Future prospects for the advancement of treatment of men with NOA: focus on gene editing, artificial sperm, stem cells, and use of imaging

Abstract

Nonobstructive azoospermia (NOA) affects about 60% of men with azoospermia, representing a severe form of male infertility. The current approach to manage NOA primarily involves testicular sperm retrieval methods such as conventional testicular sperm extraction (c-TESE) and microdissection testicular sperm extraction (micro-TESE). While combining testicular sperm retrieval with intracytoplasmic sperm injection (ICSI) offers hope for patients, the overall sperm retrieval rate (SRR) stands at around 50%. In cases where micro-TESE fails to retrieve sperm, limited options, like donor sperm or adoption, can be problematic in certain cultural contexts. This paper delves into prospective treatments for NOA management. Gene editing technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 (CRISPR/Cas9), hold potential for correcting genetic mutations underlying testicular dysfunction. However, these technologies face challenges due to their complexity, potential off-target effects, ethical concerns, and affordability. This calls for research to address key challenges associated with NOA management within the clinical settings. This also necessitate ongoing research essential for developing more sensitive diagnostic tests, validating novel treatments, and customizing current treatment strategies for individual patients. This review concluded that the future of NOA management may entail a combination of these treatment options, tailored to each patient’s unique circumstances, providing a comprehensive approach to address NOA challenges.

INTRODUCTION

Nonobstructive azoospermia (NOA) is considered as one of the most severe forms of male infertility, characterized by the absence of sperm in the semen due to minimally developed or unproduced cells in the testicles. NOA is more common compared with the obstructive azoospermia (OA) and it is affecting around 60% of men with azoospermia. This is making it one of the biggest pressing male infertility challenges estimated to be affecting 1% of the entire male population and 10%–20% of patients presenting with infertility.1 However, despite these challenges, there are limited treatment options for the ailment leaving andrologists and fertility specialists to face a significant challenge in managing NOA.

In the clinical settings, the current standards for diagnosing and treating primary NOA patients still involve conventional testicular sperm extraction (c-TESE) and microdissection testicular sperm extraction (micro-TESE). These procedures offer minimally invasive access to sperm, even in cases of severe spermatogenesis impairment.2 However, the success rate of sperm retrieval can vary depending on the underlying cause of NOA. The combination of testicular sperm retrieval with intracytoplasmic sperm injection (ICSI) has provided biological fatherhood opportunities for patients with primary NOA. This is because ICSI allows for the direct injection of a single sperm into an egg, bypassing fertilization hurdles that might exist in NOA cases and this combined approach has significantly improved chances of successful conception for couples struggling with male infertility due to NOA.3

Unfortunately, despite these approaches for managing NOA, reported overall sperm retrieval rate (SRR) is still only around 50%. This highlights the need for continued research to improve SRR and potentially develop minimally noninvasive diagnostic tools to better predict successful retrieval before surgery. Further, while ICSI offers a path to fatherhood for some men with NOA, the success rates for pregnancy and live birth remain lower compared to those with normal sperm counts. This calls for comprehensive counseling for couples considering this treatment option, including managing expectations and potential alternatives in case of failures after the treatment. This is because generally, after sperm retrieval by c-TESE or micro-TESE, to perform in vitro fertilization (IVF)/ICSI, the pregnancy rate per ICSI cycle was reported as 29%; and among these pregnancies, 24% resulted in live births, while 19% were lost due to biochemical pregnancies.3,4,5 This implies that while NOA presents a significant challenge for couples seeking fertility, ongoing research and improvement of the existing treatment options like TESE/ICSI could offer a beacon of hope for achieving pregnancy. Therefore, this review aims to introduce some possible future treatment prospects to the management of NOA that can be applied in research and clinical practice to address the needs and challenges of NOA management.

LITERATURE EVALUATION

There was an implementation of extensive literature evaluation conducted using NVivo software (https://lumivero.com/products/nvivo/) to explore the current research landscape for NOA and identify areas for further investigation. This analysis yielded valuable insights into key themes, relationships between concepts, research gaps, and treatment modalities. Table 1 summarizes these findings, including the prominent themes identified through word cloud analysis (Supplementary Figure 1 ^{(91.2KB, tif)} ) and the interconnectedness of concepts revealed by word similarity circles (Supplementary Figure 2 ^{(253.2KB, tif)} ). Additionally, Supplementary Figure 3 ^{(129.9KB, tif)} highlights crucial research gaps exposed by the matrix coding query table, alongside discussions on patient preferences, success rates of current treatments, and potential future therapeutic avenues.

Table 1.

Summary of literature evaluation on nonobstructive azoospermia

Aspect	Description	Figure
Method	Extensive literature evaluation using NVivo software	Not applicable
Key themes (word cloud)	Spermatogenesis, sperm retrieval, TESE, spermatozoa, and NOA	Supplementary Figure 1 (91.2KB, tif)
Relationships (words similarities circle)	Interconnectedness of concepts such as spermatogenesis and TESE	Supplementary Figure 2 (253.2KB, tif)
Research gaps (matrix coding query table)	Azoospermia, diagnosis, spermatogenesis, spermatozoa, TESE, and other treatments	Supplementary Figure 3 (129.9KB, tif)
Need for advanced techniques	Gene editing, artificial sperm, stem cell therapy, and imaging techniques	Not applicable
Patient preferences	Preference for natural conception with AGs	Not applicable
ICSI success rates	Lower success rates for NOA patients compared to OA patients	Not applicable
TESE/ICSI success rates	Limited success rates for unselected NOA patients	Not applicable
Causes of NOA	Often linked to genetic issues	Not applicable
Current treatments	Micro-TESE, varicocelectomy, and orchidopexy	Not applicable
Future potential treatments	Germ cells from stem cells	Not applicable
Hormonal therapy	Effective for hypogonadotropic hypogonadism	Not applicable
Spermatogenesis improvement	Use of gonadotropins and aromatase inhibitors	Not applicable

OA: obstructive azoospermia; NOA: nonobstructive azoospermia; TESE: testicular spermatozoa extraction technique; micro-TESE: microdissection TESE; ICSI: intracytoplasmic sperm injection; AGs: artificial gametes

GENE EDITING

Gene editing methods, namely clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 (CRISPR/Cas9), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), have the potential to expand therapeutic opportunities for men suffering from infertility by allowing for targeted genetic modifications. Gene editing technologies offer the potential to correct genetic defects in sperm precursor cells or even somatic cells within the testis, thus restoring spermatogenesis. Current research in gene editing for males with NOA focuses on correcting gene mutations responsible for testicular dysfunction. Many genes have been discovered to be essential for spermatogenesis and male fertility, such as spermatogenesis-associated 16 (Spata16), doublesex and mab-3-related transcription factor 1 (Dmrt1), dpy-19-like 2 (Dpy19l2), testis-specific gene A10 (Tsga10), a disintegrin and metalloproteinase with thrombospondin motifs 16 (Adamts16), ubiquitin-specific peptidase 26 (USP26), and poly(A)-specific ribonuclease-like domain-containing 1 (PNLDC1).6,7

The CRISPR/Cas9 system is the most well-known gene editing tool among various genetic editing methods.8 The CRISPR/Cas9 method allows modifying nucleic acids; through the mechanism of cutting and sewing the genome, it will be possible to identify the defective DNA locus and replace it with a functioning sequence, leading to the restoration of gene functions. According to Li et al.9, they used the CRISPR/Cas9 approach to restore fertility in mouse heterozygous mutant for a c-Kit gene (Kit^w/Kit^wv). Four months after transplantation, healthy offsprings mouse with wild type Kit gene or Kit^w mutation were obtained through natural mating. Through this study, a new treatment strategy with only three main steps, includes spermatogonial stem cell (SSC) isolation, in vitro gene editing (CRISPR/Cas9), and SSC transplantation, would bring hope for some NOA patients. This strategy is mainly aimed at the treatment of NOA caused by germ cell gene defects, especially in the NOA patients whose testes have an intact or partially intact stem cell niche and retained SSCs. In addition, men who have fertility but have other genetic disorders may be able to produce normal gametes and descendants by combining gene editing technology with assisted reproductive techniques.9

However, there are many difficulties and restrictions with gene editing use for the management of NOA due to the complex and polygenic nature of NOA making it challenging to identify all the relevant genetic factors contributing to the condition.8 According to Ormond et al.10 and Doudna,11 there are four significant problems remain unresolved with the use of gene editing in NOA. The first is whether it is possible to control the DNA-repair pathway choice without affecting the viability of clinically relevant cells. The second is how we assess the biological risk of nuclease-associated off-target activity. Third, the ethical issues surrounding germline editing are very concerning. Finally, the costs of gene editing techniques are so high making them out of reach of most couples battling infertility. Therefore, before being used within the clinical practice, gene-edited cells must also undergo a rigorous assessment of their long-term safety and effectiveness.7

While NOA remains a significant hurdle for couples seeking fertility and despite success of some conventional methods like TESE/ICSI which offer some hope in the face of limitations, a look at the current landscape shows potential avenues of gene editing tools such as Prime Editing. This novel gene editing tool which holds immense promise for NOA unlike CRISPR/Cas9, and is capable of introducing unintended mutations. Prime Editing allows for precise correction of disease-causing mutations as demonstrated in a recent study in fixing the luteinizing hormone/choriogonadotropin receptor (Lhcgr) mutation in mice, leading to restored fertility.12 Building on this success, it is possible to apply this approach in humans for fertility restoration and treatment of NOA.

The application of gene editing in male infertility has a significant limiting genetic component which affects diagnosis and management of male infertility. The standard diagnostic tools such as karyotype analysis and Y chromosome microdeletion testing offer some insight, revealing chromosomal abnormalities or missing Y chromosome segments that hinder sperm production in NOA. However, the genetic landscape of NOA is complex and diverse, with up to 15% or more of cases being attributed to genetic factors. Many of the genes involved are yet to be identified, highlighting the need for further research. Research has identified genetic factors associated with sperm development, hormone signaling, and motility as causes of NOA. In addition to genetic factors, diagnosing male infertility requires a comprehensive evaluation. This includes a semen analysis, hormone level testing, and a review of medical history and physical examination findings. In some cases, a testicular biopsy can provide a closer look at sperm development. While current tools are valuable, there are limitations to the potential use of gene editing approach. While testicular biopsies can offer a more detailed examination of sperm development, the application of gene editing techniques, such as Prime Editing, to correct specific infertility issues presents challenges. To effectively utilize these tools, a deeper understanding of the genetic landscape is essential. Researchers need to develop more targeted diagnostics and potential gene editing therapies to address the underlying causes of infertility in specific cases.

ARTIFICIAL SPERM

The use of artificial gametes, including artificial sperm and eggs, represent a novel concept in reproductive medicine.13 These gametes can be derived from mature stem cells from both male and female reproductive glands or by directly differentiating pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). After completing the process of meiosis, these cells become haploid, they can undergo fertilization and subsequent embryogenesis, transmitting their genetic and epigenetic information to the next generation.

Moreno et al.13 identified several groups of couples who could benefit from the use of artificial gametes. These groups include heterosexual couples in whom one or both partners have non-functional gametes (either congenital or acquired), patients with high-risk genetic disorders, older patients at risk of abnormal pregnancies after multiple failed ART attempts, individuals who experienced childhood cancer treatments leading to infertility, same-sex couples, and single parents. Previously, for these patient groups, donated gametes were the only option. However, many couples desire offspring with a genetic connection due to ethical, social, or emotional reasons. Thus, generating patient-specific gametes (especially artificial sperm) could be a potential treatment method for men with NOA in the future.

Most of the current knowledge about the development of artificial gametes in mammals comes from studies conducted primarily on mice. In 2006, Nayernia et al.14 first reported generating mouse pups by fertilization of murine oocytes with artificial sperm derived from mouse embryonic stem cells. However, these mice exhibited abnormalities and reduced life-span, primarily due to epigenetic defects accumulated during germline in vitro specification and maturation. Furthermore, differences in pluripotency status between mouse ESCs and human ESCs pose challenges in comparing their full developmental potential into functional germ cells. Hayashi et al.15 and Hayashi et al.16 reported that ESCs and iPSCs through the epiblast-like cell (EpiLC) state of mice can be differentiated into primordial germ cell-like cells (PGCLCs) with strong reproductive capabilities, giving rise to healthy mouse offspring when implanted in vivo. Another study has also documented successful differentiation of human pluripotent stem cells (hPSCs) into PGCLCs, and immature human egg cells from iPSCs, thereby differentiating into fully functional human sperm and egg cells, respectively.17

However, long-term safety remains a crucial factor to consider before implementing artificial gametes in clinical practice. This is because current knowledge about artificial gametes is primarily derived from mouse models, which require careful extrapolation to humans. This is coupled with the fact that present biochemical observations and tests are insufficient to assess whether cells can ensure normal human development from fertilization to adulthood.13 Therefore, ethical and societal issues associated with the use of artificial gamete must be carefully considered, regardless of its potential benefits. Further, legal regulations and social standards need to be established to ensure the ethical integrity of producing and utilizing artificial gametes, eliminating unnecessary barriers in their research and application. In this regard, issues such as (1) consent from both the cell donor and recipient, (2) the safety of methods used for ESC/iPSC derivation and differentiation, (3) human cloning capabilities, and (4) the potential for illegal or unethical donation leading to the birth of a child, need to be recognized and resolved. This implies that there is still a considerable journey ahead before artificial gametes find practical application in clinical settings.

SSCs

This is a new direction in medicine, also known as regenerative medicine, which involves the use of stem cell therapy to restore fertility in men with NOA.18 Stem cells can be derived from various cell sources, including SSCs, ESCs, iPSCs, and mesenchymal stem cells (MSCs). Once transplanted, these undifferentiated cells can self-renew and differentiate into various specialized cell types.19 The SSC, a unique type of male germ stem cell in mammals similar to hematopoietic stem cells, has the ability to self-renew, differentiate, and regenerate the seminiferous tubules, replenishing the entire depleted sperm cell lineage when transplanted.

Lim et al.20 identified SSC in the testes of NOA patients through TESE, subsequently isolating and cultivating them under laboratory conditions. The SSCs could be differentiated into male germ cells with development potential after extended culture in the laboratory conditions. The use of ESCs, iPSCs, and MSCs is another possible source of SSCs. Regarding the injection of isolated SSCs, specific injection sites have been studied, including the seminiferous tubules, rete testis, and vas deferens, with ultrasound-guided injections into rete testis appearing to be the most promising technique to date.21 Following injection, SSCs migrate to the basal membrane of the seminiferous tubules. One of the advantages of this method over testicular tissue grafting is its potential to restore fertility without the need for assisted reproductive technologies.22

The use of autologous stem cells is more ethical, safer, and non-immunogenic, making their clinical applications in the future more promising.23 Research results on SSC transplantation therapy in animals, especially in rodent models, for the restoration of spermatogenesis are promising. However, the clinical application of SSC therapy still requires optimization in several areas, including SSC culture techniques, cancer cell contamination in cancer patients, SSC preservation, ideal transplantation sites, safety during transplantation, transplantation frequency, and effective injection volume and dosage. Furthermore, due to the relatively low number of SSCs in the testes, successful isolation and cultivation remain challenging. Stem cell therapy, specifically SSC therapy, holds significant potential for treating NOA patients; however, further clinical research is essential to overcome limitations and challenges.

USE OF IMAGING

NOA is described as a condition in which there is an absence of sperm in the semen due to problems with the production of sperm within the testes.24 By implications, the absence of the sperm in the semen could be due to the testicular complications caused by the factors such as genetic disorders, hormonal imbalances, or exposure to certain medications and toxins. Therefore, NOA is a significant cause of male infertility which has been a challenging condition to diagnose and treat.25 Although the ailment could be a common male infertility challenge, the prevalence varies depending on the population being studied and the diagnostic criteria used in the population.

Hence, given the prevalence of NOA, there is a need for the early and accurate diagnosis for effective treatments to address this challenging condition to avoid highly invasive fertility treatment for infertile males. The use of imaging could play an essential role in this regard, because diagnosis and treatment of NOA using imaging can serve as part of the techniques that can provide basis for a better assessment of the structure and function of the testes, identify any abnormalities or masses present in the testes which can cause infertility, and determine the severity of the infertility condition.26 As a result of this, different imaging applications can be used as imaging modalities in the evaluation of NOA such as ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT). These can fulfill the criteria of noninvasive treatment, for example, ultrasound imaging technique that uses sound waves to produce images of the internal structures of the body can be used with little or no invasiveness. It is the most used imaging modality for evaluating the testes in patients with NOA.27 The use of ultrasound can help detect any abnormalities such as testicular atrophy or masses that may be causing the infertility condition. It can also be used to measure the size of the testis and assess blood flow to the testes, which can provide important information about the severity of the condition. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ultrasound for the diagnosis of varicocele are 90%–100%, 90%–100%, 95%–100%, and 95%–100%, respectively.28 For undescended testicles, the sensitivity, specificity, PPV, and NPV of ultrasound for the diagnosis are 95%–100%, 95%–100%, 99%–100%, and 99%–100%, respectively.29 For potential testicular tumors, the sensitivity, specificity, PPV, and NPV of ultrasound for the diagnosis are 90%–95%, 95%–100%, 95%–100%, and 95%–100%, respectively.30

MRI is another imaging modality that can be used to evaluate the testes in patients with NOA because it uses powerful magnetic fields and radio waves to produce detailed images of the internal structures of the body. It could be particularly useful in assessing the testicular blood supply and detecting small masses or lesions that may be difficult to identify using ultrasound scanning.31 While MRI cannot directly check for the viability of testicular tissue, it can be used for measuring indirect markers of viability and to identify any underlying abnormalities that may be affecting sperm production. This information can be helpful in determining the potential for successful sperm retrieval in patients with NOA. MRI can measure apparent diffusion coefficient (ADC), testicular choline and lipids, creatine, and other metabolites. These markers can be used to predict TESE success and provide information about potential sperm retrieval success, as spermatogenesis occurs only in viable cells.32 The use of CT scanning is another imaging modality that may be used to evaluate the testes in patients with NOA. Although MRI is the preferred imaging modality for testicular tumor detection in patients with NOA due to its superior sensitivity and specificity compared to CT and its lack of ionizing radiation, CT could be reserved for cases where MRI is inconclusive or where there is suspicion of a nontumorous condition.33 In summary, imaging plays a crucial role in the diagnosis and treatment of NOA. While ultrasound is the most used imaging modality for evaluating the testes in patients with NOA, MRI and CT can also provide important information in certain cases.

Imaging techniques such as photoacoustic imaging (PAI) and diffusion-weighted imaging (DWI) are current techniques that are used for the diagnosis of NOA because of their capability to differentiate between OA and NOA. This was reported that parameters derived from multimodel diffusion-weighted imaging have the potential to noninvasively differentiate OA and NOA, while discussing the potential of photoacoustic imaging in biomedical applications, including NOA diagnosis.34,35 Furthermore, Attia et al.36 provides an overview of the clinical applications of photoacoustic imaging, including NOA. This technique is capable of being used in vascular and musculoskeletal imaging, suggesting that photoacoustic and DWI techniques have potential for differential diagnosis of azoospermia.

In principle, PAI and DWI are two different imaging modalities that can be used to diagnose NOA. Although they are both noninvasive techniques, they work based on different principles and provide different types of information. PAI is an emerging imaging modality that combines the high spatial resolution of ultrasound imaging with the molecular specificity of optical imaging. PAI uses laser pulses to generate acoustic waves in the tissue, which are then detected by an ultrasound transducer. The resulting images provide information about the absorption properties of tissue, which can be used to identify the structures such as blood vessels and chromophores such as molecules that absorb light and haemoglobin.36

In the context of NOA diagnosis, PAI can be used to assess testicular blood flow and detect small blood vessels that may be associated with viable sperm production. Studies have shown that PAI can distinguish between normal testicular tissue and testicular tissue with impaired spermatogenesis, suggesting that it may be a useful tool for diagnosing NOA.34,35 DWI is an MRI technique that measures the movement of water molecules within tissues. DWI images are generated by applying gradients to the magnetic field during the MRI scan, which causes water molecules to move preferentially in certain directions. The resulting images provide information about tissue microstructure and can be used to detect abnormalities such as tumors, inflammation, or areas of restricted diffusion.37

NOA diagnosis can be assessed using DWI to check the micro-structure of the testicular tissue and identify regions with impaired spermatogenesis. Studies have shown that DWI can distinguish between normal testicular tissue and testicular tissue with NOA and can also predict the success of sperm retrieval in patients undergoing c-TESE or micro-TESE.34,35,37 In summary, PAI and DWI are two different imaging modalities that can be used to diagnose NOA. While PAI provides information about the absorption properties of tissue, DWI measures the movement of water molecules within tissues. Both techniques have shown promise in detecting abnormalities associated with NOA and may be useful in guiding treatment decisions. However, further research is needed to validate their use in clinical practice.38

Although the use of imaging presents a fascinating future for diagnosis and management of NOA, there are some challenges. While imaging techniques can identify abnormalities within the testes, they may not always pinpoint the exact cause of NOA. For instance, both ultrasound and MRI can detect masses, but they cannot definitively differentiate between a benign cyst and a cancerous tumour, which can lead to unnecessary biopsies or surgeries. Therefore, there is a need for research in this regard, by establishing a correlation between these imaging observations and quantifiable body markers for a more precise diagnostic outcome. However, newer imaging modalities such as PAI and DWI hold promise for NOA diagnosis, although these techniques are still under development and require further research to validate their accuracy and establish standardized protocols for clinical use.

Furthermore, there are ethical concerns of imaging use in NOA diagnosis. A leading concern under this category is the psychological impact of imaging procedures, especially those involving radiation like CT scans can cause anxiety and stress for patients. Hence, it is crucial to weigh the benefits of the information that will be gained using these procedures against the potential psychological burden. Finally, medical imaging generates sensitive personal data raising privacy concerns. Hence, to protect patient privacy and data security, robust procedures must be developed to prevent unauthorized access or misuse of this information.

ADDRESSING RESEARCH AND CLINICAL NEEDS AND CHALLENGES

There is a need for a better understanding of the underlying causes of NOA because this is essential for developing effective treatments. Specifically, there is a need for the development of more sensitive and specific diagnostic tests for NOA to ensure that men are correctly diagnosed and receive the appropriate treatment.24,39,40,41 The development of more effective and feasible treatments for NOA could also require research focusing on gene editing, artificial sperm, stem cell therapies, or new drug therapies, which are required validation for clinical needs of NOA treatment. While there is an ambitious research need associated with NOA, there are also huge challenges militating against achieving these ambitions including the ethical concerns surrounding the use of some new technologies, such as gene editing. It is important to address these concerns before these technologies can be widely used in the clinic. It is important to ensure that any new treatments for NOA are safe and effective through series of validation and careful preclinical and clinical testing. Since technologies for treating NOA are in the future, it is possible that new treatments for NOA are likely to be expensive. Therefore, it is important to find ways to make these treatments affordable for all families in need of the treatments in future. Finally, there is also a need to address how future treatment prospects can address the needs and challenges mentioned in the preceding paragraph.

The future treatment prospects of technologies such as gene editing, artificial sperm, and stem cells have the potential to address many research and clinical needs and challenges facing the management of NOA. For example, gene editing could be used to correct genetic mutations associated with NOA, while artificial sperm therapies could offer new and effective treatment option for men with NOA who are not eligible for other treatments, such as TESE due to clinical reasons or risk of pre- and post-TESE complications. Stem cell therapies could also offer a new and effective treatment option for men with NOA. The baseline research need is to note that these technologies are still in their early stages of development; therefore, there are needs for research focusing on preclinical and clinical validation of some of the techniques.

Hence, it is important to continue research into these technologies to support the development of new diagnostic tests and treatments for NOA. The research focusing on NOA could also consider testing and validation of combination of two or more techniques to achieve better results. For example, gene editing could correct the genetic mutations causing NOA. Afterward, stem cell therapy could be applied to restore sperm production. It is also possible that imaging technologies could be used to guide or monitor the use of other treatments. For example, imaging could be used to identify the best location to transplant stem cells into the testes. These areas required more research to determine how to combine these treatments to achieve the best results for diagnosing and treating men with NOA.

CONCLUSION

The management of NOA presents a significant challenge in the field of male infertility. This study explored various future treatment prospects, including gene editing, artificial sperm, stem cells, and the use of imaging techniques to address the needs and challenges of NOA management. The gene editing technology, particularly CRISPR/Cas9, holds great promise in correcting genetic mutations responsible for testicular dysfunction in NOA. While the potential is substantial, it is essential to overcome the challenges related to the complex and polygenic nature of NOA, the control of DNA-repair pathway choice, the assessment of off-target activity, and ethical considerations. Moreover, affordability and long-term safety must be addressed before widespread clinical application.

Artificial gametes provide a potential solution for patients with NOA who wish to have biological offspring, although they are still in the early stages of development. These therapies have the potential to address the needs of patients with genetic conditions or those who have experienced infertility due to cancer treatment or other factors. However, ethical, legal and societal considerations must be carefully weighed, and long-term safety needs rigorous evaluation. Stem cell therapies, particularly using SSCs, offer a regenerative medicine approach to restore fertility in NOA patients. These therapies are in the early stages of development and have shown promise in animal models. However, clinical translation requires addressing challenges related to isolation, culture techniques, safety, and effectiveness.

Imaging technologies, including ultrasound, MRI, and CT, play a crucial role in the diagnosis of NOA. Techniques such as PAI and DWI have shown potential for differentiating between OA and NOA, aiding in diagnosis. The comparison of these future treatment prospects highlights their different stages of development and feasibility for clinical use. While gene editing and artificial sperm therapies are advancing, stem cell therapies and imaging are already feasible for diagnosis but not yet for treatment. Addressing the research and clinical needs and challenges in NOA management is essential for the successful implementation of these treatment prospects. Continued research into the underlying causes of NOA, the development of more sensitive diagnostic tests, and the validation of new treatments are vital. Additionally, addressing ethical concerns, ensuring safety and efficacy, and finding ways to make these treatments affordable are necessary steps. In future, a combination of these treatment prospects, customized to each patient’s unique situation, may offer the most comprehensive and effective approach to managing NOA. However, rigorous research, validation, and careful consideration of ethical and societal implications will be paramount to ensure that these treatments fulfill their potential and benefit in those facing the challenges of NOA.

AUTHOR CONTRIBUTIONS

ABS carried out the initial draft of the paper outline and revised the outline based on other authors’ comments. MNT conducted critique of the manuscript layout with a focus on relevance of content in clinical settings. WZ guided the writing and editing of the paper drafts. All authors contributed to writing, review and editing, read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

Key themes in NOA research highlighted by Nvivo word cloud analysis. The figure present word cloud generated from the literature reviewed using Nvivo software, illustrating the key themes in current NOA research. Prominent terms such as spermatogenesis, sperm retrieval, TESE, spermatozoa, and NOA stand out, reflecting the primary areas of focus within the field. NOA: non-obstructive azoospermia; TESE: testicular spermatozoa extraction technique.

Supplementary Figure 2

Word similarities circle illustrating relationships between key terms in NOA research. The figure displays a word similarities circle that visualizes the relationships between key terms and common words found in the reviewed literature on NOA. This diagram reveals the intricate connections between central themes such as spermatogenesis, sperm retrieval, and TESE, emphasizing how these concepts are interrelated in the context of NOA research. The proximity and linkage of words within the circle indicate the degree of association between various terms, highlighting the interdependency of biological processes and techniques crucial for advancing treatment strategies for NOA. This visual tool aids in comprehending the complex network of ideas and methodologies that underpin current research efforts. NOA: non-obstructive azoospermia; TESE: testicular spermatozoa extraction technique.

Supplementary Figure 3

Heatmap identifying research gaps in NOA studies. The heatmap resulted from a matrix coding query analysis, highlighting research gaps in the field of NOA. The heatmap identifies areas requiring further exploration, particularly in the themes of azoospermia, diagnosis, spermatogenesis, spermatozoa, TESE, and other treatments. The varying intensities of colour represent the concentration of research efforts, with lighter areas indicating topics that are less frequently addressed and thus signify gaps in the current literature. This visual representation provides a clear overview of where additional research is needed, guiding future investigations to focus on these under-explored aspects of NOA treatment and management.3,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57 NOA: non-obstructive azoospermia; TESE: testicular spermatozoa extraction technique.