Sertoli Cell-only Syndrome Group Research Articles

P-099 Alterations in microRNA expression profiles may be implicated in increased prevalence of cancer in Sertoli cell-only syndrome cases: A systematic review and in-silico analysis

Abstract Study question Could microRNA dysregulation be an underlying molecular mechanism leading to the observed increased prevalence of cancer in patients with Sertoli-cell only syndrome (SCOS)? Summary answer Patients with SCOS are characterized by altered microRNA profiles and dysregulated gene pathways involved in SCOS pathophysiology and in cell-cycle control and therefore in carcinogenesis. What is known already Sertoli cell-only syndrome constitutes a histopathological subtype of non-obstructive azoospermia, affecting 26.3–57.8% of azoospermia patients. It is characterized by partial or complete absence of active spermatogenesis due to germ cell aplasia. Except from infertility, SCOS is associated with increased risk of testicular nodules and cancer, rendering research on the topic essential. Despite advances, the underlying molecular mechanisms connecting SCOS with cancer remain unknown. Data demonstrates that microRNAs could play crucial roles in both SCOS pathophysiology and carcinogenesis. Identifying relevant microRNAs and conducting in-silico analysis on affected pathways may lead to mapping the way forward. Study design, size, duration A systematic review was performed in PubMed/Medline and Embase up to April 2022. Only full-length original studies in humans were included. Strict inclusion-exclusion criteria were applied aiming to select studies comparing microRNA profiling between SCOS cases versus men with normal spermatogenesis or men with proven fertility. Following study selection, data on altered microRNA expression patterns were analyzed to underline differences between the abovementioned groups. Subsequently, in-silico functional analysis was performed to compare affected gene pathways. Participants/materials, setting, methods The studied population consisted of SCOS cases. Men with normal spermatogenesis or proven fertility served as the control group. Predicted microRNA–target pairs were retrieved from microT-CDS, while a 0.8 cutoff threshold was applied. The GTEx repository was used to identify microRNA-targeted genes in the testis. Annotations derived from Ensembl and miRbase. Gene-set enrichment analysis was performed employing the KEGG-database. Fisher’s exact test was performed in R package limma, setting a 0.01 p-value threshold. Main results and the role of chance Four studies reported altered microRNA expression profiles in SCOS (n = 45) versus normal spermatogenesis cases or men of proven fertility (n = 16). Functional analysis revealed that six microRNAs, which were downregulated in the SCOS cases, namely hsa-miR-34b-5p, hsa-miR-202-3p, hsa-miR-34c-5p, hsa-miR-449a, hsa-miR-141-3p, and hsa-miR-34b-3p, affected 66 statistically significant gene-targets in the testis. Two pathways were reported to be statistically significantly dysregulated from these microRNAs, namely the ‘’microRNAs in cancer’’ pathway (40 affected genes, p-value = 0.004), and the ‘’TGF-beta signaling’’ pathway (26 affected genes, p-value = 0.01). Furthermore, four microRNAs, namely hsa-miR-10b-5p, hsa-miR-4270, hsa-miR-181c-5p, and hsa-miR-605-3p, reported to be upregulated in the SCOS group. These microRNAs had 108 statistically significant gene-targets in the testis. Three pathways were statistically significantly dysregulated from these microRNAs, namely the ‘’Herpes simplex virus 1 infection’’ pathway (61 affected genes, p-value = 0.01), the ‘’microRNAs in cancer’’ pathway (28 affected genes, p-value = 0.01), and the ‘’longevity regulating’’ pathway (19 affected genes, p-value = 0.01). The molecular role of the affected gene pathways in proper cell-cycle regulation and germ cell differentiation is herein underlined as critical. In the disrupted testicular microenvironment of the SCOS cases these disrupted genes may act as inducers of carcinogenic mechanisms. Limitations, reasons for caution The main limitation is the small number of the included studies and the small number of participants investigated per study, especially with regards to the control group. Moreover, the observed heterogeneity among the studies regarding the molecular tools employed for the microRNA profiling is another reason for caution. Wider implications of the findings Our data suggest that the dysregulation of microRNAs affecting several gene pathways that control cell-cycle and differentiation may lead to increased cancer risk in SCOS cases. Further studies employing our findings as a starting point will indicate whether microRNA profiling can serve as an effective evaluation tool for cancer predisposition. Trial registration number Not applicable

Microscopic testicular sperm extraction (micro-TESE) for azoospermia at a large tertiary referral centre: Male and female factors and outcomes

Objectives: To report on the outcomes and demographics of azoospermic couples undergoing microscopic testicular sperm extraction (micro-TESE) in a large tertiary referral centre. Subjects and methods: A retrospective study of patients undergoing micro-TESE in a tertiary referral centre from March 2015 to August 2019 was undertaken. Histopathology, patient demographics, comorbidities, patient factors and live birth outcomes were evaluated. Results: A total of 102 micro-TESEs were performed with a sperm retrieval rate (SRR) of 30.3%. The successful group had a mean age of 32.7 years and a mean body mass index (BMI) of 26.8 kg/m2. Female partners in the successful group had a mean age of 32.2 years. Twenty percent of female partners had infertility factors, 86.7% had no previous pregnancy and 13.3% had a previous miscarriage. The successful group had 15 live births (50%), while 20% had frozen their sperm. 93.3% achieved live birth from single embryo transfer and currently have unutilised embryos in cryostorage to possibly create siblings in the future. Twenty percent had more than one live birth from a single micro-TESE. Sertoli cell-only syndrome (SCOS) was identified in 57.5% of all cases. This was bilateral in 80% of these cases (four cases only sampled unilaterally due to previous orchidectomies). Of the SCOS group, six men went on to have successful sperm extraction (10%). The presence of previous urologic history/surgery increased the risk of azoospermia by 13%. Conclusion: Men with previous urological conditions have an increased risk of azoospermia. SCOS was identified in 57.5% of cases making it the most common histopathological diagnosis in azoospermic men. Based on our cohort, micro-TESE offers potential sperm retrieval in azoospermic men with SCOS. Successful sperm extraction in patients was associated with 50% live births and appeared to be unaffected by the presence of male/female factors. The number of live births could be higher as 20% had frozen their sperm. Concurrent female issues did not appear to affect successful outcome after micro-TESE, although numbers are small. Level of evidence: Not applicable

Protamine 1/Protamine 2 mRNA ratio in nonobstructive azoospermic patients.

Altered protamine 1 (PRM1)/ protamine 2 (PRM2) mRNA ratio in testicular biopsy samples correlates with sperm quality and its fertilising ability. This study is planned to assess PRM1/ PRM2 mRNA ratio in subgroups of azoospermia to suggest a more reliable and accurate marker for assessing sperm quality in nonobstructive azoospermia (NOA). A cross-sectional study was done on testicular biopsy samples, taken from 106 azoospermic patients. Samples were histologically classified into subgroups: 36 obstructive azoospermia (OA), and two groups of NOA: 41 round spermatid maturation arrest (SMA) and 29 Sertoli cell-only syndrome (SCOS). OA samples showed histologically normal spermatogenesis and serve as a positive control. mRNA expression of jumonji domain-containing 1A (JMJD1A), PRM1, PRM2 and transition nuclear proteins (TNP1, TNP2) genes was determined, by RT-qPCR. Significantly lower expression of JMJD1A (p<.001), PRM1 (p=.0265) and PRM2 (p=.0032) has been seen in the SCOS group of NOA. We found significant (p<.001) increase in PRM1/PRM2 mRNA ratio in testicular biopsy samples of SCOS group of NOA patients and significant negative correlation of PRM1/PRM2 mRNA ratio with JMJD1A. Hence, PRM1/PRM2 mRNA ratio may represent a more reliable and accurate marker to assess sperm quality in NOA in addition to standard semen parameters.

Features of gonadal dysgenesis and Leydig cell impairment in testes with Sertoli cell-only syndrome.

There is evidence that disturbed spermatogenesis is associated with impaired Leydig cell function and that it may be the result of testicular dysgenesis during fetal/infant development. Sertoli cell-only syndrome (SCOS) is defined by complete lack of germ cells in the seminiferous epithelium. The pathogenesis of SCOS is still not well understood. The aim of the study is to evaluate testes with SCOS focusing on morphometric signs of testicular dysgenesis and markers of Leydig cell (LC) function in relation to hormonal status of studied infertile men. Forty-nine testicular biopsies of patients with SCOS and 15 controls with normal spermatogenesis (NOR) were studied. In each biopsy the seminiferous tubule diameter (STD), thickness of tubular membrane (TM), area fraction of intertubular space (AFIS) were measured and semi-quantitative assessment of the LC number was performed (LC-score). The results of histological examination were correlated with serum levels of FSH, LH, testosterone (T) and T/LH ratio. In SCOS group testicular volume (median [M]: 16.0 vs. 29.5; p < 0.001) and STD (M: 141.7 vs. 190.2; p < 0.001) were lower, while TM (M: 9.8 vs. 6.4; p < 0.001) and AFIS (M: 47.6 vs. 27.6; p < 0.001) were significantly higher in comparison to NOR group. LC-score was higher in SCOS than in NOR group (M: 2.2 vs. 1.1; p < 0.001). Abnormal AFIS and STD were present in 43% of SCOS biopsies and among them in 81% the increased LC-score was found. In SCOS group, the subjects had significantly higher levels of both gonadotropins (FSH, M: 19.9 vs. 3.4; p < 0.001; LH, M: 7.1 vs. 4.2; p < 0.001). Total serum testosterone level did not differ between studied groups; however, T/LH ratio was significantly lower in SCOS group (M: 2.3 vs. 3.8; p < 0.001). Negative correlation between LC-score and STD was observed in SCOS group (r = -0.48; p < 0.001). AFIS correlated positively with serum FSH level in NOR (r = 0.53; p < 0.05) and SCOS (r = 0.41; p < 0.05) group, while with LH, and negatively with T/LH ratio, only in SCOS (LH, r = 0.37; p < 0.05; T/LH, r = -0.36; p < 0.05) group. We have shown that substantial number of testes from subjects with SCOS presented abnormal morphometric features, which are recognized as the signs of testicular dysgenesis. Additionally, an increased number of Leydig cells simultaneously with abnormal T/LH ratio were found, which suggests an impaired function of these cells. Increased serum levels of LH and also FSH, may reflect dysfunction of Leydig cells. It seems that reproductive hormones levels reflect also the condition of testicular structure, and that FSH may be related to the changes in intertubular space area independently of impaired Leydig cell function.

Homo Sapiens (Human) microRNA 187 Expression Is Dysregulated in Testis of Non-obstructive Azoospermic Men

Introduction: MiRNA expression alterations are closely related to growth, proliferation, and cell development. In addition, miR-187 has been found to regulate cellular proliferation. Further, spermatogenesis is a highly complicated process which is regulated through several genes targeted by miRNAs. However, no study has completely addressed the etiology of spermatogenic impairments. Accordingly, the present study examined the expression pattern of Homo sapiens (human) microRNA (hsa-mir-187) in the testicular biopsies of infertile men with non-obstructive azoospermia (NOA) and Sertoli cell-only syndrome (SCOS) subtype in comparison with those with obstructive azoospermia (OA). Methods: Quantitative reverse‐transcription polymerase chain reaction analysis was performed to assess the expression level of hsa-mir-187 using the ΔΔCt method. Results: Our data indicated that hsa-mir-187 was significantly up-regulated in the testis of the SCOS group (P˂ 0.05) in comparison with the OA group. Conclusion: In general, the findings suggest that the differential expression of hsa-mir-187 is linked with male infertility and shed more light on molecular defects that underlie spermatogenic impairments.

Ran-binding protein 3 is associated with human spermatogenesis and male infertility.

Ran-binding protein 3 (RanBP3) is a Ran-interacting protein, which participates in the Ran GTPase system in cancer cell biology. However, the expression pattern and physiological role of RanBP3 remain largely unknown. In this study, we found that RanBP3 was expressed in human testes and localised to spermatogonium and spermatocyte of germ cells. In subcellular structure, its localisation is in the nucleus and cytoplasm. Interestingly, compared with normal groups, RanBP3 expression was lower in groups of patients with Maturation Arrest (MA) and Sertoli cell-only syndrome (SCO) when considered by the Johnson Score. RanBP3 expression in the MA group and SCO groups was dramatically lower than that in the normal control group. Studies have shown that RanBP3, which is one of the helper factors of Ran, is mainly participate in the nucleocytoplasmic transport of cells. RanBP3 helps Ran to achieve some functions such as nucleocytoplasmic transport, spindle assembly during mitosis and nuclear assembly after mitosis. Consequent changes in the expression of RanBP3 may associate with human spermatogenesis disorders and male infertility. The identification and characterisation of RanBP3 enhances our understanding of the molecular mechanisms underpinning its function in human spermatogenesis and male infertility.

Investigation of CD133 and CD24 as candidate azoospermia markers and their relationship with spermatogenesis defects

Spermatogenesis is initiated when spermatogonial stem cells (SSCs) in the mature testes enter mitosis and trigger differentiation. Thus, spermatogenesis and the ability to maintain a continuous source of spermatogonia relies on the ability to differentiate SSCs. Many studies around the world have been performed to investigate the etiology of male infertility and recent studies have focused on the presence and identification of biomarkers. CD133 and CD24 are stem cell markers locating in the testis and spermatogonia. The aim of this study was to investigate the relationship of the CD133 and CD24 genes with spermatogenesis defects and examine them as a candidate a useful biomarker for azoospermia men. The association of CD133 and CD24 with spermatogenesis defects was investigated in patients with obstructive (O) and non-obstructive azoospermia (NOA). NOA cases were histopathologically classified into Hypospermatogenesis (HS), Maturation Arrest (MA), and Sertoli Cell Only Syndrome (SCO) groups. A qRT-PCR analysis of these genes was performed and protein expression levels were measured by Western blot analysis. CD133 expression in NOA group was found to be significantly different from OA and this was confirmed by immunohistochemistry and immunocytochemical assays.The qRT-PCR analysis revealed that gene expression of CD133 and CD24 had fold changes of 0.80 ± 0.34 and 1.59 ± 0.31 compared to controls, respectively in the HS group (p > 0.05) and 0.04 ± 0.01 and 0.54 ± 0.08 in the MA group (p < 0.05). In the SCO group, CD24 showed a 1.55 ± 0.35-fold increase (p > 0.05). CD133 gene expression was not detected at the transcriptional level in the SCO group. Western blot analysis of CD133 protein expression revealed 1.83, 4.11, and 11.4-fold decreases in the HS, MA and SCO groups, respectively, compared to controls (p < 0.05). CD24 showed fold changes of 1.18, 0.38, (p < 0.05), and 0.89 in the HS, MA, and SCO groups, respectively. Immunohistochemical analysis of CD133 revealed moderate, partial staining in the HS group, compared to substantial, wide-spread staining in the OA group. No staining was detected in either the MA or SCO groups. The localization of CD133 in healthy sperm was determined to be prominent in the tail and partly expressed in the head by confocal laser scanning microscopy analysis. It was also found that the expression of CD133 protein was high in healthy commercially-sourced Sertoli cells as well as in the Sertoli cells of OA individuals. Data from this study show that CD133 exhibits different profiles in infertile patient groups and thus may be considered as a candidate biomarker. CD24 can be associated with blockage of germ cell maturation in the MA group. Curative protocols for spermatogenesis defects may be possible with the use of these markers and thus their identification is extremely valuable in terms of human reproductive health.

Association between Sertoli Cell-Only Syndrome, Varicocele and FSH Level in Azoospermic Patients

A varicocele is the most frequent causes of infertility because it causes damage to the testes that could increase the levels of FSH. The objectives of this study were to determine the relationship of varicocele history and the condition of Sertoli cell-only syndrome (SCOS) based on Johnson criteria from testicular biopsy and the relationship of SCOS with FSH levels or testicular volume in azoospermic patients. This cross-sectional study (110 samples) used data from testicular biopsy of azoospermic patients at the Biology Department FKUI and the medical records of Urology Department FKUI-RSCM in 2011-2015. Johnson’s criteria for most patients were 5 with a mean criteria of 4.42 (±1.997). The Johnson criteria of 2 in the biopsy results or SCOS is 21 (19.1%). In patients with a history of varicocele, there were only 10 (27.8%) patients with SCOS (p=0.378). There was no association between SCOS and the history of varicocele. Patients without SCOS have mean FSH levels of 14.1± 8.6IU/L and patients with SCOS have mean FSH of 21.3±7.5IU/L. There was significant difference mean of FSH level (paired t test, p<0,05), which is 7,247 in SCOS group and non-SCOS. There were 36 testes with the Johnson criteria of 2 and 157 testes having values above 2. The SCOS and non-SCOS group testicular volumes were significantly different (Mann-Whitney test, p=0.018). Varicocele could not be used as an indicator of SCOS, however high levels of FSH may indicate SCOS in azoospermic patients.

The poly(A)-binding protein genes, EPAB, PABPC1, and PABPC3 are differentially expressed in infertile men with non-obstructive azoospermia

Azoospermia is one of the major causes of male infertility and is basically classified into obstructive (OA) and non-obstructive azoospermia (NOA). The molecular background of NOA still largely remains elusive. It has been shown that the poly(A)-binding proteins (PABPs) essentially play critical roles in stabilization and translational control of the mRNAs during spermatogenesis. In the present study, we aim to evaluate expression levels of the PABP genes, EPAB, PABPC1, and PABPC3, in the testicular biopsy samples and in the isolated spermatocyte (SC) and round spermatid (RS) fractions obtained from men with various types of NOA including hypospermatogenesis (hyposperm), RS arrest, SC arrest, and Sertoli cell-only syndrome (SCO). In the testicular biopsy samples, both PABPC1 and PABPC3 mRNA expressions were gradually decreased from hyposperm to SCO groups (P < 0.05), whereas there was no remarkable difference for the EPAB expression among groups. The expression levels of cytoplasmically localized PABPC1 and PABPC3 proteins dramatically reduced from hyposperm to SCO groups (P < 0.05). In the isolated SC and RS fractions, the EPAB, PABPC1, and PABPC3 mRNA expressions were gradually decreased from hyposperm to SC arrest groups (P < 0.05). Similarly, both PABPC1 and PABPC3 proteins were expressed at higher levels in the SC and RS fractions from hyposperm group when compared to the SC and RS fractions from either RS arrest or SC arrest group (P < 0.05). Our findings suggest that observed significant alterations in the PABPs expression may have an implication for development of different NOA forms.

Single‐Nucleotide Polymorphisms in the SEPTIN12 Gene May Be a Genetic Risk Factor for Japanese Patients With Sertoli Cell–Only Syndrome

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, 10 novel genes involved in human spermatogenesis, including human SEPTIN12, were identified by expression microarray analysis of human testicular tissue. Septin12 is a member of the septin family of conserved cytoskeletal GTPases that form heteropolymeric filamentous structures in interphase cells. It is expressed specifically in the testis. Therefore, we hypothesized that mutation or polymorphisms of SEPTIN12 participate in male infertility, especially Sertoli cell-only syndrome (SCOS). To investigate whether SEPTIN12 gene defects are associated with azoospermia caused by SCOS, mutational analysis was performed in 100 Japanese patients by direct sequencing of coding regions. Statistical analysis was performed in patients with SCOS and in 140 healthy control men. No mutations were found in SEPTIN12 ; however, 8 coding single-nucleotide polymorphisms (SNP1-SNP8) could be detected in the patients with SCOS. The genotype and allele frequencies in SNP3, SNP4, and SNP6 were notably higher in the SCOS group than in the control group (P < .001). These results suggest that SEPTIN12 might play a critical role in human spermatogenesis.

HnRNPL as a key factor in spermatogenesis: Lesson from functional proteomic studies of azoospermia patients with sertoli cell only syndrome

Sertoli cell only syndrome (SCOS) is one of the main causes leading to the abnormal spermatogenesis. However, the mechanisms for abnormal spermatogenesis in SCOS are still unclear. Here, we analyzed the clinical testis samples of SCOS patients by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to find the key factors contributing to SCOS. Thirteen differential proteins were identified in clinical testis samples between normal spermatogenesis group and SCOS group. Interestingly, in these differential proteins, Heterogeneous nuclear ribonucleoprotein L(HnRNPL) was suggested as a key regulator involved in apoptosis, death and growth of spermatogenic cells by String and Pubgene bioinformatic programs. Down-regulated HnRNPL in testis samples of SCOS patients was further confirmed by immunohistochemical staining and western blotting. Moreover, in vitro and in vivo experiments demonstrated that knockdown of HnRNPL led to inhibited proliferation, increased apoptosis of spermatogenic cell but decreased apoptosis of sertoli cells. Expression of carcinoembryonic antigen-related cell adhesion molecule 1 in GC-1 cells or expression of inducible nitric oxide synthases in TM4 sertoli cells, was found to be regulated by HnRNPL. Our study first shows HnRNPL as a key factor involved in the spermatogenesis by functional proteomic studies of azoospermia patients with sertoli cell only syndrome. This article is part of a Special Issue entitled: Proteomics: The clinical link.