Emerging trends in the management of non-obstructive azoospermia

Abstract

Ten percent of infertile males have azoospermia, classified into obstructive and nonobstructive types, which require specific medical or surgical treatments. In nonobstructive azoospermia, advancements in microsurgery allow for effective sperm retrieval in about half of cases. This review aimed to highlight the most recent studies utilizing whole exome sequencing (WES) and seminal/serum biomarkers aim to predict micro-dissection testicular sperm extraction (micro-TESE) outcomes and reduce failure rates. WES identifies rare genetic mutations affecting spermatogenesis that could explain micro-TESE failures, improving preoperative assessments. Despite its potential, WES is limited by high costs. Biomarkers such as Anti-Müllerian Hormone and testis-expressed sequence 101 protein can indicate spermatogenic activity, though standardization of these measurements is needed for accuracy. Additionally, microRNAs as non-invasive markers offer potential in assessing spermatogenic reserve and classifying azoospermia types, but their efficacy in identifying specific spermatogenic disorders remains under researched, necessitating further studies on multivariate miRNA models.

Keywords

microRNAs / nonobstructive azoospermia / seminal plasma biomarkers / whole exome sequencing

1 INTRODUCTION

Azoospermia is the total lack of spermatozoa in the ejaculate. About 10% of men who are infertile and 1% of all males have this finding[1]. There are two types of azoospermia, obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). NOA constitutes 60% of cases, which may result from pre-testicular or testicular causes that affect spermatogenesis[1]. Pre-testicular causes include defects in the hypothalamus or pituitary gland, resulting in impaired production of follicular stimulating hormone (FSH) and/or luteinizing hormone (LH), resulting in secondary spermatogenic testicular failure[1]. Testicular causes are collectively referred to as primary spermatogenic failure, including congenital genetic causes (Klinefelter's syndrome (KS), Y chromosome microdeletions, myotonic dystrophy, Kennedy's syndrome, androgen insensitivity syndromes, Noonan's syndrome), varicocele, maldescended testes, tumors, infection, radiotherapy and chemotherapy[2]. The severe forms of primary spermatogenic failure present clinically by azoospermia with different causes. Eugonadotropic and hypergonadotropic NOA represent the most common forms of azoospermia[2]. In men with NOA, successful micro-dissection testicular sperm extraction (micro-TESE) followed by intra-cytoplasmic sperm injection (ICSI) may enable around half of these patients to become biological fathers[3]. Micro-TESE is now a recognized as successful sperm isolation technique, with a reported 60% success rate[3].

The American Society of Reproductive Medicine publishes the most important guidelines for managing azoospermia in collaboration with the American Urological Association, which recommends that NOA should be evaluated with karyotype and Y chromosome microdeletion studies[4]. Klinefelter syndrome (KS) exhibits one extra X chromosome for 47 chromosomes (47, XXY), the most prevalent aberrant karyotypic pattern. On micro-TESE, at least 50%–60% of 47, XXY men may have uncommon foci of spermatogenesis[4]. The second most prevalent genetic cause of infertility in men is microdeletions in the Y chromosome[4]. Around 50% of men with an AZFc deletion have a sperm retrieval rate (SRR); nevertheless, surgical intervention is not recommended for men with complete AZFa and/or AZFb microdeletions since sperm have not been recovered by testicular sperm extraction[4]. The use of hormone stimulation therapy in men with primary hypogonadism or eugonadal males is controversial, according to the European Association of Urology's division on male infertility[5]. Gonadotropins, aromatase inhibitors, and selective estrogen receptor modulators are frequently used as adjuvants to enhance surgical SRR in men with NOA.

Nevertheless, no RCTs have been conducted expressly to examine the advantages of hormone stimulation therapy for men with primary hypogonadism and NOA[5]. Men with NOA are affected emotionally and financially by failed micro-TESE. Therefore, prognostic indicators are necessary for appropriate counseling. The prediction value for sperm retrieval (SR) has been investigated using criteria such as testicular volume, patient age, serum FSH, LH, total testosterone, facial hair pattern, and gynecomastia. However, no factors have been recognized as prognostic markers[6,7]. Recent studies aimed at predicting outcomes of micro-TESE in men with nonobstructive azoospermia (NOA) and enhancing sperm retrieval rates (SRR) have found that factors like age, body mass index (BMI), luteinizing hormone (LH), prolactin (PRL), and total testosterone (TT) do not effectively predict sperm recovery in these patients[8].

Other studies have suggested that testicular volume can be used to predict the success of TESE[7]. However, the predictive significance of follicle-stimulating hormone (FSH) and estradiol remains controversial[9]. Consequently, they reduced the number of micro-TESE failures. This review highlights recent key findings that have successfully decreased micro-TESE failure rates and could significantly influence clinical practice in the coming years[6].

2 WHOLE EXOME SEQUENCING (WES)

WES is a high-throughput technology used for sequencing the subset regions of DNA that encodes proteins (also known as exome). The whole genome is the entirety of the genetic data found in a sample or individual. The introns, which are the transcribed portion of the nucleotide sequence in an mRNA, carry the non-coding portion of proteins. Conversely, an exon is the transcribed portion of the mRNA nucleotide sequence responsible for protein production. Exons are the genome's protein-coding regions and are collectively known as the exome. The sequence of introns frequently undergoes variation over time[10]. The exome represents 1%–2% of the human genome yet contains nearly 85% of the known disease-causing variants[11]. Focusing on this 1% through WES to recognize genetic variants that alter protein sequences and can be disease causing, costs far less than whole genome sequencing yet still detects a high yield of relevant variants. This technology can be applied in research and clinical diagnostics[12]. As a genomic analysis method, WES looks for genetic alterations linked to changes in protein function. The fundamental idea consists of:

• DNA capture and enrichment: First, DNA sequences are captured and enriched using exon-specific DNA or RNA probes. This is usually accomplished via liquid-phase hybrid capture technology, which uses base pairing to hybridize biotin-labeled RNA probes with adapter-sequence-containing DNA libraries before enriching the target region's DNA via magnetic bead binding[13,14].

• High-throughput sequencing: High-throughput sequencing technology is subsequently used to sequence the enhanced DNA sequences. The method of sequencing involves identifying every deoxyribonucleotide configuration in the exome, which could aid in developing an understanding of possible pathophysiological alterations in particular diseases[15].

• Data analysis: Bioinformatic analysis of the sequencing data is performed to identify genetic mutations related to alterations in protein function. The detected variants are then analyzed by geneticists and clinicians to determine their pathogenicity using databases and literature to classify variants as pathogenic, likely pathogenic, or of uncertain significance[16].

2.1 Diagnostic role of WES in medicine

Exome sequencing is an economical, effective method for analyzing the genetic foundations of Mendelian diseases and detecting de novo mutations in rare monogenic disorders. It has proven valuable in diagnosing patients who remain unidentified after traditional testing[17]. Additionally, WES is increasingly utilized for rapid prenatal and neonatal diagnoses[18], and for characterizing genetic mutations in various cancers, providing clinically relevant insights[19]. WES also surpasses targeted methods in identifying clinically significant genetic alterations and has revealed actionable secondary findings in cancer genomes[20,21].

In addition to complex diseases resulting from the interaction of several behavioral, environmental, and genetic factors, exome sequencing has become a technically viable and more economical method of determining the genetic basis of Mendelian disease and identifying de novo mutations underlying rare monogenic diseases[17]. WES is a proper diagnostic technique for finding uncommon causal genetic alterations in rare instances and for patients who have not been diagnosed despite extensive traditional testing and workup[18]. In addition, WES is increasingly used in prenatal diagnosis of genetic conditions and in neonatal intensive care units rapid diagnosis of critically ill neonates[19]. Furthermore, genome-wide sequencing made it possible to characterize the landscape of genetic changes in numerous malignancies, which produced molecular insights of clinical significance.

WES aids in identifying genetic alterations of potential clinical interest more than targeted approaches for known hotspot mutations. Moreover, germline WES data has led to identification of clinically actionable secondary findings in cancer[20,21].

2.2 WES in azoospermia

WES has revolutionized the field of genetic diagnostics, offering a powerful tool for understanding the genetic basis of diseases, including azoospermia[22]. For many men with azoospermia, the cause of their condition remains unknown even after standard diagnostic evaluations[2]. After the exclusion of all known acquired causes through routine genetic testing, unclear etiology in a considerable proportion of patients is related to genetic/epigenetic causes[23].

WES can provide a molecular diagnosis by identifying genetic mutations that affect sperm production and maturation in genes responsible for spermatogenesis; CFTR, TEX11, NR5A1, and DMRT1 among others, these mutations have been associated with different forms of azoospermia. Rare mutations which are not detected using targeted gene panels in genes like SPATA16 and SYCP3 can be identified highlighting the extensive genetic heterogeneity of azoospermia. The condition can result from mutations in a wide array of genes, each playing a role in different aspects of spermatogenesis, from meiosis to sperm motility. Owing to WES, the number of genes involved in cases of NOA is rapidly increasing[24]. Exome analysis is very efficient in understanding the etiology of meiotic arrest, with potential implications for testicular sperm extraction (TESE) prognosis[25]. By identifying the full spectrum of genetic mutations involved, WES contributes to the development of a more comprehensive understanding of the condition, paving the way for new therapeutic targets and interventions. The use of WES in azoospermia research can identify potential targets for gene therapy or other molecular-based treatments[26]. Also, this helps in genetic counseling of the affected individuals and guiding decisions regarding assisted reproduction, TESE and in vitro fertilization (IVF) and the potential of their success[27]. Interestingly, sperm retrieval failed in all people with meiotic gene abnormalities, suggesting that genetic diagnostics before TESE could assist in identifying those with low or null likelihood of successful SR. Therefore, stay away from unsuccessful procedures[22].

2.3 Limitations of WES

Despite its advantages, WES has several limitations that include; increased cost, incomplete coverage of entire genome, low efficiency in detecting structural large chromosomal rearrangements and copy number variations and complicated interpretation[15]. Each one is detailed in Table 1. Many clinicians are not familiar with this quickly developing technical field. WES challenges traditional clinical methods, and the interpretation of exome data requires analysis by genomic informaticians who often lack clinical experience. There is a critical need for clinicians to receive training in genetic informatics to bridge the divide between these two fields[15].

3 PREDICTIVE BIOMARKERS FOR SPERMATOGENESIS

3.1 Anti-Müllerian hormone (AMH)

AMH is a secreted dimeric glycoprotein hormone with critical roles in reproductive development and regulation[31]. Prior to gonadal differentiation, male and female mammalian embryos had two sets of paired reproductive ducts: the paramesonephric (Müllerian) and mesonephric (Wolffian) ducts[32]. In response to AMH and testosterone, which are secreted by immature Sertoli cells (SCs) and Leydig cells, respectively, the mesonephric ducts develop into the epididymis, vasa deferentia and seminal vesicles, while the paramesonephric ducts regress[33]. In males, AMH is secreted by immature SCs, and its circulating concentration before puberty is exceptionally high compared to that of women[34]. As puberty progresses, immature SCs differentiate into mature SCs, and AMH levels drop significantly[35]. The AMH gene resides on the short arm of chromosome 19, coded between the regions p13.2 and p13.3[36,37]. Two types of transmembrane receptors control the signal pathway of AMH[38,39]. The loss of function of AMH receptor type 2 (gene mutation) or even the AMH ligand causes persistent Müllerian duct syndrome in humans[40].

3.2 AMH and azoospermia

Some studies had explored AMH level as a predictor for SRR of TESE or micro-TESE NOA patients[41]. Fenichel found a positive association between seminal AMH and normal spermatogenesis, suggesting its secretion towards seminiferous lumen[42]. Another cross-sectional study in 2023 found that men with NOA who had lower amounts of circulating AMH had a higher chance of a successful SR during micro-TESE, which may indicate that their SCs were mature and that they were, therefore, more likely to have spermatogenic foci upon surgery[43,44].

KS is the most common genetic cause of human male infertility representing 10%–14% of azoospermic men. In patients with KS, AMH was normal till puberty then declined to subnormal levels after puberty. This could be explained as SCs are not affected till puberty with the onset of tubular hyalinization[45]. Furthermore, the fall of AMH associated with puberty is delayed in boys with KS (characterized by accelerated germ cell depletion from puberty), and the levels of AMH, inhibin B, and testosterone decrease rapidly in adulthood[31,46].

Chemotherapy often causes azoospermia during treatment, but whether this persists after treatment is unknown. AMH serves as a suitable marker for assessing long-term ovarian damage caused by chemotherapy, helping to establish its role in evaluating chemotherapy-induced ovarian toxicity[47]. Dynamic changes to serum AMH levels pre- and post-chemotherapy could be used to evaluate testicular damage[48]. AMH levels were reported to rise shortly after chemotherapy[48].

A brief rise in AMH levels following testicular damage has been documented in several studies. For instance, men with severe varicocele who were sub fertile showed a substantial drop in AMH[49]. These instances demonstrate both the long-term decline in SC function brought on by severe testicular damage and the compensatory increase in SC function in the early stages of testicular injury[46].

Hypogonadotropic hypogonadism is a pre-testicular cause of azoospermia. AMH can be used for differential diagnosis of constitutive pubertal delay and congenital hypogonadotropic hypogonadism (HH) in prepubertal boys with delayed sexual maturation, providing that AMH is a marker of immature SCs and the clinical value of serum gonadotropins (Gn) and testosterone is limited, because of their low serum levels in both conditions[50]. Decreased numbers of SCs were reported in patients with congenital HH, accompanied by low levels of serum AMH. However, SC function is expected in boys with constitutive pubertal delay, so the serum AMH levels are also normal[51].

Pozzi et al.'s recent meta-analysis demonstrated the importance of serum AMH in predicting successful SR in patients with lower AMH levels. This was attributed to higher production of AMH from more immature Sertoli cells reflecting lower levels of spermatogenesis. A cut-off of < 4 ng/mL serum level of AMH could predict mTESE success with a probability area under the curve (AUC) of 70.3%[44]. Inversely, lower levels of AMH predicts failure of SR in azoospermic men with KS. Tubular hyalinosis and atrophy are hypothesized to cause decreasing in levels of serum AMH and chances of successful sperm retrieval by micro-TESE[52].

AMH, according to the abovementioned studies, showed a promising predictive factor for spermatogenic activity, yet further studies are needed to standardize the proper cut-off threshold below which SR is unlikely.

4 SEMINAL PLASMA BIOMARKERS

Sperm interactions with the different surroundings along the male and female genital tracts are related to the proteins of seminal plasma (SP), which are important for sperm function[53]. A proteomic study of SP is essential as a noninvasive clinical diagnostic tool for disorders of the male reproductive system since the molecular changes in SP can impact male fertility[54]. A total of 2545 SP proteins had been identified, of which 83 proteins from the testis, 42 from the epididymis, 7 from the seminal vesicles and 17 from the prostate[55,56]. The proteomes of pooled SP from post-vasectomy (PV) males and fertile controls were studied to determine which SP proteins came from the testicular and epididymal fluids[55]. Thirty-two proteins were only found in the control group (testicular and epididymal proteins), and 49 inadequately displayed proteins in PV men (proteins expressed in the testis and epididymis, but also in other organs from the male reproductive tract)[56]. Some of the identified testicular and epididymal proteins were testis-expressed sequence 101 protein (TEX101), The extracellular matrix protein 1 (ECM1), phosphoglycerate kinase 2 (PGK2), histone H2B type 1-A (HIST1H2BA) and glyceraldehyde-3-phosphate dehydrogenase, testis-specific (GAPDHS)[57].

4.1 Testis-expressed protein 101 (TEX101)

The TEX101 gene is located on the long arm of chromosome 19 at position 19q13.31 in humans[58]. Germ cells are the only cells that express human TEX101. The testicular tissue contains two forms of TEX101: one that is secreted and one that is membrane-bound[59]. Next, TEX101 protein is expressed by haploid secondary spermatocytes, haploid spermatids, and haploid testicular sperm. These cells are primarily found on the plasma membrane and have a weak presence in the cytoplasm thereafter[60].

According to other studies, TEX101 remains in the lipid rafts of mature sperm and is only partially cleaved from the surface of testicular spermatozoa during epididymal maturation[61]. Immunofluorescence analysis of mouse and human sperm showed that TEX101 is localized to the post-acrosomal region of mature sperm[62,63]. Recently, TEX101 and the extracellular matrix protein 1 (ECM1) were used to develop an algorithm for non-invasive differential diagnosis of OA versus NOA[62]. The SP levels of TEX101 could also distinguish different NOA subtypes. Normal spermatogenesis is indicated by TEX101 levels of 120 ng/mL or greater, while hypospermatogenesis (HS) or maturation arrest (MA) are linked to values of 5 ng/mL–120 ng/mL, and Sertoli cell only (SCO) syndrome is indicated by levels below 5 ng/mL (theoretically zero)[62]. In such cases, TESE, under general anesthesia, could be avoided[64].

4.2 Lectin galactoside-binding, soluble 3 binding protein (LGALS3BP)

The male genital tract exhibits levels of LGALS3BP expression that can be identified using the ELISA confirmation method. Although it is not a marker specific to germ cells, its physiological function in cell-to-cell interaction through the extracellular matrix raises the possibility of its involvement in spermatogenesis, especially in the late phases of spermatogenic activity[65].

LGALS3BP is expressed in the whole genital tract and seminal plasma. Its role in spermatogenesis remains unclear. The findings of Freour et al. illustrated its predictive role in azoospermic men[66].

According to some studies, patients with a positive TESE result had significantly greater levels of LGALS3BP in the SP. A cut-off of 153 ng/mL demonstrated a 100% sensitivity and a 45% specificity[66]. Araujo and Bertolla concluded that LGALS3BP may be able to predict eventful TESE in NOA patients prior to ICSI[67].

4.3 The extracellular matrix protein 1 (ECM1)

The ECM1 gene, located near the epidermal differentiation complex on chromosome 1q21.2, produces four different isoforms of the 85 kDa secreted glycoprotein essential for angiogenesis, cancer biology, and endochondral bone formation[68,69]. The ECM1 protein includes a 19-amino-acid signal peptide and is detectable via ELISA, with levels ranging from 31 to 2000, typically higher in men with non-obstructive azoospermia (NOA) than those with obstructive azoospermia (OA)[70,71]. ECM1, recognized as an azoospermia biomarker, effectively differentiates between vasectomized men and non-vasectomized men with a threshold of 2.3 μg/mL, exhibiting 100% specificity. Additionally, this threshold distinguishes OA from NOA with 73% specificity and 100% sensitivity[62,72,73].

4.4 Lipocalin-type prostaglandin D synthase (L-PGDS)

Another important plasma protein surrounding spermatozoa as they go through the male genital tract is L-PGDS[74]. The exact physiological and biochemical function of L-PGDS in the male genital tract is still unclear. L-PGDS is thought to play a key role in the maturation of spermatozoa in the epididymis and the development of germ cells in the seminiferous tubules by acting as a carrier of retinoids, thyroid hormones, and essential fatty acids[75]. In 1975, Olsson discovered L-PGDS in trace amounts within seminal fluid, highlighting a significant issue: this protein, usually found in very high concentrations, was dramatically reduced in the semen of men with oligozoospermia[76]. This important finding has unfortunately been overlooked for many years. However, recent research has begun to shed light on this phenomenon, linking it to post-testicular obstructions[77]. Men with azoospermia may benefit from using the amount of L-PGDS in seminal plasma as a valuable biomarker to assess the seminal tract's patency[75].

4.5 Seminal biomarkers in routine clinical practice

Knowledge of the seminal plasma proteome enhances our understanding of proteins and their interactions related to normal gametogenesis and other biological processes. Using seminal biomarkers, which offer potential thresholds for directing patient management, could be integrated into routine clinical protocols. The protein TEX101 was quantified using liquid chromatography and selected reaction monitoring (LC-SRM), with concentrations ≥ 120 ng/mL correlating with normal spermatogenesis, levels between 5 ng/mL and 120 ng/mL suggesting maturation arrest or hypospermatogenesis, and < 5 ng/mL indicating Sertoli cell-only syndrome (SCO syndrome)[62]. Additionally, ECM1 has been validated as a sensitive biomarker for distinguishing between obstructive azoospermia (OA) and non-obstructive azoospermia (NOA)[65].

ELISA studies revealed that levels of L-PGDS are under 100 μg/L in individuals with obstructive azoospermia, suggesting that NOA can be diagnosed without a biopsy when

L-PGDS concentrations exceed 100 μg/L[77]. Further, males with oligozoospermia exhibit significantly lower L-PGDS levels compared to normal males, as demonstrated through sandwich ELISA and quantitative immunofluorometry[78,79]. These findings suggest a correlation between reduced L-PGDS levels and diminished sperm production.

5 SMALL NON-CODING RNAs (miRNAs)

5.1 miRNA synthesis and physiology

Physiologically synthesized in eukaryotic cells, microRNAs (miRNAs) are small, non-coding, single-stranded RNA molecules that pair with their complementary base sequence in related mRNA molecules in the cytoplasm to regulate or mostly down-regulate genes[78]. The involvement of miRNAs in the development of numerous human disorders has been thoroughly investigated and documented, in addition to the physiological regulation of gene expression in various biological processes, including cell cycle control and differentiation, cell growth and death, and embryo development[79]. Apart from the cellular level, it has been documented that miRNAs can also be found in extracellular fluids such as plasma, saliva, vaginal secretions, menstrual blood, and semen[80].

5.2 miRNA and spermatogenesis

Numerous distinct genes are involved in spermatogenesis, and their expression is assumed to be partially regulated by miRNA suppression, as it is translationally uncoupled from the production of proteins in germ cell development[81,82]. Functional spermatogenesis has been discovered to depend on miRNA's control of genes involved in meiosis and spermatid differentiation[83]. It is interesting to note that the expression profile of miRNA in the testis is distinct; for instance, Linsen et al. in 2010 discovered that although there were 35 testis-specific miRNAs, there were fewer miRNAs in rat testis tissue than in the other tissues they examined[84]. Ro et al. in 2007 discovered 141 miRNAs expressed in the mouse testis by cloning; 35% were preferentially expressed in the testis, and 5% were exclusive to the testis[85].

5.3 miRNA and diagnosis of male infertility and azoospermia

In 2009, Lian et al. presented the first evidence of a change in miRNA expression in the testis of patients with NOA. Notably, testis-derived NOA exhibits a broad spectrum of abnormalities, including sperm maturation arrest, hypospermatogenesis, and Sertoli-cell-only syndrome[86]. Therefore, it is necessary to evaluate the differentiating expressions of miRNAs in these infertile people[86]. miRNAs are non-invasive molecular biomarkers that can be used to predict testicular SR outcomes for assisted reproduction. They can also be used to classify azoospermia by origin into either OA or NOA and to determine the spermatogenic reserve of the testis of patients with non-obstructive/secretory azoospermia[87]. In semen samples from infertile men and normal fertile individuals as controls, Wang et al. in 2011 discovered changes in miRNA profiles by Solexa Sequencing in both azoospermia and asthenozoospermia. They regarded a 50-fold increase or decrease in expression as significant, and real-time RT-qPCR was used to confirm this for seven different miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p). Compared to the control, the levels of these seven miRNAs were much more significant in asthenozoospermia and significantly lower in azoospermia. The stability of the aforementioned miRNAs under various settings was another noteworthy finding of the previously described study[88]. They explained this phenomenon by pointing to the miRNAs’ small size and capacity to attach to intricate chemical compounds in semen samples devoid of cells. Last but not least, these seven miRNAs might be helpful for molecular diagnostic confirmation of male infertility[88]. Because miRNA plays various roles in spermatogenesis, any variation in associated genes may result in infertility[89].

5.4 Mechanisms by which miRNA affects spermatogenesis

Single-nucleotide polymorphisms (SNPs) in rs10719, rs12323635, and rs642321 were linked to male infertility in the Han Chinese population under investigation, according to genotyping using RT-PCR[90]. Consequently, azoospermia and infertility may arise from a malfunction in miRNA processing. Infertility may also be linked to other abnormalities, such as dysregulation of the expression of specific miRNAs, SNPs in the miRNA binding region, or SNPs in the genes involved in miRNA synthesis[91]. In order to distinguish azoospermia by origin and identify samples with a high probability of SR, Larriba et al. showed that the expression levels of individual and/or combined canonical isoform miRNAs (miR-10a-5p, miR-146a-5p, miR-31-5p, and miR-181b-5p) in small extracellular vesicles have significant clinical value. Multivariate miRNA models in semen with small extracellular vesicles can identify those individuals with remaining spermatogenesis, even if no single miRNA demonstrated enough discriminating power to detect severe spermatogenic disorders with focal spermatogenesis[92].

6 FUTURE DIRECTIONS

Our primary goal is to minimize unsuccessful treatments by employing the most effective predictive technologies. New diagnostic methods are now being used in the diagnosis and treatment of infertility, particularly non-obstructive azoospermia (NOA). Recent large-scale genomic studies, including WES, have uncovered numerous genetic variations among individuals[93].

These methods can pinpoint every mutation responsible for singular tumors in genetic diseases, such as cancer. Recent findings also suggest that genetic abnormalities could underlie cases of idiopathic NOA. By determining each patient's genetic profile, precision medicine provides tailored, sophisticated treatments, ensuring optimal care based on individual genetic data[93].

Artificial intelligence (AI) and machine learning (ML) are playing a crucial role in advancing precision medicine, impacting various medical fields including reproductive health[94]. Techniques such as CRISPR/Cas9 gene editing are being explored to correct genetic anomalies that lead to testicular dysfunction, potentially restoring normal spermatogenesis by targeting genetic defects in sperm precursor or somatic testicular cells[93,95].

However, a major challenge with these advanced treatments, especially for NOA, is their potential high cost, making it imperative to develop strategies to make these therapies more accessible. Further research is necessary to establish precise thresholds for sperm retrieval likelihood[96].

7 CONCLUSION

The management of NOA has advanced significantly through recent discoveries in genetics, biomarkers, and microsurgical techniques. WES has emerged as a powerful diagnostic tool, uncovering genetic mutations such as CFTR, TEX11, and DMRT1, which influence spermatogenesis and may predict sperm retrieval success.

Non-invasive biomarkers, including seminal plasma proteins like TEX101 and microRNAs, offer promising potential for improving diagnostic accuracy and guiding treatment strategies. These tools can minimize unsuccessful surgical attempts and provide more tailored interventions. Despite these advancements, challenges remain, including cost, accessibility, and the need for large-scale validation of predictive models.

Future directions in NOA management, such as precision medicine, gene editing technologies like CRISPR/Cas9, and artificial intelligence, hold immense promise for enhancing sperm retrieval rates and developing innovative therapeutic approaches. Continued research and integration of these advancements into clinical practice are essential for optimizing outcomes for NOA patients.

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