This is a primary sample used for the study of NOA. It can provide valuable information through the analysis of sperm DNA, RNA (including microRNAs and other non-coding RNAs), cell free DNA (cfDNA), proteins and metabolic profiles. Seminal plasma analysis can reveal insights into sperm function, spermatogenesis, and the underlying causes of NOA (21).

Urine can be used to detect biomarkers associated with reproductive health. It can contain DNA, RNA, hormones, and metabolites that reflect the physiological state of the reproductive system (22).

Peripheral blood is a common non-invasive sample source for biomarker research. Blood tests can measure hormone levels, genetic material, and other circulating molecules that could be indicative of NOA or related conditions (23).

Although less commonly used than other samples, saliva can contain hormones and other biomolecules that might reflect reproductive health status, including conditions like NOA (24).

In some cases fluid from the epididymis can be collected and analyzed, particularly when looking at sperm maturation processes.

Please separate this paragraph each of these samples can offer unique information about the reproductive system and all these biological samples may be useful in identifying biomarkers for the diagnosis and treatment of NOA. The choice of sample often depends on the specific biomarker of interest, the ease and feasibility of collection, and the invasiveness of the procedure.

Collectively, genetic factors are believed to underlie approximately 30% of NOA cases (25). Karyotype anomalies, and Y chromosome microdeletions associated with NOA are summarized in Table 2 . Among them Klinefelter syndrome (47,XXY also in a mosaic condition) represents the most frequent cytogenetic abnormality associated with NOA.

Routine clinical assessment of NOA patients typically includes karyotyping, evaluation of microdeletions in the Y chromosome AZF region, and, in selected cases, gene sequencing to reveal possible monogenic conditions. When sperm retrieval is possible, genetic alterations identification in NOA patients is crucial for understanding potential impact on offspring, therefore it is important to discuss the chance of genetic defect transmission during genetic counseling, along with options for pre-implantation or prenatal diagnosis. For example, in case of balanced chromosomal translocations, the production of unbalanced gametes, resulting in infertility, offspring with severe phenotypes, or miscarriages, should be deeply discussed with the couple before ART procedures.

Another chromosomal cause of NOA relates in structural alterations of Y chromosome, which can be cytogenetically visible (e.g. translocation involving Y; deletions or duplications of the entire Yq arm) or submicroscopic, as in case of deletions affecting the AZF regions on the Yq. The AZF is classically divided in three regions (AZFa, AZFb, AZFc), harboring genes essential for spermatogenesis. Identifying specific AZF deletions helps to predict the success rate of surgical sperm retrieval (32) ( Table 2 ). A proposed mechanism for the different outcomes associated with deletions of AZFa, AZFb, AZFc focuses on the importance of the DAZ gene, characterized by several copy number variants (CNVs) and located in the AZFc region (DAZ1, DAZ2, DAZ3, and DAZ4). This genomic structure of AZF regions, enriched in CNVs, predisposes them to microdeletions (33). The complete deletion of the AZF regions is also present in males with a female karyotype, a case belonging to the Disorders of Sex Development (DSD), XX subgroup. The male phenotype is related to the presence of the SRY gene on one X homologue, due to an incorrect crossing-over in the father of the proband involving the pseudoautosomal regions on the p arm of X and Y chromosomes (29).

Regarding gene variants in NOA, the introduction of high-throughput sequencing methods has significantly increased the identification of candidate genes associated with genetic disorders, including male infertility.

Pathogenic variants in genes associated with infertility have been reported in both isolated and syndromic NOA cases (6).

A recent systematic review of the validated monogenic causes of human male infertility is reported by Houston et al. (34). Of the hundreds of genes expressed in testis, the most notable group belongs to the “TEX genes” (i.e. male-germ-cell-specific genes). This term, coined by Wang et al. (35), refers to the about 60 human genes distributed throughout the genome, playing roles in spermatogenesis and fertilization (36–39).

In a recent study using WES to improve the diagnosis of men with NOA (14), the authors employed a straightforward strategy based on a set of candidate genes to aid in interpreting WES data. This study identified causative mutations in genes with predicted or well-established impact on spermatogenesis in the 23% of NOA subjects, thus doubling the detection rate of NOA patients with a genetic defect. In addition, another study (40) evaluating 35 patients with spermatogenic failure reported causative mutations in 20 different genes in the 5.4% of analyzed subjects.

These findings highlight the potential of the genetic diagnosis, when combined with other clinical data, to provide strong guidance regarding the suitability of TESE. They support the use of NGS in assisting clinicians in offering more precise and relevant advice to patients, potentially influencing treatment decisions. Among the genes mutated in NOA reported by Kherraf et al. (14), and others (41), several (e.g. C14orf39, HFM1, MCM8, MEIOB, MSH4, DMC142, STAG3, KASH5) are also associated with female infertility, underscoring common mechanisms in male and female meiosis. A comparative evaluation of recent studies (14, 34, 40, 42) has reported pathogenic or likely pathogenic variants in genes like M1AP, TDRD9, TEX11, and KASH5 in NOA patients. Some of the more relevant genes found mutated in NOA patients are summarized in Table 3 . The list includes:

The M1AP gene (Meiosis 1-Associated Protein, OMIM: 619098) that is crucial for meiosis, and its dysfunction can lead to primary testicular failure, a major contributor to NOA. Variants in M1AP have been associated with cryptozoospermia and oligozoospermia in three different studies (43, 48, 49).

TDRD9 (Tudor Domain-Containing Protein 9, OMIM: 617963), is essential for transposon silencing in spermatogenesis. Biallelic variants in TDRD9 have been linked to maturation arrest in azoospermic men (44).

The X-linked TEX11 (Testis-Expressed gene 11, OMIM: 300311) gene, expressed in late-pachytene spermatids, is absent in Sertoli and interstitial cells, indicating its specific role in germ cells. Specifically, TEX11 is involved in the association of homologous chromosomes and meiotic recombination. Hemizygous mutations in TEX11 were found in 2.4% of azoospermic men (45).

The TEX15 (Testis Expressed 15, OMIM: 605795) gene encodes a protein that is required for DNA double-strand break repair, chromosome synapsis, and meiotic recombination in spermatocytes. Males caring homozygous variants in TEX15 show spermatogenesis defects that result in severe oligozoospermia or azoospermia (50). However, TEX15 mutations are very rare in NOA.

Rare recessive forms of NOA involve KASH5 (Coiled-Coil Domain Containing 155, OMIM: 618125), which protein links the nucleoskeleton and cytoskeleton by interacting with the inner nuclear membrane protein SUN1 and recruiting the cytoplasmic protein dynein (LINC complex). The nucleocytoplasmic interactions established by the LINC complex is required for telomere attachment to the nuclear envelope in the prophase of meiosis and for rapid telomere prophase movements in both male and female gametogenesis (51). Pathogenic variants in KASH5 have been reported in consanguineous families. Male infertility associated with KASH5 mutations is due to prepachytene meiotic arrest (46).

The MSH4 gene (MutS Protein Homolog 4, OMIM: 602105) encodes for a meiosis-specific protein that is involved in DNA mismatch correction and is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. Biallelic variants are associated with premature ovarian failure and spermatogenic failure. Limited NOA cases have been reported to carry MSH4 variants and were characterized by no round spermatids or spermatozoa in the seminiferous tubules (47).

In addition to genes involved in isolated NOA, syndromic forms have been also recognized. Among them: dynein genes known for their roles in ciliary movement, have been associated with syndromic forms of azoospermia, including Primary Ciliary Dyskinesia and Kartagener Syndrome (OMIM: 244400). These conditions feature additional symptoms beyond reproductive issues, such as respiratory system abnormalities. Dynein mutations disrupt axonemal structure, essential for sperm motility, highlighting a syndromic link to azoospermia (52).

The Wilms Tumor 1 (WT1) gene, critical for kidney and gonadal development, has been linked to syndromic NOA. WT1 mutations can lead to disorders such as Denys-Drash syndrome (OMIM: 194080) and Frasier syndrome (OMIM: 136680), which include genital tract malformations and predisposition to Wilms tumor, alongside NOA (53).

Kallmann syndrome (OMIM: 308700) is a developmental genetic disorder characterized by the association of congenital hypogonadotropic hypogonadism (CHH) due to GnRH deficiency, and anosmia or hyposmia (with hypoplasia or aplasia of the olfactory bulbs).

Fanconi anemia (OMIM: 227650) can lead to reproductive issues, including NOA as part of its broad spectrum of clinical manifestations. This connection is primarily due to the role of the Fanconi anemia pathway in DNA repair, critical for healthy spermatogenesis. Krausz et al. (54), identified a high-risk subgroup of infertile men with NOA for undiagnosed Fanconi anemia, highlighting the importance of assessing FA-related genes mutations in selected NOA patients.

The diverse range of loci identified in various studies underscores the complex genetics of NOA, with these genes representing significant contributors to the condition, although in limited cases. Their identification and study offer insights into the molecular mechanisms of NOA and pave the way for potential therapeutic interventions and stratification of patients on the bases of possible genetic causes. Understanding the genetic alterations underpinning NOA has also broader implications in the field of reproductive medicine; the identification of genetic defects shared between male and female infertility suggests a more interconnected approach to understanding and treating reproductive disorders.

In conclusion, an extensive genetic evaluation of NOA subjects should be incorporated into the clinical assessment of patients not only in selected cases ( Table 1 ), as it offers more chances for understanding the etiology of infertility, which can directly influence management and treatment strategies. The integration of karyotyping, AZF microdeletion analysis, and targeted gene panels assays should be considered the baseline for genetic assessment. In addition, with the advent of high-throughput sequencing technologies, the diagnostic yield is improving in NOA, enabling the identification of novel genetic factors contributing to NOA.

The genetic assessment can provide valuable insights into the likelihood of successful sperm retrieval by TESE, despite the complex and not fully elucidated relationship between mutations in genes associated with NOA and their specific histological manifestations.

In addition, genetic counseling becomes paramount in cases in which genetic abnormalities are detected. Patients and their partners should be informed of the implications for their offspring, including the risk of transmitting genetic abnormalities and the available options for pre-implantation genetic diagnosis (PGD) or prenatal testing.

Considering genes with strong evidence as well as those found vary rarely affected in NOA males, it might be more efficient the use of WES to identify pathogenic or likely pathogenic variants in NOA. This approach allows for the comprehensive analysis of genetic variants across all coding regions of the genome. Subsequently, virtual selection of genes known or suspected to be involved in NOA can further refine the search for causal mutations, streamlining the diagnostic process and enhancing our understanding of the genetic basis of NOA. This strategy optimizes the use of genetic information, facilitating the identification of potential targets for therapeutic intervention expanding the knowledge on the genetic basis of NOA. Furthermore, mutational analysis should be preceded by counseling, to collect the clinical family history to facilitate understanding of inheritance patterns in each family.

Various studies have shown significantly higher mean cfDNA levels in seminal plasma from patients with sperm abnormalities compared to controls. The levels of cfDNA are particularly elevated in men with azoospermia, including NOA, and teratozoospermia, suggesting a strong association between cfDNA levels and sperm abnormalities. Two primary hypotheses for elevated cfDNA levels in NOA are: i) abortive spermatogenesis: in NOA, germinal stem cells may begin differentiation but are halted by checkpoints, leading to apoptosis and increased cfDNA levels; ii) infections and inflammatory phenomena: inflammation, whether specific or nonspecific, can cause cell lysis, releasing nucleic acids into the seminal plasma.

The majority of cfDNA in seminal plasma is believed to derived from apoptotic and necrotic cells within the male reproductive tract (55, 56). During apoptosis, cells undergo programmed cell death, leading to DNA fragmentation. Similarly, necrosis resulting from cellular injury can release DNA into the extracellular environment (57). Additionally, during spermatogenesis, germ cells undergo extensive remodeling, and DNA that is not incorporated into spermatozoa can be shed into the seminal fluid (58). Cells from the epididymis and prostate, which significantly contribute to seminal fluid, may release DNA during their physiological turnover or in response to inflammation or pathology. Moreover, cfDNA can be directly released from spermatozoa, particularly from those that are damaged or undergo cell death (16).

​The concentration of cfDNA in seminal plasma may serve as a predictive biomarker for the success of sperm retrieval in NOA. Elevated levels of cfDNA can, indeed, indicate abortive spermatogenesis or infections​ (59, 60).

Regarding cell free RNA (cfRNA), a significant portion of cfRNAs in seminal plasma is packaged within extracellular vesicles such as exosomes. These vesicles are secreted by various cells in the reproductive tract, including prostate epithelial cells, seminal vesicles and Sertoli cells (61).

RNAs are also released from cells at different stages of spermatogenesis. As these cells progress through various stages of maturation, they may shed RNAs into the seminal plasma. Like cfDNA, apoptosis of germ cells and somatic cells within the testes can lead to the release of RNAs into seminal plasma (62). In addition, prostate and seminal vesicles can release RNA molecules in the seminal plasma, thus reflecting the physiological and pathological state of these tissues (63).

Limitations: despite the growing interest in cell-free nucleic acid as non-invasive biomarkers for the diagnosis and prognosis of disease in various clinical contexts, including NOA, its clinical use in standard practice is limited for several reasons:

Although data on cfDNA are more extensive compared with those on cfRNA, additional clinical studies are needed to confirm the utility of cfDNA in various contexts of male infertility, to validate specific biomarkers, and to integrate cfDNA results with other diagnostic methods. It is conceivable that the search on cfDNA and cfRNA markers in seminal plasma might not yield consistent indicators due to the heterogeneous nature of NOA.

More comprehensive studies are crucial to establish the clinical utility of cfDNA and cfRNA as biomarkers in NOA. However, these studies should be meticulously designed considering the patient stratification on clinical and genetic background. This approach acknowledges that cfDNA and cfRNA may serve as specific indicators for different impediments in the spermatogenesis process, reflecting the unique pathophysiological conditions of each NOA case.

In conclusion, while cfDNA has significant potential as a diagnostic and prognostic tool in NOA, its practical application requires further research and development in the field of reproductive medicine. Understanding the origins of cfDNA and cfRNA in seminal plasma is crucial for interpreting their levels and modifications in different pathological or physiological conditions.

Many genes are expressed during meiosis, and their transcription profile was explored in NOA patients as biomarkers. They can be classified in: i) microRNAs (miRNAs), small non-coding RNAs that regulate gene expression, found in seminal plasma, urine, and blood; ii) messenger RNAs (mRNAs), reflective of gene expression patterns relevant to spermatogenesis; iii) circular RNAs (circRNAs), covalently closed RNA molecules, with roles in gene regulation; and iv) long non-coding RNAs (lncRNAs), involved in chromatin remodeling, transcriptional and post-transcriptional regulation.

Expression analyses in NOA are significantly improving our understanding on the molecular mechanisms underlying this condition, mainly by the evaluation of testes biopsies, which results can be tested and translated in semen plasma. In the past years, studies on single potential biomarkers have been performed, now overtaken by omics technologies. One example was the evaluation of ESX1 gene transcript in NOA (64, 65).

The development of single-cell RNA sequencing has been, indeed, pivotal in this regard, enabling the investigation of individual cell populations in the testis at high resolution. Such studies have provided a deeper insight into the pathogenesis of NOA and identified key genes and transcription factors involved in this process (63).

The Transcriptional Regulatory Networks (TRNs) in single-cell RNA-Seq data sets, has identified specific transcription factors in Leydig cells and testicular macrophages of iNOA patients, such as LHX9, KLF8, KLF4, ARID5B, and RXRG in iNOA Leydig cells, and POU2F2, SPIB, IRF5, CEBPA, ELK4, and KLF6 in iNOA testicular macrophages. Interestingly, about 256 differentially expressed genes (DEGs), including the upregulation of TEX38, FAM71F, PRR30, FAM166A, LYZL6, TPPP2, ARMC12, and SPACA4, have been identified in NOA. These genes are involved in crucial biological processes like spermatogenesis, cell differentiation, flagellated sperm motility, and spermatid development. These findings underscore the complexity of NOA and the diverse molecular pathways involved. The identified transcription profile and their regulatory mechanisms add knowledge in NOA pathophysiology and highlight potential areas for targeted interventions.

Translating these findings into the identification of non-invasive biomarkers for predicting the success of sperm retrieval in patients with NOA involves several steps: i) the identification of specific biomarkers in liquid biopsy, mainly in seminal fluid, since most studies have identified specific changes in the Sertoli cells of NOA patients. This might involve the detection of specific mRNAs or miRNAs that reflect the changes observed in testicular tissues. In addition, further studies are essential for their validation to verify their specificity and sensitivity in predicting sperm retrieval success across different patient groups; ii) the integration with other clinical findings: biomarkers derived from these studies should be considered alongside existing clinical indicators, such as hormone levels, genetic analysis, and physical examination, to provide a comprehensive and personalized assessment of sperm retrieval potential; iii) the technological development and accessibility: the implementation of non-invasive tests requires advanced technologies for the accurate and sensitive detection of biomarkers at very low levels. The accessibility and feasibility of such tests are crucial for their widespread clinical application.

In summary, while these results offer promising perspectives, their translation into non-invasive diagnostic tools for predicting sperm retrieval success requires additional research, technological development, and clinical validation.

Recently, a comprehensive analysis of the potential use of seminal plasma for biomarker detection in NOA patients to predict sperm retrieval was carried out by Li et al. (17). The authors evaluated about 80 studies ranging in quality from medium to high. The biomarkers examined were diverse, including RNAs (miRNAs, lncRNAs, circRNAs, tsRNAs). Within the vast array of RNA found in seminal plasma, specific types have been highlighted for their roles in cellular processes that may mirror the status of spermatogenesis. These include miRNAs, lncRNAs, circRNAs, and tRNA-derived small RNAs (tsRNAs). Several miRNAs are implicated in the development of germ cells and the process of spermatogenesis. Their stability in seminal plasma, due to their packaging in microvesicular bodies or protein complexes, makes them promising non-invasive markers for spermatogenesis. The study has shown a different expression of specific miRNAs in NOA patients with both positive and negative TESE outcomes, suggesting their potential in predicting spermatozoa retrieval, though further validation is needed.

LncRNAs are involved in various regulatory functions within the cell and have been associated with spermatogenesis. Recent research using seminal plasma from TESE procedures has identified a panel of lncRNAs that could predict spermatozoa recovery more accurately than sex hormone levels, offering a new avenue for pre-surgical evaluation in NOA patients (64).

CircRNAs have a role in spermatogenesis and are detectable in seminal plasma. They are resistant to degradation by RNA exonucleases, making them stable markers. A recent study identified three circRNAs with the potential to predict TESE outcomes, although their functions and connections to spermatogenesis require further investigation (65).

Finally, tsRNAs, emerging as key players in gene regulation, are involved in sperm maturation and are abundant in seminal plasma. A recent study found higher levels of a specific tsRNA (RF-Val-AAC-010) in NOA patients with successful micro-TESE, suggesting its utility as a biomarker for predicting TESE outcome. This tsRNA was also shown to participate in spermatogenesis, strengthening its potential as a predictive tool (66).

Overall, these RNA molecules in seminal plasma can be a non-invasive way to forecast the success of TESE in NOA patients, thus significantly impacting on clinical decision-making and patient counseling. However, due to the variability and preliminary nature of current studies, larger and more comprehensive investigations are needed to establish their clinical utility.

Limitations: the application of transcriptomics for predicting sperm retrieval in clinical settings faces several limitations:

These limitations indicate that while transcriptomic approaches hold promise for improving sperm retrieval predictions in NOA patients, more research and development are needed to enhance their clinical utility.

Over the past two decades, there have been numerous proteomics studies investigating potential biomarkers for azoospermia. These studies have systematically examined human samples, aiming to identify biomarkers that can provide insights into azoospermia without the need for invasive procedures​​. Proteomic biomarkers include proteins in the seminal plasma that correlate with sperm health and spermatogenesis, and reproductive hormones, like testosterone, FSH, LH, that can be detected in blood and saliva.

Seminal plasma contains proteins in a concentration of 35–55 mg/mL, with a mix of testicular, epididymal, seminal vesicle, and prostate proteins. About 60% of these proteins contribute to its enzymatic activity, including hydrolases and peptidases (67, 68)​​.

Various proteins of testicular and epididymal origin have been identified in seminal plasma, which can help distinguish between OA and NOA​​. Significant proteins that have been studied in this context include:

Limitations:

Overall, while significant progress has been made in proteomic analysis for identifying biomarkers of male sterility in seminal plasma, these challenges highlight the need for continued research and development in this field.