Single-cell transcriptomics reveals a spermatogonial stem cell-centered spermatogenic microenvironment: Pathophysiological mechanisms and therapeutic targets in male infertility

Abstract

Male infertility affects approximately 17.5% of the global reproductive-age population, with disruption of the testicular spermatogenic microenvironment - particularly the spermatogonial stem cell niche - representing a central pathogenic mechanism. Single-cell RNA sequencing has enabled unprecedented resolution of cellular heterogeneity within the human testis; however, its translational value remains constrained by population data imbalance and limited integration across molecular layers. Here, we synthesize single-cell RNA sequencing studies published between January 2018 and September 2025 to construct a microenvironment-centered mechanistic framework linking inflammaging, NLRP3 inflammasome activation, and progressive spermatogonial stem cell depletion in non-obstructive azoospermia, supported by convergent single-cell, bulk transcriptomic, and emerging preclinical evidence. We further discuss transgenerational epigenetic effects mediated by sperm small non-coding RNAs and critically evaluate translational barriers, including ancestry bias, technical limitations of single-cell platforms, and ethical challenges of germline intervention. We propose that future clinical translation will require multiethnic cohort construction and artificial intelligence-driven multi-omics integration to enable stem cell-oriented precision diagnosis and therapy for male infertility.

Key Words: Single-cell transcriptomics; Spermatogonial stem cell; Testicular microenvironment; Male infertility; NLRP3 inflammasome

Core Tip: This review integrates single-cell transcriptomics data from 2018 to 2025 to reconstruct the pathological landscape of male infertility, identifying the spermatogonial stem cell niche as a core therapeutic target. We elucidate a novel mechanistic axis linking “inflammaging” and NLRP3 inflammasome activation to spermatogonial stem cell depletion in non-obstructive azoospermia. Furthermore, we critically evaluate translational barriers, including geographic data bias and technical limitations, and propose a precision medicine framework integrating multiethnic cohorts to bridge the gap between single-cell atlases and clinical andrology.

INTRODUCTION

Male infertility affects approximately 15%-20% of couples worldwide[1,2] and accounts for nearly half of all cases of reproductive failure[3], posing a substantial yet often underestimated public health burden. In addition to genetic and endocrine factors, increasing evidence indicates that disruption of the testicular spermatogenic microenvironment is a decisive driver of impaired spermatogenesis. In particular, the spermatogonial stem cell (SSC) niche plays a central role in maintaining continuous sperm production, as SSC self-renewal and differentiation are tightly regulated by surrounding Sertoli cells, Leydig cells, immune components, vascular structures, and extracellular matrix-mediated signaling[4-6].

The advent of single-cell RNA sequencing (scRNA-seq) has revolutionized the study of human spermatogenesis[7-10] by enabling high-resolution characterization of cellular heterogeneity and developmental trajectories that cannot be resolved by conventional bulk transcriptomic approaches. Large-scale single-cell atlases of the human testis have delineated distinct germ cell states, somatic cell subtypes, and dynamic intercellular communication networks under both physiological and pathological conditions[11-18]. These advances have shifted the conceptual framework of male infertility from a purely germ cell-intrinsic disorder toward one involving coordinated dysfunction of the stem cell-supporting microenvironment.

However, several critical gaps remain insufficiently integrated. Most existing reviews primarily emphasize atlas construction or germ cell differentiation programs, whereas the mechanistic consequences of microenvironmental remodeling for SSC maintenance and exhaustion remain incompletely synthesized. Emerging evidence suggests that chronic low-grade inflammation associated with aging and metabolic stress (“inflammaging”) and activation of the NLRP3 inflammasome contribute to Sertoli cell dysfunction, blood-testis barrier disruption, and progressive SSC depletion, particularly in non-obstructive azoospermia (NOA)[19-21]. Moreover, current scRNA-seq datasets are heavily skewed toward limited geographic populations, raising concerns regarding ancestry-dependent molecular signatures and translational generalizability[22]. In this review, we integrate scRNA-seq studies published between 2018 and 2025 with spatial transcriptomic and epigenetic evidence[23-38] to establish a microenvironment-centered and SSC-focused pathogenic framework, elucidate the inflammaging-NLRP3-SSC axis, and critically evaluate key translational barriers to precision diagnosis and therapy in male infertility.

METHODOLOGICAL FRAMEWORK

This review was conducted as a narrative and integrative synthesis of recent advances in human testicular single-cell research, rather than a formal systematic review[39]. We focused on original studies published between January 2018 and September 2025 that applied scRNA-seq and, where available, spatial transcriptomic technologies to characterize the cellular composition and microenvironmental regulation of human spermatogenesis. Literature searches were performed primarily in PubMed and Web of Science using combinations of keywords including “single-cell RNA sequencing”, “testis”, “spermatogenesis”, “spermatogonial stem cell”, and “male infertility”.

Studies were included if they: (1) Analyzed human testicular tissue; (2) Generated original scRNA-seq or spatial transcriptomic datasets; and (3) Provided biologically interpretable results relevant to germ cell development, somatic cell function, or microenvironmental regulation, with particular attention to SSCs and NOA. We excluded studies based exclusively on animal models, technical reports without substantive biological analysis, conference abstracts, and narrative commentaries. Reviews were used only to contextualize findings but were not considered primary sources of evidence.

To address population-related bias, we recorded the reported geographic origin of study cohorts whenever available and considered this factor when interpreting conserved vs population-specific molecular signatures. Given the substantial heterogeneity across sequencing platforms, tissue dissociation protocols, and computational pipelines, no quantitative meta-analysis was attempted. Instead, we adopted a qualitative comparative approach, emphasizing reproducible cellular patterns, convergent signaling pathways, and mechanistic consistency across independent datasets[40,41].

OVERVIEW OF SINGLE-CELL TRANSCRIPTOMIC TECHNOLOGIES

scRNA-seq in testicular research

scRNA-seq has become the principal technology for dissecting cellular heterogeneity within the human testis, enabling unbiased identification of rare cell populations, developmental intermediates, and disease-associated transcriptional states. In contrast to bulk transcriptomics, which averages signals across mixed cell types, scRNA-seq resolves cell type-specific gene expression patterns and reconstructs differentiation trajectories, thereby providing a powerful framework for studying SSCs, somatic support cells, and their coordinated regulation within the spermatogenic niche[42,43].

Despite these advantages, several technical limitations should be acknowledged when interpreting scRNA-seq-based findings. Tissue dissociation can introduce transcriptional stress responses and preferentially affect fragile cell populations, while RNA dropout and sequencing depth variability may obscure low-abundance transcripts[44]. In addition, batch effects arising from differences in sample preparation, sequencing platforms, and computational pipelines remain a major source of cross-study heterogeneity, although integration algorithms such as Seurat-based alignment and Harmony correction have partially mitigated these issues[45]. These factors underscore the importance of cautious biological interpretation and cross-validation across independent datasets. Here, “pseudotime” refers to a computationally inferred ordering of individual cells along a continuous developmental trajectory, for example from undifferentiated spermatogonia and SSCs to spermatocytes and post-meiotic spermatids[46,47].

Spatial transcriptomics and integration with scRNA-seq

More recently, spatial transcriptomic technologies have complemented scRNA-seq by preserving the anatomical context of gene expression, enabling mapping of cellular interactions within seminiferous tubules and interstitial compartments[48]. This spatial information is particularly valuable for characterizing the organization of the SSC niche and its interactions with supporting somatic and immune cell populations[49]. However, current platforms remain constrained by limited spatial resolution, signal mixing within capture spots, and substantial computational complexity during data integration. Consequently, most available studies adopt a combined strategy in which scRNA-seq defines cell identities and spatial transcriptomics provides positional information[50,51]. Together, these technologies form the methodological foundation for investigating microenvironmental remodeling in male infertility, while their technical constraints highlight the need for continued methodological refinement[52].

SINGLE-CELL ATLAS OF THE HUMAN TESTIS AND MICROENVIRONMENTAL HETEROGENEITY

Cellular composition of the human testis revealed by scRNA-seq

Single-cell transcriptomic profiling has delineated the major cellular constituents of the adult human testis, including SSCs and differentiating germ cells, Sertoli cells, Leydig cells, peritubular myoid cells, endothelial cells, and diverse immune populations. Within the germline compartment, scRNA-seq has resolved discrete transcriptional states spanning undifferentiated spermatogonia, differentiating spermatogonia, spermatocytes, and post-meiotic spermatids, enabling reconstruction of spermatogenic trajectories with unprecedented resolution[11,13,14]. Importantly, SSCs are now recognized as a transcriptionally heterogeneous population whose fate is tightly regulated by niche-derived paracrine and metabolic cues[4,5].

Heterogeneity of somatic and immune cell populations in the spermatogenic niche

Beyond germ cells, single-cell atlases have uncovered substantial heterogeneity among somatic and immune cell compartments. Sertoli cells exhibit multiple functional states associated with metabolic support, immune modulation, and blood-testis barrier maintenance, whereas Leydig cells display variable steroidogenic and inflammatory transcriptional profiles[16,19,21]. Testicular macrophages, T cells, and mast cells form an immunologically specialized microenvironment that balances immune tolerance with local inflammatory surveillance[53-55]. These findings highlight that spermatogenesis is orchestrated by a complex multicellular network rather than by germ cells in isolation.

Microenvironmental remodeling in male infertility

Comparative analyses between fertile controls and patients with NOA have consistently demonstrated profound microenvironmental remodeling, characterized by altered Sertoli cell maturation states, increased inflammatory signaling, endothelial dysfunction, and depletion or transcriptional reprogramming of SSC populations[19-21]. Disruption of intercellular communication pathways, including cytokine, growth factor, and extracellular matrix signaling, further compromises niche stability[4,5]. Collectively, these alterations suggest that failure of spermatogenesis reflects a breakdown of coordinated microenvironmental regulation, providing the cellular context for subsequent mechanistic links to inflammaging and inflammasome activation. Detailed information on study design, platforms, and geographic distribution is provided in Table 1.

Table 1 Representative human testis single-cell RNA sequencing studies (2018-2025).

Ref.	Journal	Sample size & type	Platform (cells analyzed)	Key findings	Clinical relevance	Geographic location
Wang et al[23], 2018	Cell Stem Cell	n = 3 normal; adult donors	10X Genomics (approximately 6490 cells)	First comprehensive atlas of human testis; identified 5 germ cell states; characterized niche-specific gene signatures; discovered PLPPR3+ spermatogonia subtype; state 0 SSC defined	Established baseline reference for normal spermatogenesis; identified SSC markers for potential therapeutic targeting	China (Beijing)
Hermann et al[24], 2018	Cell Reports	n = 7 normal; adult testis (18-36 years)	Fluidigm C1 + 10X (approximately 62000 total cells, 182 SSCs)	Focused analysis of SSC heterogeneity; defined ID4+/UTF1+ SSC population; revealed FGFR3 as functional SSC marker; complete spermatogenesis transcriptome mapping	Potential for SSC isolation and expansion for fertility preservation	United States (Pittsburgh)
Guo et al[25], 2018	Cell Research	n = 3 normal; adult males	10X Genomics (approximately 6500 cells)	Adult human testis transcriptional cell atlas; state 0 SSC identification; RNA velocity reveals spermatogonial plasticity; epigenetic landscape analysis	Understanding SSC self-renewal and differentiation balance; SSC-specific marker identification	United States (UT)
Sohni et al[26], 2019	Cell Reports	Neonatal (n = 2) and adult (n = 3) human testis	10X Genomics (approximately 17000 cells)	Defined neonatal and adult human testis at single-cell level; identified four undifferentiated spermatogonia clusters; characterized protein markers for primitive SPG state; mapped timeline from PGCs to adult SPG	SSC-enriched cell subset purification; understanding developmental trajectories	United States (CA)
Guo et al[27], 2020	Cell Stem Cell	n = 4 normal; juvenile males (7-14 years)	10X Genomics (approximately 10000 cells)	Pubertal testis development atlas; identified common pre-pubertal progenitor for Leydig and myoid cells; two distinct pre-pubertal Sertoli cell states; testosterone roles via transfemale testis analysis	Insights into pubertal developmental disorders; critical windows for testicular maturation	United States (UT)
Guo et al[28], 2021	Cell Stem Cell	n = 10 normal; embryonic to infant (6 weeks to 5 months)	10X Genomics (approximately 32500 cells)	Fetal testis development atlas; sertoli and interstitial cells from common progenitor at 6-7 weeks; PGCs transition to state 0-like cells at 14-16 weeks; somatic niche specification precedes germline transition	Understanding fetal gonadal development; insights into congenital testicular disorders	United States (UT/UCLA)
Shami et al[29], 2020	Developmental Cell	n = 8 normal; young adults (17-25 years)	10X Genomics (> 35000 cells)	Comprehensive human-mouse comparison; species-specific gene expression programs; extended meiotic progression in humans; novel markers for meiotic stages	Caution for translating mouse models; human-specific therapeutic targets	United States (MI)
Zhao et al[30], 2020	Nature Communications	n = 10 NOA, n = 10 normal (infant to adult)	10X Genomics (> 88000 cells)	First large-scale NOA single-cell analysis; three-stage Sertoli cell maturation roadmap; Sertoli cell maturation blockade in iNOA; inflammatory microenvironment signature; loss of SSC niche factors (GDNF, FGF2)	Identified targetable pathways in NOA; potential biomarkers for diagnosis; Sertoli cell-centered therapeutic approach	China (Nanjing)
Di Persio et al[31], 2021	Cell Reports Medicine	n = 5 NOA, n = 5 cryptozoospermia, n = 5 normal	10X Genomics (> 24000 cells)	Comparative analysis of impaired spermatogenesis; major alterations in cryptozoospermia SSC compartment; increased PIWIL4+ undifferentiated spermatogonia; transcriptional switch driven by EGR4 overexpression; reduced UTF1+ reserve spermatogonia (Adark)	Distinct pathological mechanisms guide personalized treatment	Germany (Münster)
Alfano et al[32], 2021	Nature Communications	n = 8 infertile, n = 3 normal (TESE samples)	Smart-seq2 (1246 cells)	Aging, inflammation and DNA damage in somatic niche; idiopathic germ cell aplasia pathology; senescence and immune activation in Sertoli cells; testicular M1 macrophage polarization; chronic inflammation signature	Hormonal therapy optimization; testosterone production defects in infertility	Italy (Milan)
Chen et al[33], 2021	Cell Reports	n = 3 normal adult testis (mouse + human)	10X Genomics + slide-seq spatial transcriptomics	Spatial transcriptomic atlas of mammalian spermatogenesis; near-single-cell resolution spatial gene expression; identified spatially patterned genes along seminiferous tubules; Habp4 as chromatin remodeling regulator; compared WT vs diabetic mouse testis	Understanding spatial organization for targeted therapy; zone-specific molecular signatures	United States (Harvard/Broad Institute)
Mahyari et al[34], 2021	American Journal of Human Genetics	n = 3 Klinefelter syndrome, n = 3 normal controls	10X Genomics (approximately 13000 cells)	First single-cell analysis of Klinefelter testis; identified immature sertoli and Leydig cells; revealed pro-inflammatory macrophage enrichment; discovered altered microenvironment in KS	Understanding Klinefelter-specific pathology; potential therapeutic targets for KS	Estonia (Tartu)
Nie et al[35], 2022	Developmental Cell	n = 12 normal (young + older), n = 6 with elevated BMI	10X Genomics (> 44000 cells)	Human testis aging study; age-related changes in spermatogenesis and somatic cells; altered pathways: Inflammation, metabolic signaling in Sertoli cells, hedgehog/testosterone in Leydig cells; BMI correlation with dysregulation in older men; cell-cell communication changes during aging	Age-specific fertility preservation strategies; BMI management for reproductive health in aging men	United States (UT)
Di Persio and Neuhaus[36], 2023	Human Reproduction	Review article covering multiple NOA subtypes	N/A (comprehensive review)	Comprehensive scRNA-seq review; novel concepts on SSC subtypes (state 0, state 1); SSC niche crosstalk mechanisms; transcriptional alterations in NOA, cryptozoospermia, Klinefelter syndrome, AZF deletions	Precision medicine approaches based on genetic etiology; marker genes for SSC subsets	Germany (Münster)
Amodio et al[37], 2025	Nature Communications	NOA and OAT patients vs controls	scRNA-seq + multiparameter phenotyping	Different infertility subtypes correlated with T cell exhaustion/senescence signatures; young infertile men show pro-inflammatory milieu (similar to healthy elderly men); immune alterations in seminal fluid and peripheral blood; interferon-gamma and -alpha response upregulation	Identifies infertility-specific immune signatures; suggests personalized immunomodulatory treatment strategies; links infertility to systemic health	Italy (Milan)
Cui et al[38], 2025	Nature Aging	n = 35 normal donors (21-69 years)	10X Genomics (214369 cells)	Machine learning reveals somatic cells show stronger aging response than germ cells; two waves of aging-related changes: Age 30-39 years old (peritubular cells, basement membrane thickening), Age 50-59 years old (functional changes in Leydig cells and macrophages); BMI impact on spermatogenic capacity after age 45	Age-specific fertility preservation; potential diagnostic markers and therapeutic targets; BMI management critical for reproductive health in aging men	China (multi-center)

This table summarizes representative human testicular single-cell RNA sequencing and spatial transcriptomic studies included in this review, including sequencing platforms, sample types, major findings, and cohort origins. Notably, currently available datasets exhibit a pronounced geographic imbalance, with most studies derived from European and East Asian populations and limited representation from South Asia, Africa, and Latin America. This uneven distribution may restrict cross-population generalizability of molecular signatures and therapeutic targets and highlights the need for broader multiethnic cohort construction in future research. PLPPR3: Plasma membrane protein phospholipid phosphatase-related protein 3; SSC: Spermatogonial stem cell; FGFR3: Fibroblast growth factor receptor 3; SPG: Spermatogonia; PGCs: Primordial germ cells; NOA: Non-obstructive azoospermia; iNOA: Idiopathic non-obstructive azoospermia; GDNF: Glial cell line-derived neurotrophic factor; FGF2: Fibroblast growth factor 2; EGR4: Early growth response 4; UTF1: Undifferentiated cell transcription factor 1; TESE: Testicular sperm extraction; WT: Wild type; KS: Klinefelter syndrome; BMI: Body mass index; scRNA-seq: Single-cell RNA sequencing; AZF: Azoospermia factor; OAT: Oligoasthenoteratozoospermia.

MICROENVIRONMENTAL DYSFUNCTION IN NOA

Single-cell landscape of NOA

Non-obstructive azoospermia represents the most severe form of spermatogenic failure and is a major cause of male infertility[3]. Single-cell transcriptomic studies have revealed that NOA is not merely characterized by the absence of mature germ cells, but rather by profound reorganization of the entire testicular microenvironment[19-21]. Compared with fertile controls, testes from patients with NOA exhibit marked alterations in Sertoli cell maturation states, impaired metabolic support programs, endothelial dysfunction, and expansion of inflammatory immune cell subsets. A schematic overview of these interconnected pathological mechanisms is provided in Figure 1.

Figure 1 Multi-layered pathological mechanisms of male infertility revealed by single-cell transcriptomics. The pathological cascade initiates with testicular niche dysfunction (Sertoli cell maturation arrest, M1-like macrophage polarization, Leydig cell decline), amplified by endogenous aging and environmental exposures, converging on NLRP3 inflammasome and mammalian target of rapamycin pathway activation. This leads to spermatogonial stem cell depletion and spermatogenic failure. Genetic variants and transgenerational epigenetic inheritance via sperm small non-coding RNAs further modulate susceptibility. Red denotes pathological states; blue indicates therapeutic intervention points. SSC: Spermatogonial stem cell; NOA: Non-obstructive azoospermia.

The pathological cascade initiates with testicular niche dysfunction (Sertoli cell maturation arrest, M1-like macrophage polarization, Leydig cell decline), amplified by endogenous aging and environmental exposures, converging on NLRP3 inflammasome and mammalian target of rapamycin pathway activation. This leads to SSC depletion and spermatogenic failure. Genetic variants and transgenerational epigenetic inheritance via sperm small non-coding RNAs further modulate susceptibility. Red denotes pathological states; blue indicates therapeutic intervention points.

Within the germline compartment, SSCs display reduced abundance, transcriptional instability, and impaired differentiation trajectories. These changes are accompanied by disruption of key paracrine signaling pathways, including glial cell line-derived neurotrophic factor, KIT-KITLG, and extracellular matrix-mediated interactions, suggesting that SSC exhaustion in NOA reflects failure of niche support rather than an isolated intrinsic defect.

Inflammaging and chronic inflammatory remodeling of the testicular niche

Accumulating evidence indicates that chronic low-grade inflammation associated with aging, metabolic disorders, and environmental stress - commonly referred to as “inflammaging” - plays a central role in reshaping the testicular microenvironment[56]. Single-cell analyses consistently demonstrate upregulation of pro-inflammatory cytokines, chemokines, and stress-response pathways in Sertoli cells, Leydig cells, and resident immune populations in NOA testes[57,58].

This persistent inflammatory milieu disrupts blood-testis barrier integrity, alters redox homeostasis, and interferes with cellular metabolic coupling between somatic cells and germ cells[59]. Importantly, SSCs are highly sensitive to oxidative stress and inflammatory signaling, which can impair self-renewal capacity and promote premature differentiation or apoptosis. Thus, inflammaging provides a systemic and local context that predisposes the spermatogenic niche to progressive functional decline.

NLRP3 inflammasome as a mechanistic hub linking inflammation to SSC exhaustion

Among inflammatory pathways, activation of the NLRP3 inflammasome has emerged as a key molecular node connecting chronic inflammatory stress to testicular dysfunction. Single-cell and bulk transcriptomic studies have reported increased expression of NLRP3, CASPASE-1, interleukin-1β, and interleukin-18 in Sertoli cells, macrophages, and endothelial cells from NOA samples, indicating inflammasome activation within the niche[60]. Functionally, NLRP3-driven cytokine release amplifies local inflammation, promotes endothelial permeability, and perturbs Sertoli cell-mediated metabolic and structural support[61]. Experimental models further suggest that inflammasome activation directly compromises SSC survival by inducing mitochondrial dysfunction, DNA damage responses, and apoptotic signaling. Together, these findings support a pathogenic cascade in which inflammaging triggers sustained NLRP3 activation, leading to progressive deterioration of niche integrity and depletion of the SSC pool.

Other convergent mechanisms beyond inflammation

Although chronic inflammation represents a central driver of microenvironmental dysfunction in NOA, additional pathological mechanisms also contribute to SSC impairment. Single-cell and bulk transcriptomic studies have consistently reported increased oxidative stress signatures, mitochondrial dysfunction, and activation of DNA damage response pathways in both somatic and germ cell populations[62]. Excessive reactive oxygen species disrupt mitochondrial energy metabolism in Sertoli cells and SSCs, thereby compromising cellular homeostasis and self-renewal capacity[63-65].

Furthermore, mitochondrial injury and genomic instability may sensitize SSCs to inflammatory cytokines and inflammasome-mediated apoptosis, acting synergistically with NLRP3 activation to accelerate niche deterioration. These non-inflammatory pathways likely operate in parallel with chronic immune remodeling, collectively driving progressive collapse of the spermatogenic microenvironment. Recognition of these convergent mechanisms underscores the need for therapeutic strategies that target not only inflammatory signaling but also metabolic and genomic stress responses within the SSC niche.

Transgenerational epigenetic effects and sperm small non-coding RNAs

Beyond direct impairment of spermatogenesis, microenvironmental stress may exert longer-term biological consequences through epigenetic reprogramming of male germ cells. Experimental studies have demonstrated that metabolic disorders, chronic inflammation, and toxic environmental exposures can alter the composition of sperm small non-coding RNAs, including microRNAs and tRNA-derived fragments, which are capable of transmitting phenotypic traits to offspring[66-68].

Although evidence in humans remains limited, emerging data suggest that dysregulation of inflammatory and metabolic pathways within the testicular niche may influence the epigenetic landscape of SSC-derived germ cells[69]. These observations raise the possibility that chronic microenvironmental injury not only compromises fertility but may also contribute to intergenerational disease susceptibility. Genetic factors, including copy number variations such as DAZ gene deletions, further modulate individual vulnerability to microenvironmental stress[70]. Further large-scale human studies integrating single-cell transcriptomics with sperm epigenomic profiling will be required to clarify the clinical relevance and mechanistic basis of this phenomenon.

THERAPEUTIC TARGETS AND TRANSLATIONAL PERSPECTIVES

NLRP3 inflammasome inhibition as a potential therapeutic strategy

Given the central role of NLRP3 inflammasome activation in linking chronic inflammation to SSC dysfunction, pharmacological targeting of this pathway represents a promising therapeutic direction[60,61]. Preclinical studies in models of metabolic syndrome, testicular inflammation, and systemic inflammatory disorders have demonstrated that genetic or pharmacological inhibition of NLRP3 can attenuate cytokine release, preserve blood-testis barrier integrity, and improve germ cell survival[71]. In parallel, several small-molecule NLRP3 inhibitors, including dapansutrile, have entered clinical trials for inflammatory and metabolic diseases, showing favorable safety profiles and anti-inflammatory efficacy[72,73]. Key translational barriers between single-cell discoveries and clinical implementation are summarized in Figure 2.

Figure 2 Translational barriers from single-cell discoveries to clinical implementation. The translational gap comprises three interconnected barriers: Technical limitations (batch effects, spatial information loss), data biases (geographic underrepresentation of non-European populations risking therapy failures in neglected groups), and ethical/socioeconomic challenges (germline editing concerns, healthcare inequity, data privacy). Bridging this gap requires globally diverse cohorts, bias-aware artificial intelligence integration, and equitable governance frameworks. scRNA-seq: Single-cell RNA sequencing; RCT: Randomised controlled trial.

The translational gap comprises three interconnected barriers: Technical limitations (batch effects, spatial information loss), data biases (geographic underrepresentation of non-European populations risking therapy failures in neglected groups), and ethical/socioeconomic challenges (germline editing concerns, healthcare inequity, data privacy). Bridging this gap requires globally diverse cohorts, bias-aware artificial intelligence integration, and equitable governance frameworks.

Although direct clinical evidence in male infertility is currently lacking, these convergent findings support the biological plausibility of NLRP3 inhibition as a niche-preserving strategy. Importantly, targeting inflammasome signaling may offer advantages over hormone-based therapies by addressing upstream microenvironmental pathology rather than downstream spermatogenic failure. Future proof-of-concept trials will be required to determine whether suppression of chronic testicular inflammation can stabilize SSC populations and restore spermatogenic capacity in selected patient subgroups.

Gene editing and stem cell-based interventions: Opportunities and limitations

Advances in CRISPR-Cas9 technology and stem cell biology have raised the prospect of directly correcting genetic defects or regenerating impaired germline compartments[74,75]. In theory, genome editing could be applied to patient-derived SSCs followed by autologous transplantation, thereby avoiding systemic genetic modification[76]. However, multiple obstacles currently limit clinical translation, including inefficient delivery to target cells, off-target mutagenesis, genomic mosaicism, and long-term safety concerns[77].

Beyond technical barriers, germline manipulation raises profound ethical and regulatory challenges due to the potential for heritable genetic alterations[78]. International consensus statements and regulatory frameworks continue to emphasize strict limitations on clinical germline editing, particularly in non-life-threatening conditions[79,80]. Consequently, while gene editing and SSC transplantation represent powerful experimental tools, their near-term clinical application in male infertility remains highly constrained.

Nanomedicine and targeted modulation of the spermatogenic microenvironment

Nanotechnology-based drug delivery systems offer an alternative strategy for modulating the spermatogenic niche without direct genetic intervention[81]. Nanoparticles engineered to cross or accumulate near the blood-testis barrier have shown potential for targeted delivery of anti-inflammatory agents, antioxidants, and small interfering RNAs in preclinical models[82]. Such approaches may enhance local drug concentration while minimizing systemic exposure and adverse effects. Nevertheless, substantial challenges persist, including biocompatibility, long-term tissue accumulation, immune activation, and incomplete understanding of nanoparticle transport dynamics within testicular tissue. At present, nanomedicine should be regarded as an enabling platform rather than a standalone therapy, with its ultimate value dependent on the identification of robust molecular targets such as the NLRP3 inflammasome and inflammatory signaling networks within the SSC niche.

ETHICAL CONSIDERATIONS AND GLOBAL HEALTH IMPLICATIONS

Ethical challenges of germline and stem cell-oriented interventions

The increasing feasibility of manipulating SSCs and their microenvironment raises important ethical considerations[78]. Although autologous SSC editing and transplantation theoretically avoid systemic genetic modification, any intervention targeting the germline carries the potential for heritable genomic alterations. Off-target mutations, mosaicism, and unpredictable long-term consequences remain substantial unresolved risks, particularly in the context of complex polygenic disorders such as male infertility[79,80].

International scientific and regulatory bodies currently maintain strict limitations on clinical germline editing, emphasizing that such approaches should be confined to basic research settings until safety, efficacy, and societal implications are more clearly established[83,84]. In this context, therapeutic strategies that modulate the testicular microenvironment - such as pharmacological suppression of chronic inflammation or niche restoration - may represent a more ethically acceptable and clinically realistic pathway for near-term translation.

Global health equity and population representation in precision andrology

Another critical consideration is the pronounced geographic and socioeconomic imbalance in current single-cell datasets and translational research pipelines[85,86]. Most available scRNA-seq studies of the human testis originate from a limited number of high-income regions, whereas male infertility imposes a substantial burden in low- and middle-income countries where access to advanced reproductive technologies is often restricted[87].

This disparity raises two major concerns. First, molecular signatures and therapeutic targets derived from homogeneous populations may not generalize across diverse genetic backgrounds and environmental contexts[88]. Second, the high cost and technical complexity of single-cell technologies risk further widening existing gaps in reproductive healthcare[89]. Addressing these challenges will require coordinated international efforts to establish multiethnic cohorts, promote data sharing, and develop cost-effective diagnostic and therapeutic platforms that are scalable beyond specialized research centers[90,91].

CONCLUSION

Single-cell transcriptomics has fundamentally reshaped the conceptual framework of male infertility, revealing it to be a disorder driven not only by germ cell defects but by coordinated dysfunction of the spermatogenic microenvironment and its resident SSC compartment. Evidence synthesized in this review supports a pathogenic model in which chronic inflammatory remodeling, particularly through activation of the NLRP3 inflammasome, contributes to progressive niche deterioration and SSC depletion in NOA. By integrating single-cell, spatial transcriptomic, and emerging epigenetic data, this work highlights the central role of microenvironmental regulation in sustaining spermatogenesis and identifies inflammatory signaling pathways as rational targets for therapeutic intervention. Although substantial technical, ethical, and population-related challenges remain, continued integration of multi-omics technologies and broader representation of diverse populations may facilitate the development of stem cell-oriented precision strategies for the diagnosis and treatment of male infertility.