OBJECTIVE: Non-obstructive azoospermia (NOA) is a severe and common cause of male infertility. Currently, the most reliable predictor of sperm retrieval success in NOA is histopathology, but preoperative testicular biopsy often increases the difficulty of sperm retrieval surgery. This study aims to explore the characteristics of N6-methyladenosine (m6A) modification in NOA patients and investigate the potential biomarkers and molecular mechanisms for pathological diagnosis and treatment of NOA using m6A-related genes.
METHODS: NOA-related datasets were downloaded from the GEO database. Based on the results of LASSO regression analysis, a prediction model was established from differentially expressed m6A-related genes, and the predictive performance of the model was evaluated using ROC curves. Cluster analysis was performed based on differentially expressed m6A-related genes to evaluate the differences in different m6A modification patterns in terms of differentially expressed genes (DEGs), biological features, and immune features.
RESULTS: There were significant differences in eight m6A-related genes between NOA samples and healthy controls. The ROC curves showed excellent predictive performance for the diagnostic models constructed with ALKBH5 and FTO. DEGs of two m6A modification subtypes indicated the influence of m6A-related genes in the biological processes of mitosis and meiosis in NOA patients, and there were significant immune differences between the two subtypes.
CONCLUSIONS: The NOA pathological diagnostic models constructed with FTO and ALKBH5 have good predictive ability. We have identified two different m6A modification subtypes, which may help predict sperm retrieval success rate and treatment selection in NOA patients.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH\'s role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

BACKGROUND: Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia (NOA). Testicular specimens possess poor motility and low population of viable spermatozoa. Therefore, sperm selection methods such as applying pentoxifylline (PTX) may improve motility in these cases. The main aim of this study was to evaluate the protective effects of PTX on testicular spermatozoa before and after performing SSC.
METHODS: Thirty testicular samples were obtained from men with azoospermia. This study was conducted in two phases. Phase 1 evaluated the effect of PTX for sperm selection before SSC. Twenty testicular samples were divided to two experimental groups: SSC without (I) and with PTX treatment (II). For PTX treatment spermatozoa were incubated with PTX at 37°C for 30 min and only motile spermatozoa were selected for SSC. In phase 2, ten testicular samples were cryopreserved with SSC and warming procedure was carried out in droplet with and without PTX. Motility and viability rates, morphology by motile sperm organelle morphology examination (MSOME), DNA fragmentation by sperm chromatin dispersion test (SCD) and mitochondrial membrane potential (MMP) were evaluated.
RESULTS: In phase 1, post warm motility rate was higher in PTX exposed group compared to the unexposed group (25.6 ± 8.13 vs. 0.85 ± 2.1) (p > 0.00). Recovery rate, viability and morphology were not significantly different between groups. DNA integrity and MMP were also similar between both groups. In phase 2 although motility increased in PTX group compared to without PTX group (29.30 ± 12.73 vs. 1.90 ± 2.64) (p > 0.00), the viability rate was not different (70.40 ± 12.12 vs. 65.30 ± 11.87). All above mentioned parameters were similar between the two SSC groups.
CONCLUSIONS: Supplementation of testicular spermatozoa with PTX before cryopreservation increases motility and did not have adverse effects on viability, morphology, DNA integrity and MMP. PTX could be used as sperm selection method before single sperm cryopreservation, but PTX could not maintain motile the most of viable testicular sperms.

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.

OBJECTIVE: The objective of this study was to discern ferroptosis-related genes (FRGs) linked to non-obstructive azoospermia and investigate the associated molecular mechanisms.
METHODS: A dataset related to azoospermia was retrieved from the Gene Expression Omnibus database, and FRGs were sourced from GeneCards. Ferroptosis-related differentially expressed genes (FRDEGs) were discerned. Subsequently, these genes underwent analyses encompassing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, as well as protein-protein interaction (PPI) networks and assessments of functional similarity. Following the identification of hub genes, an exploration of immune infiltration, single-cell expression, diagnostic utility, and interactions involving hub genes, RNA-binding proteins (RBPs), transcription factors (TFs), microRNAs (miRNAs), and drugs was conducted.
RESULTS: A total of 35 differentially expressed FRGs were discerned. These genes demonstrated enrichment in functions and pathways associated with ferroptosis. From the PPI network, eight hub genes were selected. Functional similarity analysis highlighted the potential pivotal roles of HMOX1 and GPX4 in azoospermia. Analysis of immune cell infiltration indicated a significant decrease in activated dendritic cells in the azoospermia group, with notable correlations between hub genes, particularly SAT1 and HMGCR, and immune cell infiltration. Unique expression patterns of hub genes across various cell types in the human testis were observed, with GPX4 prominently enriched in spermatid/sperm. Eight hub genes exhibited robust diagnostic value (AUC > 0.75). Lastly, a comprehensive hub gene-miRNA-TF-RBP-drug network was constructed.
CONCLUSIONS: In summary, our investigation unveiled eight FRDEGs associated with azoospermia, which hold potential as biomarkers for the diagnosis and treatment of azoospermia.

Aim: Azoospermia accounts for 10-20% of male infertility. In 20-30% of affected males, genetic abnormalities are the leading cause of azoospermia. LncRNAs can regulate spermatogenic cell development. Methods: This study chose 76 azoospermia patients and 36 healthy males. The gene expression was examined using the qRT-PCR technique. Results: IGSF11-AS1 and BVES-AS appeared to be considerably underexpressed in the patients; however, only IGSF11-AS1 demonstrated a significant biomarker role. Additionally, IGSF11-AS1 expression was positively correlated with testosterone but was negatively correlated with follicle-stimulating hormone (FSH) and luteinizing hormone (LH). For the BVES-AS gene, however, FSH and LH had a negative correlation. Conclusion: As a result of its low expression level in tissue samples, IGSF11-AS1 has a biomarker role for early azoospermia detection.
[Box: see text].

OBJECTIVE: To investigate cryopreserved testicular spermatozoa among patients with azoospermia.
METHODS: In this retrospective study spanning from October 1993 to December 2021, we examined men diagnosed with azoospermia who underwent testicular spermatozoa cryopreservation. Data from medical records included utilization and disposal of sperm samples, age at initial cryopreservation. We analyzed the data over 20 years using Kaplan-Meier curves, compared age with the log-rank test, and assessed hazard ratios (HR) with 95% confidence intervals (CI) using Cox regression analysis.
RESULTS: A total of 356 patients with a mean age of 32.1 ± 6 were included. Of these, 225 patients utilized thawed testicular sperm for fertility treatments, with 118 patients using all their frozen straws and 107 patients partially using their stored straws. Additionally, 29 patients opted for disposal (six patients partially used their testicular spermatozoa before disposal), resulting in 108 patients who neither used nor disposed of their straws. From a laboratory standpoint, nearly 90% of patients contributed a single testicular sample, which was subsequently divided and cryopreserved as straws, with a median of 4 straws per sample. Notably, in the older age group (> 35 years old), there were a significantly lower usage rate and a higher disposal rate compared to the younger age groups (p < 0.05 for both), corroborated by univariable Cox analysis.
CONCLUSIONS: This extensive study unveils unique patterns in the preservation and disposal of testicular spermatozoa among azoospermic patients. Most patients utilize a significant portion of their stored samples, while older patients tend to use their testicular spermatozoa less frequently.

Non-obstructive azoospermia (NOA) is the most severe form of male factor infertility. It results form from either primary or secondary testicular failure. Here, we report cases of two patients with NOA due to maturation arrest and increased serum FSH, treated with GnRH agonist and gonadotrophins. The two NOA patients underwent a pharmacological treatment consisting of pituitary desensibilization using a GnRH agonist and testicular stimulation using menotropin. Testicular stimulation started one month after the beginning of GnRH agonist treatment. The female partner underwent controlled ovarian stimulation (COS) followed by intracytoplasmic sperm injection (ICSI). On the third day of the cycle, menotropin daily doses was administered. When at least one follicle ≥14 mm was visualized, pituitary blockage was performed using GnRH antagonist ganirelix. When three or more follicles attained a mean diameter of ≥17 mm, triptorelin acetate was administered to trigger final follicular maturation. Oocyte retrieval was performed 35 hours later. After treatment, male partner blood levels of the FSH, LH, decreased and total testosterone were increased. Spermatozoa was observed after semen collection in both cases. After COS, oocytes were retrieved and ICSI was performed. Embryos were biopsied for preimplantation genetic testing (PGT) and those considered euploidy were transferred resulting in positive implantation, ongoing pregnancy, and livebirth on both cases. In this report we present a successful strategy for hypergonadotropic hypogonadism AOA men, as an alternative approach to the surgical testicular sperm recovery. Nevertheless, prospective randomized trials are needed to confirm our findings.

BACKGROUND: Male infertility has become a global health problem, and genetic factors are one of the essential causes. Y chromosome microdeletion is the leading genetic factor cause of male infertility. The objective of this study is to investigate the correlation between male infertility and Y chromosome microdeletions in Hainan, the sole tropical island province of China.
METHODS: We analyzed the semen of 897 infertile men from Hainan in this study. Semen analysis was measured according to WHO criteria by professionals at the Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University, where samples were collected. Y chromosome AZF microdeletions were confirmed by detecting six STS markers using multiple polymerase chain reactions on peripheral blood DNA. The levels of reproductive hormones, including FSH, LH, PRL, T, and E2, were quantified using the enzyme-linked immunosorbent assay (ELISA).
RESULTS: The incidence of Y chromosome microdeletion in Hainan infertile men was 7.13%. The occurrence rate of Y chromosome microdeletion was 6.69% (34/508) in the oligozoospermia group and 7.71% (30/389) in the azoospermia group. The deletion of various types in the AZF subregion was observed in the group with azoospermia, whereas no AZFb deletion was detected in the oligozoospermia group. Among all patients with microdeletions, the deletion rate of the AZFc region was the higher at 68.75% (44 out of 64), followed by a deletion rate of 6.25% (4 out of 64) for the AZFa region and a deletion rate of 4.69% (3 out of 64) for the AZFb region. The deletion rate of the AZFa region was significantly higher in patients with azoospermia than in patients with oligozoospermia (0.51% vs. 0.39%, p < 0.001). In comparison, the deletion rate of the AZFc region was significantly higher in patients with oligozoospermia (3.08% vs. 6.30%, p < 0.001). Additionally, the AZFb + c subregion association deletion was observed in the highest proportion among all patients (0.89%, 8/897), followed by AZFa + b + c deletion (0.56%, 5/897), and exclusively occurred in patients with azoospermia. Hormone analysis revealed FSH (21.63 ± 2.01 U/L vs. 10.15 ± 0.96 U/L, p = 0.001), LH (8.96 ± 0.90 U/L vs. 4.58 ± 0.42 U/L, p < 0.001) and PRL (263.45 ± 21.84 mIU/L vs. 170.76 ± 17.10 mIU/L, p = 0.002) were significantly increased in azoospermia patients with microdeletions. Still, P and E2 levels were not significantly different between the two groups.
CONCLUSIONS: The incidence of AZF microdeletion can reach 7.13% in infertile men in Hainan province, and the deletion of the AZFc subregion is the highest. Although the Y chromosome microdeletion rate is distinct in different regions or populations, the regions mentioned above of the Y chromosome may serve an indispensable role in regulating spermatogenesis. The analysis of Y chromosome microdeletion plays a crucial role in the clinical assessment and diagnosis of male infertility.

OBJECTIVE: This study aimed to investigate the correlation between testicular shear wave elastography (SWE) values and semen analysis results in men with infertility.
METHODS: This was a retrospective case-control study. Patients were categorized as normal, abnormal, or azoospermic based on sperm analysis results. Testicular volume was measured using B-mode ultrasonography using the Lambert formula. Subsequently, 40-80 regions of interest measuring 1.5 × 1.5 mm were manually positioned in both testicles based on their size, and two-dimensional SWE was applied through virtual touch imaging quantification software.
RESULTS: The patients had a mean age of 33.79 ± 6.3 years, with semen analysis revealing normal results in 15 patients (22.4%), pathological findings in 35 patients (52.2%), and azoospermia in 17 patients (25.4%). Right, left, total, and mean testicular volumes were significantly lower in patients with azoospermia compared to those in both normal and impaired semen parameters (P < .05). Conversely, testicular elastography scores were higher in patients with azoospermia than in the other groups (P < .05). The significant negative correlation between volume and elastographic findings remained independent of age (r = 0.4, P < .001). The accuracy rates for detecting impaired semen parameters and azoospermia were 94.3% and 94.1%, respectively, after considering factors such as age, testicular volume (right/left/total), and elastography (right/left/total). Notably, the total mean elastography score ranked first, with 100% in the independent normalized importance distribution of these variables.
CONCLUSIONS: SWE can be used effectively alone or in combination with other diagnostic tools to evaluate histopathological changes in the testicles of male patients with infertility.