FANCM Gene Variants in a Male Diagnosed with Sertoli Cell-Only Syndrome and Diffuse Astrocytoma.

Azoospermia is a form of male infertility characterized by a complete lack of spermatozoa in the ejaculate. Sertoli cell-only syndrome (SCOS) is the most severe form of azoospermia, where no germ cells are found in the tubules. Recently, FANCM gene variants were reported as novel genetic causes of spermatogenic failure. At the same time, FANCM variants are known to be associated with cancer predisposition. We performed whole-exome sequencing on a male patient diagnosed with SCOS and a healthy father. Two compound heterozygous missense mutations in the FANCM gene were found in the patient, both being inherited from his parents. After the infertility assessment, the patient was diagnosed with diffuse astrocytoma. Immunohistochemical analyses in the testicular and tumor tissues of the patient and adequate controls showed, for the first time, not only the existence of a cytoplasmic and not nuclear pattern of FANCM in astrocytoma but also in non-mitotic neurons. In the testicular tissue of the SCOS patient, cytoplasmic anti-FANCM staining intensity appeared lower than in the control. Our case report raises a novel possibility that the infertile carriers of FANCM gene missense variants could also be prone to cancer development.

Enhancement of Vascularization and Ovarian Follicle Survival Using Stem Cells in Cryopreserved Ovarian Tissue Transplantation-A Systematic Review.

The increase in cancer survival rates has put a focus on ensuring fertility preservation procedures for cancer patients. Ovarian tissue cryopreservation presents the only option for prepubertal girls and patients who require immediate start of treatment and, therefore, cannot undergo controlled ovarian stimulation. We aimed to provide an assessment of stem cells' impact on cryopreserved ovarian tissue grafts in regard to the expression of growth factors, angiogenesis promotion, tissue oxygenation, ovarian follicle survival and restoration of endocrine function. For this systematic review, we searched the Scopus and PubMed databases and included reports of trials using murine and/or human cryopreserved ovarian tissue for transplantation or in vitro culture in combination with mesenchymal stem cell administration to the grafting site. Of the 1201 articles identified, 10 met the criteria. The application of stem cells to the grafting site has been proven to support vascular promotion and thereby shorten the period of tissue hypoxia, which is reflected in the increased number of remaining viable follicles and faster recovery of ovarian endocrine function. Further research is needed before implementing the use of stem cells in OT cryopreservation and transplantation procedures in clinical practice. Complex ethical dilemmas make this process more difficult.

Monocytes expressing activin A and CCR2 exacerbate chronic testicular inflammation by promoting immune cell infiltration.

STUDY QUESTION: Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER: Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY: The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION: EAO was induced in 10-12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2-/-) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7-9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4-5) and untreated (n = 4-6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE: Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P < 0.001), Ccr2 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P < 0.0001), Ccr3 (P < 0.0001), Ccr5 (P < 0.01), Cxcr3 (P < 0.001), and Cx3cr1 (P < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P < 0.01), CCR1 (P < 0.05), CCR2 (P < 0.001), and CCR5 (P < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P < 0.05) and negatively with the mean spermatogenesis score (P < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1, Ccr2, and Ccr5 while reducing their expression of Ccl2, Ccl3, Ccl4, Ccl6, Ccl7 Ccl8, and Ccl12. These findings were validated in vivo, by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P < 0.05) and Ccr5 (P < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P < 0.05), CCL3/4 (P < 0.01), and CCL12 (P < 0.05) in the medium and attenuated the production of TNF (P < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests.

Global Practice Patterns in the Evaluation of Non-Obstructive Azoospermia: Results of a World-Wide Survey and Expert Recommendations.

PURPOSE: Non-obstructive azoospermia (NOA) represents the persistent absence of sperm in ejaculate without obstruction, stemming from diverse disease processes. This survey explores global practices in NOA diagnosis, comparing them with guidelines and offering expert recommendations. MATERIALS AND METHODS: A 56-item questionnaire survey on NOA diagnosis and management was conducted globally from July to September 2022. This paper focuses on part 1, evaluating NOA diagnosis. Data from 367 participants across 49 countries were analyzed descriptively, with a Delphi process used for expert recommendations. RESULTS: Of 336 eligible responses, most participants were experienced attending physicians (70.93%). To diagnose azoospermia definitively, 81.7% requested two semen samples. Commonly ordered hormone tests included serum follicle-stimulating hormone (FSH) (97.0%), total testosterone (92.9%), and luteinizing hormone (86.9%). Genetic testing was requested by 66.6%, with karyotype analysis (86.2%) and Y chromosome microdeletions (88.3%) prevalent. Diagnostic testicular biopsy, distinguishing obstructive azoospermia (OA) from NOA, was not performed by 45.1%, while 34.6% did it selectively. Differentiation relied on physical examination (76.1%), serum hormone profiles (69.6%), and semen tests (68.1%). Expectations of finding sperm surgically were higher in men with normal FSH, larger testes, and a history of sperm in ejaculate. CONCLUSIONS: This expert survey, encompassing 367 participants from 49 countries, unveils congruence with recommended guidelines in NOA diagnosis. However, noteworthy disparities in practices suggest a need for evidence-based, international consensus guidelines to standardize NOA evaluation, addressing existing gaps in professional recommendations.

Testicular Germ Cell Tumor Tissue Biomarker Analysis: A Comparison of Human Protein Atlas and Individual Testicular Germ Cell Tumor Component Immunohistochemistry.

The accurate management of testicular germ cell tumors (TGCTs) depends on identifying the individual histological tumor components. Currently available data on protein expression in TGCTs are limited. The human protein atlas (HPA) is a comprehensive resource presenting the expression and localization of proteins across tissue types and diseases. In this study, we have compared the data from the HPA with our in-house immunohistochemistry on core TGCT diagnostic genes to test reliability and potential biomarker genes. We have compared the protein expression of 15 genes in TGCT patients and non-neoplastic testicles with the data from the HPA. Protein expression was converted into diagnostic positivity. Our study discovered discrepancies in three of the six core TGCT diagnostic genes, POU5F1, KIT and SOX17 in HPA. DPPA3, CALCA and TDGF1 were presented as potential novel TGCT biomarkers. MGMT was confirmed while RASSF1 and PRSS21 were identified as biomarkers of healthy testicular tissue. Finally, SALL4, SOX17, RASSF1 and PRSS21 dysregulation in the surrounding testicular tissue with complete preserved spermatogenesis of TGCT patients was detected, a potential early sign of neoplastic transformation. We highlight the importance of a multidisciplinary collaborative approach to fully understand the protein landscape of human testis and its pathologies.

Circulating Cell-Free Tumor Deoxyribonucleic Acid Analysis as a Tool for the Diagnosis and Monitoring of Pediatric Solid Tumors.

The utility of cell-free tumor deoxyribonucleic acid analysis is currently being evaluated in a wide range of clinical studies. The validity of cell-free tumor deoxyribonucleic acid analysis methods used for screening and detecting malignant diseases, monitoring the effectiveness of treatment and disease progression, and identifying potential relapse is tested. Molecular technologies used for cell-free tumor deoxyribonucleic acid analysis include targeted polymerase chain reaction assays and next-generation sequencing approaches along with newly introduced epigenetic analysis methods such as methylation-specific polymerase chain reaction. The aim of this review was to compare the methods, pitfalls, and advantages of tests developed for the analysis of cell-free tumor deoxyribonucleic acid in the diagnosis and treatment of pediatric solid tumors. For this purpose, the PubMed database was searched for articles published in the last 10 years, in English, that investigated a human cohort aged 0 to 18 years. A total of 272 references were analyzed. A total of 33 studies were included in the review. Cell-free tumor deoxyribonucleic acid analysis has emerged as a promising novel approach that could potentially bring a significant improvement in the field of pediatric oncology, but the implementation of cell-free tumor deoxyribonucleic acid in clinical practice is largely hindered by the lack of standardized methods for processing and analysis.

Expression of Androgen and Estrogen Receptors in the Human Lacrimal Gland.

Lacrimal gland dysfunction causes dry eye disease (DED) due to decreased tear production. Aqueous-deficient DED is more prevalent in women, suggesting that sexual dimorphism of the human lacrimal gland could be a potential cause. Sex steroid hormones are a key factor in the development of sexual dimorphism. This study aimed to quantify estrogen receptor (ER) and androgen receptor (AR) expression in the human lacrimal gland and compare it between sexes. RNA was isolated from 35 human lacrimal gland tissue samples collected from 19 cornea donors. AR, ERα, and ERß mRNA was identified in all samples, and their expression was quantified using qPCR. Immunohistochemical staining was performed on selected samples to evaluate protein expression of the receptors. ERα mRNA expression was significantly higher than the expression of AR and ERß. No difference in sex steroid hormone (SSH) receptor mRNA expression was observed between sexes, and no correlation was observed with age. If ERα protein expression is found to be concordant with mRNA expression, it should be investigated further as a potential target for hormone therapy of DED. Further research is needed to elucidate the role of sex steroid hormone receptors in sex-related differences of lacrimal gland structure and disease.

Analysis of 29 Targeted Genes for Non-Obstructive Azoospermia: The Relationship between Genetic Testing and Testicular Histology.

PURPOSE: To analyze the presence of potentially pathogenic variants of 29 candidate genes known to cause spermatogenic failure (SPGF) in patients with non-obstructive azoospermia (NOA) who underwent testicular histology. MATERIALS AND METHODS: Forty-eight patients with unexplained NOA referred to the Department of Transfusion Medicine and Transplantation Biology, University Hospital Center Zagreb, Zagreb, Croatia for testicular biopsy. They were divided into three groups: those who had cryptorchidism (n=9), those with varicocele (n=14), and those with idiopathic NOA (n=25). All included patients underwent blood withdrawal for next-generation sequencing (NGS) analysis and gene sequencing. RESULTS: We found a possible genetic cause in 4 patients with idiopathic NOA (16%) and in 2 with cryptorchidism (22%). No pathogenic or possibly pathogenic mutations were identified in patients with varicocele. Variants of undetermined significance (VUS) were found in 11 patients with idiopathic NOA (44%), 3 with cryptorchidism (33%), and 8 patients with varicocele (57%). VUSs of the USP9Y gene were the most frequently as they were found in 14 out of 48 patients (29%). In particular, the VUS USP9Y c.7434+14del was found in 11 patients. They showed varied histological pictures, including Sertoli cell-only syndrome, mixed atrophy, and hypospermatogenesis, regardless of cryptorchidism or varicocele. No direct correlation was found between the gene mutation/variant and the testicular histological picture. CONCLUSIONS: Different mutations of the same gene cause various testicular histological pictures. These results suggest that it is not the gene itself but the type of mutation/variation that determines the testicular histology picture. Based on the data presented above, it remains challenging to design a genetic panel with prognostic value for the outcome of testicular sperm extraction in patients with NOA.

Trends in the treatment of undescended testes: a pediatric tertiary care center experience from Croatia.

Objective: Undescended testes (UDT) is the most common anomaly of the male genitourinary tract. The guidelines suggest that orchidopexy in congenitally UDT should be performed between 6 months and 18 months of age, while in acquired UDT, orchidopexy should be performed before puberty. Delay in treatment increases the risk of cancer and infertility. The main aim of this study was to determine whether we meet international standards in the treatment of UDT. Methods: The present study included all boys who underwent orchidopexy either due to congenital or acquired UDT in 2019 (from January 1 to December 31). For each group, laterality, location, associated anomalies, premature birth and in how many cases ultrasound was applied were determined. Additionally, for each group, the types of surgery, the number of necessary reoperations, and in how many cases atrophy occurred were determined. Finally, ages of referral, of clinical examination, and of orchidopexy were determined. Results: During this period, 198 patients with 263 UDT underwent orchidopexy. The median time of orchidopexy for the congenital group was 30 months, while that for the acquired group was 99 months. In the congenital group up to 18 months of age, orchidopexy was performed in 16 (16%) boys, while in the acquired group up to 13 years of age, orchidopexy was performed in 95 (96.94%) boys. Conclusion: Given the well-known risks of late treatment of UDT, orchidopexy needs to be performed much earlier, especially in the congenital group.

Activin A and CCR2 regulate macrophage function in testicular fibrosis caused by experimental autoimmune orchitis.

Experimental autoimmune-orchitis (EAO), a rodent model of chronic testicular inflammation and fibrosis, replicates pathogenic changes seen in some cases of human spermatogenic disturbances. During EAO, increased levels of pro-inflammatory and pro-fibrotic mediators such as TNF, CCL2, and activin A are accompanied by infiltration of leukocytes into the testicular parenchyma. Activin A levels correlate with EAO severity, while elevated CCL2 acting through its receptor CCR2 mediates leukocyte trafficking and recruits macrophages. CCR2 + CXCR4 + macrophages producing extracellular matrix proteins contribute widely to fibrogenesis. Furthermore, testicular macrophages (TMs) play a critical role in organ homeostasis. Therefore, we aimed to investigate the role of the activin A/CCL2-CCR2/macrophage axis in the development of testicular fibrosis. Following EAO induction, we observed lower levels of organ damage, collagen deposition, and leukocyte infiltration (including fibronectin+, collagen I+ and CXCR4+ TMs) in Ccr2-/- mice than in WT mice. Furthermore, levels of Il-10, Ccl2, and the activin A subunit Inhba mRNAs were lower in Ccr2-/- EAO testes. Notably, fibronectin+ TMs were also present in biopsies from patients with impaired spermatogenesis and fibrotic alterations. Overexpression of the activin A antagonist follistatin reduced tissue damage and collagen I+ TM accumulation in WT EAO testes, while treating macrophages with activin A in vitro increased the expression of Ccr2, Fn1, Cxcr4, and Mmp2 and enhanced migration along a CCL2 gradient; these effects were abolished by follistatin. Taken together, our data indicate that CCR2 and activin A promote fibrosis during testicular inflammation by regulating macrophage function. Inhibition of CCR2 or activin A protects against damage progression, offering a promising avenue for therapeutic intervention.

The utility of cfDNA in TGCT patient management: a systematic review.

Background: Testicular germ cell tumors (TGCTs) are the most common young male malignancy with a steadily rising incidence. Standard clinical practice is radical orchidectomy of suspicious lumps followed by histopathological diagnosis and tumor subtyping. This practice can lead to complications and quality of life issues for the patients. Liquid biopsies, especially cell-free DNA (cfDNA), promised to be true surrogates for tissue biopsies, which are considered dangerous to perform in cases of testicular tumors. In this study, we have performed a systematic review on the potential of cfDNA in TGCT patient management, its potential challenges in translation to clinical application and possible approaches in further research. Materials & Methods: The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on EuropePMC and PUBMED electronic databases, with the last update being on October 21, 2021. Due to the high heterogeneity in identified research articles, we have performed an overview of their efficacy. Results: Eight original articles have been identified on cfDNA in TGCT patients published from 2004 to 2021, of which six had more than one TGCT patient enrolled and were included in the final analysis. Three studies investigated cfDNA methylation, one has investigated mutations in cfDNA, two have investigated cfDNA amount, and one has investigated cfDNA integrity in TGCT. The sensitivity of cfDNA for TGCT was found to be higher than in serum tumor markers and lower than miR-371a-3p, with comparable specificity. cfDNA methylation analysis has managed to accurately detect teratoma in TGCT patients. Conclusion: Potential challenges in cfDNA application to TGCT patient management were identified. The challenges relating to the biology of TGCT with its low mutational burden and low cfDNA amounts in blood plasma make next-generation sequencing (NGS) methods especially challenging. We have also proposed possible approaches to help find clinical application, including a focus on cfDNA methylation analysis, and potentially solving the challenge of teratoma detection.

Reinke crystals: Hallmarks of adult Leydig cells in humans.

BACKGROUND: Reinke crystals are structures pathognomonic for Leydig cells, which have the important function of testosterone production and are vital for male reproductive health. These crystalline inclusions are thought to be of protein origin; however, the molecular composition has not yet been resolved. OBJECTIVES: This review summarizes all available information regarding Reinke crystal's characteristics and aims to produce a comprehensive guide for research on this topic as well as to determine and discuss potential Reinke-protein candidates. METHODS: Pubmed was thoroughly searched for all publications regarding Reinke crystals and 137 publications were identified. All publications were surveyed and all relevant information was included in the review. RESULTS: Along with the cytoplasm, structures that resemble Reinke crystals were also observed in the nucleus, suggesting that their formation depends only on protein concentration. Variations in tissue processing protocols could impact Reinke crystal microscopic visualization, which is an important factor in diagnosing Leydig cell disorders such as Leydig cell tumors. Reinke crystals appear to be hallmarks of normally differentiated, adult, Leydig or Leydig-like cells in humans, while some abnormal and nonhuman Leydig cells contain Reinke-like paracrystalline inclusions or crystalloids. CONCLUSIONS: These characteristics point to some differentially expressed proteins, which could be involved in Reinke crystal formation. Differential Reinke crystal and paracrystalline inclusion presence could also be due to small changes in protein structure or the cell environment. Further research is needed to solve the ongoing mystery of the Reinke crystal, which would enhance our knowledge of Leydig cell contribution in the pathogenesis of various male reproductive disorders and improve their diagnosis and treatment.

Astaxanthin Relieves Testicular Ischemia-Reperfusion Injury-Immunohistochemical and Biochemical Analyses.

Testicular torsion potentially leads to acute scrotum and testicle loss, and requires prompt surgical intervention to restore testicular blood flow, despite the paradoxical negative effect of reperfusion. While no drug is yet approved for this condition, antioxidants are promising candidates. This study aimed to determine astaxanthin's (ASX), a potent antioxidant, effect on rat testicular torsion-detorsion injury. Thirty-two prepubertal male Fischer rats were divided into four groups. Group 1 underwent sham surgery. In group 2, the right testis was twisted at 720° for 90 min. After 90 min of reperfusion, the testis was removed. ASX was administered intraperitoneally at the time of detorsion (group 3) and 45 min after detorsion (group 4). Quantification of caspase-3 positive cells and oxidative stress markers detection were determined immunohistochemically, while the malondialdehyde (MDA) value, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities were determined by colorimetric assays. The number of apoptotic caspase-3 positive cells and the MDA value were lower in group 4 compared to group 2. A significant increase in the SOD and GPx activity was observed in group 4 compared to groups 2 and 3. We conclude that ASX has a favorable effect on testicular ischemia-reperfusion injury in rats.

cfDNA methylation in liquid biopsies as potential testicular seminoma biomarker.

Background: Seminoma is a testicular tumor type, routinely diagnosed after orchidectomy. As cfDNA represents a source of minimally invasive seminoma patient management, this study aimed to investigate whether cfDNA methylation of six genes from liquid biopsies, have potential as novel seminoma biomarkers. Materials & methods: cfDNA methylation from liquid biopsies was assessed by pyrosequencing and compared with healthy volunteers' samples. Results: Detailed analysis revealed specific CpGs as possible seminoma biomarkers, but receiver operating characteristic curve analysis showed modest diagnostic performance. In an analysis of panels of statistically significant CpGs, two DNA methylation panels emerged as potential seminoma screening panels, one in blood CpG8/CpG9/CpG10 (KITLG) and the other in seminal plasma CpG1(MAGEC2)/CpG1(OCT3/4). Conclusion: The presented data promote the development of liquid biopsy epigenetic biomarkers in the screening of seminoma patients.

Seminoma belongs to testicular cancer, which represents a common malignancy among men of reproductive age. Diagnosis of seminoma is a multistep process that also includes checking tumor biomarkers from blood. However, these biomarkers are not specific for seminoma and to conclude a definite diagnosis of seminoma immunohistochemical analysis is needed, which requires the removal of a whole or partial testicle. Therefore, there is a need for novel, noninvasive biomarkers. cfDNA is the most extensively investigated source of minimally invasive tumor markers. Therefore, this study investigated cfDNA methylation of six genes as potential noninvasive biomarkers for the management of seminoma patients. By examining CpG sites of selected genes by pyrosequencing, the authors detected significant differences. However, receiver operating characteristic curve analysis showed modest results. Therefore, the authors tested possible panels of significantly different CpGs and detected two possible DNA methylation panels for seminoma screening. These findings suggest the further investigation of possible epigenetic biomarkers for seminoma patient management from liquid biopsies.

Stereological properties of seminiferous tubules in infertile men with chromosomal and genetic abnormalities.

BACKGROUND: Male infertility is caused by genetic anomalies in 15-30% of cases. This study aimed to determine stereological properties of seminiferous tubules in infertile men with genetic anomalies, including Klinefelter Syndrome (KS), Y chromosome microdeletions (MYC) and CFTR gene mutations (CFTR); and to compare them to seminiferous tubules of men with obstructive azoospermia of non-genetic origin (control group). METHODS: The study was conducted on 28 human testis biopsy specimens obtained from 14 patients with MYC, 18 samples from 9 patients with KS, and 6 samples from 3 patients with CFTR. Whenever possible, a bilateral biopsy was included in the study. The control group had 33 samples from 18 patients (3 of them with a solitary testis). Qualitative and quantitative (stereological) analysis of seminiferous tubules (including the status of spermatogenesis, volume, surface area, length and number of tubules) were performed in all groups. RESULTS: Qualitative histological analysis revealed significant impairment of spermatogenesis in KS and MYC, whereas testicular parenchyma was fully maintained in CFTR and control groups. Spermatogenesis was most seriously impaired in KS. All stereological parameters were significantly lower in KS and MYC, compared to the CFTR and control groups. The total volume, surface and length of seminiferous tubules were significantly lower in KS compared with MYC. CONCLUSIONS: Stereological analysis is valuable in evaluating male infertility, whereas qualitative histological analysis can be helpful in assessing sperm presence in testicular tissue of patients with KS or MYK undergoing TESE.

Leydig Cells in Patients with Non-Obstructive Azoospermia: Do They Really Proliferate?

BACKGROUND: Non-obstructive azoospermia (NOA) is a form of male infertility caused by disorders of the testicular parenchyma and impaired spermatogenesis. This study aimed to investigate the nature of Leydig cell changes in patients with NOA, especially whether their actual proliferation occurred. METHODS: 48 testicular biopsies from infertile patients with NOA and 24 testicular biopsies originating from azoospermic patients suffering from obstructive azoospermia (OA) were included in the study. Leydig cells and their possible proliferative activity were analysed by immunohistochemistry and morphometry (stereology). RESULTS: Unlike in the OA group, Leydig cells in NOA patients were sometimes organised into larger clusters and displayed an abundant cytoplasm/hypertrophy. Moreover, significant fibrosis of the interstitial compartment was demonstrated in some NOA samples, often accompanied by inflammatory cells. Stereological analysis showed no increase/proliferation of Leydig cells; on the contrary, these cells decreased in number in the NOA group. CONCLUSIONS: The decrease in the number of Leydig cells can be explained by previous inflammatory changes within the testicular interstitium and consequent interstitial fibrosis. The interstitial fibrosis might have a deteriorating effect on Leydig cells.

THE ROLE OF ULTRASOUND ELASTOGRAPHY IN THE DIAGNOSIS OF PATHOLOGIC CONDITIONS OF TESTICLES AND SCROTUM.

Several years ago, elastography emerged as a potentially very useful ultrasound technique that is currently used in diagnostic workup of the breast, liver and some other organ systems, whereas for other ones it is still mainly in the phase of research. The aim of the study was to compare elasticity index (EI) of testicles using strain elastography in healthy subjects and those with pathologic changes of testicles/scrotum. A total of 117 patients were included in the study. Measurements were performed on a Logiq E9 ultrasound system using strain elastography. In healthy subjects, the mean EI value was 1.34±0.35 for right testis and 1.49±0.47 for left testis. Increased mean EI values were found in the following six conditions: patients with varicocele, infertile patients, solitary testis after orchidectomy of the other testicle because of tumor, patients with testicular tumors, patients after orchidopexy of undescended testicle, and patients with congenitally smaller testicle. There is a paucity of literature data on the use of elastography in testes, as well as on normal elastography values in testicular tissue. Strain elastography was demonstrated to be a valuable method to acquire additional information in patients with pathologic changes in testicles/scrotum. These data provide reference values for further research in a larger sample of subjects.

Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics.

While tissue biopsy has for the longest time been the gold-standard in biomedicine, precision/personalized medicine is making the shift toward liquid biopsies. Cell-free DNA (cfDNA) based genetic and epigenetic biomarkers reflect the molecular status of its tissue-of-origin allowing for early and non-invasive diagnostics of different pathologies. However, selection of preanalytical procedures (including cfDNA isolation) as well as analytical methods are known to impact the downstream results. Calls for greater standardization are made continuously, yet comprehensive assessments of the impact on diagnostic parameters are lacking. This study aims to evaluate the preanalytic and analytic factors that influence cfDNA diagnostic parameters in blood and semen. Text mining analysis has been performed to assess cfDNA research trends, and identify studies on isolation methods, preanalytical and analytical impact. Seminal and blood plasma were tested as liquid biopsy sources. Traditional methods of cfDNA isolation, commercial kits (CKs), and an in-house developed protocol were tested, as well as the impact of dithiothreitol (DTT) on cfDNA isolation performance. Fluorimetry, qPCR, digital droplet PCR (ddPCR), and bioanalyzer were compared as cfDNA quantification methods. Fragment analysis was performed by qPCR and bioanalyzer while the downstream application (cfDNA methylation) was analyzed by pyrosequencing. In contrast to blood, semen as a liquid biopsy source has only recently begun to be reported as a liquid biopsy source, with almost half of all publications on it being review articles. Experimental data revealed that cfDNA isolation protocols give a wide range of cfDNA yields, both from blood and seminal plasma. The addition of DTT to CKs has improved yields in seminal plasma and had a neutral/negative impact in blood plasma. Capillary electrophoresis and fluorometry reported much higher yields than PCR methods. While cfDNA yield and integrity were highly impacted, cfDNA methylation was not affected by isolation methodology or DTT. In conclusion, NucleoSnap was recognized as the kit with the best overall performance. DTT improved CK yields in seminal plasma. The in-house developed protocol has shown near-kit isolation performance. ddPCR LINE-1 assay for absolute detection of minute amounts of cfDNA was established and allowed for quantification of samples inhibited in qPCR. cfDNA methylation was recognized as a stable biomarker unimpacted by cfDNA isolation method. Finally, semen was found to be an abundant source of cfDNA offering potential research opportunities and benefits for cfDNA based biomarkers development related to male reproductive health.