Predictors of successful salvage microdissection testicular sperm extraction (mTESE) after failed initial TESE in patients with non-obstructive azoospermia: A systematic review and meta-analysis.

BACKGROUND: There has been no systematic review and meta-analysis to analyze and summarize the predictive factors of successful sperm extraction in salvage microdissection testicular sperm extraction. OBJECTIVES: We aimed to investigate the factors predicting the result of salvage microdissection testicular sperm extraction in patients with non-obstructive azoospermia who failed the initial microdissection testicular sperm extraction or conventional testicular sperm extraction. MATERIALS AND METHODS: We conducted a systematic literature search in PubMed, Web of Science, EMBASE, and the Cochrane Library for literature that described the characteristics of patients with non-obstructive azoospermia who underwent salvage microdissection testicular sperm extraction after failing the initial microdissection testicular sperm extraction or conventional testicular sperm extraction published prior to June 2022. RESULTS: This meta-analysis included four retrospective studies with 332 patients with non-obstructive azoospermia who underwent a failed initial microdissection testicular sperm extraction and three retrospective studies with 177 non-obstructive azoospermia patients who underwent a failed conventional testicular sperm extraction. The results were as follows: among non-obstructive azoospermia patients whose first surgery was microdissection testicular sperm extraction, younger patients (standard mean difference: -0.28, 95% confidence interval [CI]: -0.55 to -0.01) and those with smaller bilateral testicular volume (standard mean difference: -0.55, 95% CI: -0.95 to -0.15), lower levels of follicle-stimulating hormone (standard mean difference: -0.86, 95% CI: -1.18 to -0.54) and luteinizing hormone (standard mean difference: -0.68, 95% CI: -1.16 to -0.19), and whose testicular histological type was hypospermatogenesis (odds ratio: 3.52, 95% CI: 1.30-9.53) were more likely to retrieve spermatozoa successfully, while patients with Sertoli-cell-only syndrome (odds ratio: 0.41, 95% CI: 0.24-0.73) were more likely to fail again in salvage microdissection testicular sperm extraction. Additionally, in patients who underwent salvage microdissection testicular sperm extraction after a failed initial conventional testicular sperm extraction, those with testicular histological type of hypospermatogenesis (odds ratio: 30.35, 95% CI: 8.27-111.34) were more likely to be successful, while those with maturation arrest (odds ratio: 0.39, 95% CI: 0.18-0.83) rarely benefited. CONCLUSION: We found that age, testicular volume, follicle-stimulating hormone, luteinizing hormone, hypospermatogenesis, Sertoli-cell-only syndrome, and maturation arrest were valuable predictors of salvage microdissection testicular sperm extraction, which will assist andrologists in clinical decision-making and minimize unnecessary injury to patients.

EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: State of the art 2023.

Testing for AZoospermia Factor (AZF) deletions of the Y chromosome is a key component of the diagnostic workup of azoospermic and severely oligozoospermic men. This revision of the 2013 European Academy of Andrology (EAA) and EMQN CIC (previously known as the European Molecular Genetics Quality Network) laboratory guidelines summarizes recent clinically relevant advances and provides an update on the results of the external quality assessment program jointly offered by both organizations. A basic multiplex PCR reaction followed by a deletion extension analysis remains the gold-standard methodology to detect and correctly interpret AZF deletions. Recent data have led to an update of the sY84 reverse primer sequence, as well as to a refinement of what were previously considered as interchangeable border markers for AZFa and AZFb deletion breakpoints. More specifically, sY83 and sY143 are no longer recommended for the deletion extension analysis, leaving sY1064 and sY1192, respectively, as first-choice markers. Despite the transition, currently underway in several countries, toward a diagnosis based on certified kits, it should be noted that many of these commercial products are not recommended due to an unnecessarily high number of tested markers, and none of those currently available are, to the best of our knowledge, in accordance with the new first-choice markers for the deletion extension analysis. The gr/gr partial AZFc deletion remains a population-specific risk factor for impaired sperm production and a predisposing factor for testicular germ cell tumors. Testing for this deletion type is, as before, left at the discretion of the diagnostic labs and referring clinicians. Annual participation in an external quality control program is strongly encouraged, as the 22-year experience of the EMQN/EAA scheme clearly demonstrates a steep decline in diagnostic errors and an improvement in reporting practice.

Azoospermia factor c microdeletions and outcomes of assisted reproductive technology: a systematic review and meta-analysis.

OBJECTIVE: To investigate whether Azoospermia Factor c (AZFc) microdeletions affect Assisted Reproductive Technology (ART) outcomes. DESIGN: Systematic review and meta-analysis. SETTING: Not applicable. PATIENTS: Infertile men with and without AZFc microdeletions. INTERVENTION(S): Electronic databases were searched for case-control studies reporting sperm retrieval rates and outcomes of ART in infertile men with and without AZFc microdeletions from inception to April 2023. Study quality was assessed using the Newcastle-Ottawa Scale. Summary effect sizes (odds ratio [OR] with 95% confidence interval [CI]) were calculated for both categories of infertile men. MAIN OUTCOME MEASURES: The primary outcome was successful sperm retrieval and the secondary outcomes were outcomes of ART. RESULTS: Case-control studies reporting sperm retrieval rates and ART outcomes in men with AZFa and AZFb deletions were unavailable. On the basis of the data from 3,807 men, sperm retrieval rates were found to be higher in men with AZFc microdeletions compared to their non-deleted counterparts [OR = 1.82, 95% CI 0.97, 3.41], but the difference was not statistically significant. A significantly lower fertilization rate (OR = 0.61, 95% CI [0.50, 0.74]), clinical pregnancy rate (OR = 0.61, 95% CI [0.42, 0.89]), and live birth rate (OR = 0.54, 95% CI [0.40, 0.72]) were observed in men with AZFc deletions compared with men without deletions. There was no statistically significant difference in rates of embryo cleavage, blastocyst formation, good-quality embryos, implantation, and miscarriage between the two groups. On correcting for female factors, the fertilization rate (OR = 0.76, 95% CI [0.71, 0.82]), cleavage rate (OR = 0.54, 95% CI [0.41, 0.72]), clinical pregnancy rate (OR = 0.39, 95% CI [0.30, 0.52]), and live birth rate (OR = 0.48, 95% CI [0.35, 0.65]) were significantly lower in men with AZFc deletions compared with controls. CONCLUSIONS: Presence of AZFc microdeletions adversely affects outcomes of ART in infertile men. Further in-depth studies delineating the role of the AZF genes in embryonic development are necessary to understand the full-impact of this finding. CLINICAL TRIAL REGISTRATION NUMBER: CRD42022311738.

Identification and verification of potential biomarkers in sertoli cell-only syndrome via bioinformatics analysis.

Sertoli cell-only syndrome (SCOS), a severe testicular spermatogenic failure, is characterized by total absence of male germ cells. To better expand the understanding of the potential molecular mechanisms of SCOS, we used microarray datasets from the Gene Expression Omnibus (GEO) and ArrayExpress databases to determine the differentially expressed genes (DEGs). In addition, functional enrichment analysis including the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed. Protein-protein interaction (PPI) networks, modules, and miRNA-mRNA regulatory networks were constructed and analyzed and the validation of hub genes was performed. A total of 601 shared DEGs were identified, including 416 down-regulated and 185 up-regulated genes. The findings of the enrichment analysis indicated that the shared DEGs were mostly enriched in sexual reproduction, reproductive process, male gamete generation, immune response, and immunity-related pathways. In addition, six hub genes (CCNA2, CCNB2, TOP2A, CDC20, BUB1, and BUB1B) were selected from the PPI network by using the cytoHubba and MCODE plug-ins. The expression levels of the hub genes were significantly decreased in patients with SCOS compared to that in normal spermatogenesis controls as indicated by the microarray data, single-cell transcriptomic data, and clinical sample levels. Furthermore, the potential miRNAs were predicted via the miRNA-mRNA network construction. These hub genes and miRNAs can be used as potential biomarkers that may be related to SCOS. However, it has not been proven that the differential expression of these biomarkers is the molecular pathogenesis mechanisms of SCOS. Our findings suggest that these biomarkers can be serve as clinical tool for diagnosis targets and may have some impact on the spermatogenesis of SCOS from a testicular germ cell perspective.

Testis cell pyroptosis mediated by CASP1 and CASP4: possible sertoli cell-only syndrome pathogenesis.

BACKGROUND: Sertoli cell-only syndrome (SCOS) is the most serious pathological type of non-obstructive azoospermia. Recently, several genes related to SCOS have been identified, including FANCM, TEX14, NR5A1, NANOS2, PLK4, WNK3, and FANCA, but they cannot fully explain the pathogenesis of SCOS. This study attempted to explain spermatogenesis dysfunction in SCOS through testicular tissue RNA sequencing and to provide new targets for SCOS diagnosis and therapy. METHODS: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry. RESULTS: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 ß, IL-18, LDH, and ROS) were significantly increased in the SCOS group. CONCLUSIONS: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.

DDX58 expression promotes inflammation and growth arrest in Sertoli cells by stabilizing p65 mRNA in patients with Sertoli cell-only syndrome.

Sertoli cell -only syndrome (SCOS) is a type of testicular pathological failure that causes male infertility and no effective treatment strategy, is available for this condition. Moreover, the molecular mechanism underlying its development remains unknown. We identified DExD/H-Box helicase 58 (DDX58) as a key gene in SCOS based on four datasets of testicular tissue samples obtained from the Gene Expression Synthesis database. DDX58 was significantly upregulated in SCOS testicular Sertoli cells. Moreover, high expression of DDX58 was positively correlated with the expression of several testicular inflammatory factors, such as IL -1ß, IL-18, and IL-6. Interestingly, DDX58 could be induced in the D-galactose (D-gal)-stimulated TM4 cell injury model. Whereas silencing of DDX58 inhibited D-gal -mediated p65 expression, inflammatory cytokine release, and growth arrest. Mechanistically, we found that DDX58 acts as an RNA-binding protein, which enhances p65 expression by promoting mRNA stability. Furthermore, p65 gene silencing decreased the expression of inflammatory cytokines and inhibition of cell growth in D-gal-induced cells. In conclusion, our findings demonstrate that DDX58 promotes inflammatory responses and growth arrest in SCOS Sertoli cells by stabilizing p65 mRNA. Accordingly, the DDX58/p65 regulatory axis might be a therapeutic target for SCOS.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

Y-chromosome haplogroups and Azoospermia Factor (AZF) analysis in Tunisian infertile male.

The aim of the present study was to clarify the implication of Y chromosome genetic variations and haplogroups in Tunisian infertile men. A total of 27 Y-chromosomal binary markers partial microdeletions (gr/gr, b1/b3 and b2/b3) and copy number variation of DAZ and CDY genes in the AZFc region were analysed in 131 Tunisian infertile men with spermatogenic failure and severe reduced sperm concentrations and in 85 normospermic men as controls. Eleven different haplogroups in the overall population study (E3b2; J1J*, E1, E3b*, F, G, K, P/Q, R*, R1* and R1a1) were found. Interestingly, the J1J* haplogroup was significantly more frequent in azoo/oligospermic patients than in normospermic men (35.1% and 22.3%, respectively (p value = 0.04)). Results showed also that patients without DAZ/CDY1 copies loss and without partial microdeletions belonged to the R1 haplogroup. The relative high frequencies of two haplogroups, E3b2 (35.1%) and J (30%) was confirmed in Tunisia. We reported in the present study and for the first time, that J1J* haplogroup may confer a risk factor for infertility in the Tunisian population and we suggested that R1 haplogroup may ensure certain stability to Y-chromosome in Tunisian men.

Microdeletions and vertical transmission of the Y-chromosome azoospermia factor region.

Spermatogenesis is regulated by several Y chromosome-specific genes located in a specific region of the long arm of the Y chromosome, the azoospermia factor region (AZF). AZF microdeletions are the main structural chromosomal abnormalities that cause male infertility. Assisted reproductive technology (ART) has been used to overcome natural fertilization barriers, allowing infertile couples to have children. However, these techniques increase the risk of vertical transmission of genetic defects. Despite widespread awareness of AZF microdeletions, the occurrence of de novo deletions and overexpression, as well as the expansion of AZF microdeletion vertical transmission, remains unknown. This review summarizes the mechanism of AZF microdeletion and the function of the candidate genes in the AZF region and their corresponding clinical phenotypes. Moreover, vertical transmission cases of AZF microdeletions, the impact of vertical inheritance on male fertility, and the prospective direction of research in this field are also outlined.

Y-chromosome genes associated with sertoli cell-only syndrome identified by array comparative genome hybridization.

BACKGROUND: The precise contribution of each chromosome gene or gene family in achieving male fertility is still the subject of debate. Most studies have examined male populations with heterogeneous causes of infertility, and have therefore reached controversial or uncertain conclusions. This study uses Y-chromosome array-based comparative genomic hybridization (aCGH) to examine a population of males with a uniform sertoli cell-only syndrome (SCOS) infertility phenotype. METHODS: Initial analysis of gene copy number variations in 8 SCOS patients, with determination of the log-ratio of probe signal intensity against a DNA reference, was performed using the Y-chromosome NimbleGen aCGH. To confirm the role of candidate genes, real-time quantitative RT-PCR was used to compare 19 patients who had SCOS non-obstructive azoospermia with 15 patients who had obstructive azoospermia but normal spermatogenesis. RESULTS: Our initial aCGH experiments identified CDY1a and CDY1b double deletions in all 8 patients who had total germ cell depletion. However, 5 patients had DAZ1/2 and DAZ3/4 deletions, 1 patient had a DAZ2 and DAZ3/4 deletion, and 2 patients had no DAZ1/2 or DAZ3/4 deletions. Examination of testicular mRNA expression in another 19 patients with SCOS indicated all patients had no detectable levels of CDY1. CONCLUSIONS: Our findings indicate that CDY1 deletion in SCOS patients, and analysis of the expression of DAZ and CDY1 genes using aCGH and quantitative RT-PCR, may be useful to predict the presence of mature spermatozoa.

Microdeletions and vertical transmission of the Y-chromosome azoospermia factor region / 亚洲男科学杂志(英文版)

Spermatogenesis is regulated by several Y chromosome-specific genes located in a specific region of the long arm of the Y chromosome, the azoospermia factor region (AZF). AZF microdeletions are the main structural chromosomal abnormalities that cause male infertility. Assisted reproductive technology (ART) has been used to overcome natural fertilization barriers, allowing infertile couples to have children. However, these techniques increase the risk of vertical transmission of genetic defects. Despite widespread awareness of AZF microdeletions, the occurrence of de novo deletions and overexpression, as well as the expansion of AZF microdeletion vertical transmission, remains unknown. This review summarizes the mechanism of AZF microdeletion and the function of the candidate genes in the AZF region and their corresponding clinical phenotypes. Moreover, vertical transmission cases of AZF microdeletions, the impact of vertical inheritance on male fertility, and the prospective direction of research in this field are also outlined.

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome.

BACKGROUND: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. METHODS: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. RESULTS: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2-/- hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2-/- group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. CONCLUSIONS: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

Evaluation of cell-free seminal mRNA for the diagnosis of obstruction as the cause of azoospermia in infertile men: A prospective cohort study.

Differentiating obstructive (OA) from non-obstructive (NOA) azoospermia is clinically important in managing infertile men. Classically, the differentiation has been based on clinical, hormonal and histological analysis. Histological tests are invasive and may miss spermatogenic areas. Seminal fluid can serve as a medium to assess the status of spermatogenesis and presence or absence of certain markers can help diagnosing and differentiating azoospermia. We evaluated the role of cell-free seminal markers: DDX4, PRM1 and PRM2 in diagnosing and differentiating between OA and NOA and classifying their subtypes. We observed DDX4 was more sensitive for NOA compared with OA. Among various subtypes of NOA, DDX4 positivity was higher in patients with maturation arrest and hypospermatogenesis compared with Sertoli cell only syndrome. PRM1 and PRM2 had very low positivity rate for any meaningful comparison. Seminal cell-free markers can serve as non-invasive tests in diagnosing and differentiating etiologies of azoospermia but their validity needs to be proved in long-term trials with more refined molecular techniques.

Aberrant Gene Expression Profiling in Men With Sertoli Cell-Only Syndrome.

Sertoli cell-only syndrome (SCOS) is the most severe and common pathological type of non-obstructive azoospermia. The etiology of SCOS remains largely unknown to date despite a handful of studies reported in this area. According to the gene expression of testicular tissue samples in six datasets from the Gene Expression Omnibus, we detected 1441 differentially expressed genes (DEGs) between SCOS and obstructive azoospermia (OA) testicular tissue samples. Enriched GO terms and KEGG pathways for the downregulated genes included various terms and pathways related to cell cycle and reproduction, while the enrichment for the upregulated genes yielded many inflammation-related terms and pathways. In accordance with the protein-protein interaction (PPI) network, all genes in the most critical module belonged to the downregulated DEGs, and we obtained nine hub genes, including CCNB1, AURKA, CCNA2, BIRC5, TYMS, UBE2C, CDC20, TOP2A, and OIP5. Among these hub genes, six were also found in the most significant SCOS-specific module obtained from consensus module analysis. In addition, most of SCOS-specific modules did not have a consensus counterpart. Based on the downregulated genes, transcription factors (TFs) and kinases within the upstream regulatory network were predicted. Then, we compared the difference in infiltrating levels of immune cells between OA and SCOS samples and found a significantly higher degree of infiltration for most immune cells in SCOS than OA samples. Moreover, CD56bright natural killer cell was significantly associated with six hub genes. Enriched hallmark pathways in SCOS had remarkably more upregulated pathways than the downregulated ones. Collectively, we detected DEGs, significant modules, hub genes, upstream TFs and kinases, enriched downstream pathways, and infiltrated immune cells that might be specifically implicated in the pathogenesis of SCOS. These findings provide new insights into the pathogenesis of SCOS and fuel future advances in its theranostics.

The molecular characteristics in different procedures of spermatogenesis.

Spermatogenesis is a multistep biological process. In addition to somatic cells, it involves the orderly differentiation of dozens of spermatogenic cells. In this process, the regulatory networks between different spermatogenic cell populations are significantly different. RNA m6A regulators and miRNAs have been found to be closely related to spermatogenesis in recent years, and they are an important part of the above regulatory networks. Understanding gene expression and its rules in different spermatogenic cell populations will help in the in-depth exploration of their detailed roles in spermatogenesis. This study collected a public dataset of nonobstructive azoospermia (NOA). Based on the Johnson score, the testicular samples of NOA were divided into three types, Sertoli-cell only syndrome, meiotic arrest and postmeiotic arrest, which represented the loss of three germ cell populations, including whole spermatogenic cells, postmeiotic spermatogenic cells, and a mixture of late spermatids and spermatozoa, respectively. The aforementioned three types of testis data were compared with normal testis data, and the molecular expression characteristics of the abovementioned three germ cell populations were obtained. Our study showed that different germ cell populations have different active molecules and their pathways. In addition, RNA m6A regulators, including METTL3, IGF2BP2 and PRRC2A, and miRNAs, including hsa-let-7a-2, hsa-let-7f-1, hsa-let-7g, hsa-miR-15a, hsa-miR-197, hsa-miR-21, hsa-miR-30e, hsa-miR-32, hsa-miR-503 and hsa-miR-99a, also presented regulatory roles in almost all germ cells.

Copy number variants within AZF region of Y chromosome and their association with idiopathic male infertility in Serbian population.

Results of numerous studies gave contradictory conclusions when analysing associations between copy number variants (CNVs) within the azoospermia factor (AZF) locus of the Y chromosome and idiopathic male infertility. The aim of this study was to identify the presence and possible association of CNVs in the AZF region of Y chromosome with idiopathic male infertility in the Serbian population. Using the multiplex ligation-dependent probe amplification technique, we were able to detect CNVs in 24 of 105 (22.86%) infertile men and in 11 of 112 (9.82%) fertile controls. The results of Fisher's exact test showed a statistically significant difference between cases and controls after merging g(reen)-r(ed)/g(reen)-r(ed) and b(lue)2/b(lue)3 partial deletions identified in the AZFc region (p = 0.024). At the same time, we observed a trend towards statistical significance for a deletion among gr/gr amplicons (p = 0.053). In addition to these, we identified a novel complex CNV involving inversion of r2/r3 amplicons, followed by b2/b3 duplication and b3/b4 deletion, respectively. Additional analyses on a larger study group would be necessary to draw meaningful conclusions about associations among CNVs that presented with higher frequency in the infertile men than the fertile controls.

A systemic review and meta-analysis exploring the predictors of sperm retrieval in patients with non-obstructive azoospermia and chromosomal abnormalities.

To identify the most prevalent chromosomal abnormalities in patients with non-obstructive azoospermia (NOA), consolidate their surgical sperm retrieval (SSR) rates and determine the significant predictors of positive SSR in this patient population. A systematic review and meta-analysis were performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Fifty-three studies including 2965 patients were identified through searching the PubMed database. Klinefelter Syndrome (KS) was the most prevalent chromosomal abnormality reported in 2239 cases (75.5%). Azoospermia factor c (AZFc) microdeletions were the second most common (18.6%), but men with these deletions had higher SSR rates than patients with KS (41.95% with AZFc vs. 38.63% with KS). When examining predictors of SSR in KS patients, younger age was a significant predictor of positive SSR in patients undergoing microsurgical testicular sperm extraction (micro-TESE). Higher testosterone was a favourable predictor in those undergoing micro-TESE and conventional TESE. Lower luteinizing hormone (LH) and follicular stimulating hormone (FSH) values were significantly associated with positive SSR with testicular sperm aspiration (TESA). No parameter predicted SSR rates in patients with AZFc microdeletions. Overall, genetic abnormalities have significant implications on SSR success in patients with NOA.

Identification and Functional Investigation of Novel Heterozygous HELQ Mutations in Patients with Sertoli Cell-only Syndrome.

Background: Male infertility is a major health concern in couples of childbearing ages. Nonobstructive azoospermia (NOA) is an extreme form of male infertility that affects ∼1% of adult men, and the etiology remains unknown in most cases. Sertoli cell-only syndrome (SCOS) is the most severe type of NOA. Aims: To explore novel human candidate variants that cause SCOS. Methods: (1) Whole exome sequencing (WES) of 20 men with SCOS, (2) Sanger sequencing of the HELQ gene in an additional 163 men with SCOS, (3) in vitro functional assays, and (4) in vivo studies. Results: WES of 20 patients with SCOS led to the identification of two heterozygous missense mutations (M1 and M2) in two unrelated Chinese patients with infertility. Using subsequent Sanger sequencing covering all the coding regions of the HELQ gene for 163 additional SCOS cases, we identified four additional heterozygous mutations (M3-M6) in unrelated patients. In vitro functional analyses revealed that two of these mutations (M5, c.2538T > G and M6, c.2945G > T) might affect the function of the HELQ protein. Two heterozygous mutant mouse models with mutations similar to those of two patients (M5 and M6) did not show any considerable spermatogenic defects. Conclusion: Assuming that the mouse models accurately reflect the impact of the mutations, heterozygous HELQ variants alone did not lead to the development of the SCOS phenotype in mice. However, we cannot rule out the risk variants in Chinese or other human populations, and a larger dataset is needed to confirm the association between HELQ mutations with SCOS.

Identification of genomic imbalances (CNVs as well as LOH) in sertoli cell only syndrome cases through cytoscan microarray.

Sertoli cell only syndrome (SCOS) is characterized by complete absence of germ cells in seminiferous tubules of testis. SCOS is multifactorial but genetic factors play a major role in pathogenesis of the disorder with idiopathic origin. Genetic factors majorly include sex chromosomal aneuploidy and Yq Microdeletion. But a large number of cases are still idiopathic. The study aimed to evaluate the genomic imbalances (CNVs and LOH) in idiopathic SCOS patients. The study is based on 28 apparent idiopathic SCOS cases and 10 controls. Molecular cytogenetic techniques viz., FISH, STS-Multiplex PCR and Affymetrix cytoscan microarray (750 K) were used. The microarray screened whole genomic imbalances in DNA from peripheral blood of 25 cases (excluded Klinefelter syndrome patients) and testicular FNAC sample of 2 cases. High FSH and low Inhibin B were observed in cases as compared to control controls groups. Four cases of sex chromosomal abnormality (i.e., three non-mosaic 47, XXY males and one non-mosaic 46, XX male) as well as four cases of Yq microdeletion (i.e., three cases with AZFc deletion and one case with complete AZFa, b and c deletion) were identified. Microarray detected unbalanced translocation of two segments of Y-chromosome i.e., Yp11.31-p11.2 (~4.o mb region, involving SRY) and Yp11.2 (~2.5 mb region) on X-chromosome in XX male. Also, loss of segment on same X-chromosome involving PAR1 region was identified. We have identified both autosomal and sex chromosomal CNVs (recurrent as well as private) involving candidate genes like SYCE1, ZFPM2, SRPK1, DAZ1, BPY2, HSFY1, VCY1 etc. All these CNVs are possibly associated with SCOS pathogenesis. CNVs identified in cases were already reported as pathogenic variant in clinical database DECIPHER. Microarray also detected many LOH (all autosomal, >3.0 mb size) that covered genes with spermatogenesis related function. The mechanism of action of LOH in pathogenesis of SCOS still remains unravelled. CNVs and LOH related to spermatogenesis identified from two different sample types (blood vs. testicular tissue) were discordant. This study should be extended for larger cohort of patients.

Trace the profile and function of circular RNAs in Sertoli cell only syndrome.

Studies increasingly show the involvement of circular RNAs (circRNAs) in several diseases. This study aims to explore the circRNA expression pattern in the testicular tissues of patients with Sertoli only cell syndrome (SCOS) and their potential functions. High throughput circRNA microarray analysis indicated that 399 circRNAs were upregulated and 1195 were down-regulated (fold change >2, P < 0.05) in SCOS relative to obstructive azoospermia (OA). The hsa_circRNA_101222, hsa_circRNA_001387, hsa_circRNA_001153, hsa_circRNA_101373 and hsa_circRNA_103864 were validated by qRT-PCR. Furthermore, the hosting genes of the differentially expressed circRNAs (DEcircRNAs) were enriched in biological processes related to cell cycle and intercellular communication. Also, the overlapping genes between the hosting genes of SCOS-related DEcircRNAs and those highly expressed in Sertoli cells of non-obstructive azoospermia (NOA) were enriched in immune cell development and cell communication. Taken together, aberrantly expressed circRNAs likely mediate SCOS development by regulating the function of Sertoli cells and the spermatogenic microenvironment.