage (r = 0.493, p = 0.001; r = 0.477, p = 0.001; r = 0.353, P = 0.001, respectively), and sperm normal forms percentage (r = 0.578, p = 0.001; r = 0.520, p = 0.001; r = 0.430, p = 0.001, respectively). DISCUSSION AND CONCLUSION: Seminal Ca and Mg levels and Ca/Mg ratio are significantly decreased in infertile men associated with Vx compared with fertile men with significant increases after varicocelectomy.