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

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.