Open research questions in Sperm and Testicular Function

What the literature leaves open

• Abstract Diabetes mellitus (DM) is a growing global health crisis affecting male reproductive function, yet the enhanced combined effects of diabetes with pre-existing fertility abnormalities remain poorly understood.

  The link between diabetes and male infertility: mechanisms and implications · 2026 · DOI

• Alginate oligosaccharides (AOS) exhibit antioxidant and reproductive protective effects, but the molecular mechanism by which AOS improves HS-induced declines in semen quality remains unclear.

  Alginate oligosaccharides alleviate heat stress-induced damage to tight junctions and regulate autophagy in sertoli cells · 2026 · DOI

• Various epigenomic studies reported conflicting results on the distribution of residual nucleosomes in mammalian sperm1-9, questioning their potential role in mediating intergenerational inheritance of paternal epigenetic information10,11.

  Single molecule footprinting measures low nucleosome occupancy in mature spermatozoa of mice and men · 2026 · DOI

• In particular, maximum dosing, delivery strategies, clinical efficacy during cryo- preservation conditions, and the long-term effects on ART are still under-investigated. However, a brief comment is warranted on whether such pharmacological stimulation might merely improve motility while masking underlying genetic damage.

  Technological and clinical advances in sperm cryopreservation: A literature review · 2026 · DOI

• A key strength is the within-ejaculate, five-arm design, which reduces confounding from interindividual semen variability. In addition, we assessed conventional parameters together with functional (ARIC) and genomic (TUNEL-based DFI) outcomes, enabling a more comprehensive evaluation. This study has several limitations, including the modest sample size (n=30), the inclusion of only normozoospermic cases, and the use of a single assay (TUNEL) to assess DNA fragmentation without parallel validation by other methods, such as SCD or Comet. In addition, the labor-intensive nature of CCECM isolation may limit its immediate clinical applicability unless simplified and standardized protocols are established. In normozoospermic ejaculates processed for ART, adding cumulus cell extracellular matrix to standard sperm preparation media did not alter the expected reduction in sperm concentration after processing. Still, it was associated with higher motility and viability (protocol-dependent) and, significantly, with quality. improved genomic Specifically, CCECM supplementation increased ARIC scores compared with raw semen and produced the most reductions substantial significant benefit observed with the swim-up protocol. These findings support CCECM as a promising, more physiologic additive to routine sperm preparation; however, validation in larger cohorts— including oligo/teratozoospermic populations—and with additional DNA integrity assays and clinical endpoints development, pregnancy outcomes) is warranted. in DFI, with (fertilization, functional embryo and Researcher Contribution Statement: Idea and design: D.G.Y., C.Ç.; Data collection and processing: S.A., T.Y.D., K.A., I.K.; Analysis and interpretation of data: D.G.Y., S.A., C.Ç.; Writing of significant parts of the article: D.G.Y., S.A. Support and Acknowledgement Statement: This study was conducted within the framework of the Bursa Uludağ University Scientific Research Projects Coordination Unit (BAP) project TYL-2025-2129.

  Effects of Cumulus Cell Extracellular Matrix–Assisted Sperm Preparation Protocols on In Vitro Sperm Quality · 2026 · DOI

• Paternal contributions to embryogenesis extend beyond DNA, yet the molecular cargo carried by sperm and its impact on development remain poorly defined.

  Aggregation of misfolded proteins in the sperm head impairs preimplantation embryo development · 2026 · DOI

• We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined.

  The RNA helicase DDX53 (CAGE) contributes to RNA metabolism in a human germ cell model · 2026 · DOI

• It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear.

  The RNA helicase DDX53 (CAGE) contributes to RNA metabolism in a human germ cell model · 2026 · DOI

• Taken together, we suggest that synergistic combinations of antioxidants that are economically feasible, such as resveratrol with vitamins C or E, could be the way forward, but warrant further investigations into dosage versus toxicity, standardised extraction protocols, and breed-specificity in their supplementation in semen extenders.

  Cryopreservation-induced oxidative damage in ram sperm and the role of natural antioxidants as cryopreserved sperm quality enhancers · 2026 · DOI

• Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear.

  Ectopic hAMH-driven SOX17 expression induces hyperplastic Sertoli valve formation in mouse testes · 2026 · DOI

• Although limited by a small sample size, this study elucidates the molecular mechanisms underlying male reproductive maturation in Kazakh horses at the post-transcriptional regulatory network level, providing preliminary theoretical support and potential markers for evaluating stallion reproductive performance and molecular breeding.

  miRNA Sequencing and Differential Analysis of Testes from 1-Year-Old and 3-Year-Old Kazakh Horses · 2026 · DOI

• This review has several limitations, including the small number of included studies (n = 7) and comparatively small sample sizes ( d”100), with resultant reduce statistical power and increase susceptibility to bias. Methodological weaknesses were also common, including inadequate randomization, limited or absent blinding, and incomplete outcome reporting increasing the risk of bias. Follow-up periods were typically short as most trials lasted less than six months, limiting the ability to assess longterm efficacy and safety. The findings may also have been influenced by variability in the plant species used, extraction and preparation methods, dosage, treatment duration, and lack of standardization across herbal formulations. Additionally, as all studies were conducted the generalizability of the results to other populations may be limited.

  Therapeutic Potential of Herbal Medicine for Male Infertility in Pakistan from 1995 to 2025: A Systemic Review and Meta Analysis · 2026 · DOI

• The main limitation of this study resides in its relatively modest sample size (n = 337), which may compromise the stability and generalizability of the findings. These results contribute to our understanding of the role of ZAG in male reproductive physiology and warrant further investigation into its potential as a biomarker for semen quality assessment.

  ZAG and Zn/ZAG Ratio in Seminal Plasma: Dual Roles in Male Semen Quality · 2026 · DOI

• Semen leukocytes and albumin are commonly measured in routine care, and their consistent associations with DFI suggest that they may warrant further study as complementary markers in the evaluation of sperm DNA integrity.

  Association of semen leukocytes with sperm DNA fragmentation in a clinical cohort · 2026 · DOI

• Overall, this study reports the first LC–MS/MS method for the simultaneous determination of free 2′-dG and 8-OHdG in seminal plasma. Validated according to FDA bioanalytical guidelines, the developed method combines solid-phase extraction (SPE) with TABLE 2 Concentrations of 2′-dG and 8-OHdG in seminal plasma by group, with intra-sample RSD (%).

  Simultaneous LC–MS/MS quantification of free deoxyguanosines 2′-dG and 8-OHdG in ovine seminal plasma as biomarkers of oxidative DNA damage · 2026 · DOI

• Neverthe- less, further research is warranted, particularly in semen samples with different abnormalities and in exploring combinations of selec- tion techniques to optimize sperm recovery. The potential applicability of this sperm preparation strategy should be explored in abnormal semen profiles, such as oligozoospermia and asthenozoospermia.

  Integrating zeta potential with conventional sperm preparation: Comparative effects of swim-up–zeta and density gradient-zeta methods on motility and DNA integrity · 2026 · DOI

• Future research should focus on optimizing ferroptosis inhibitor doses and delivery in semen extenders, evaluating the contribution of ferroptosis relative to other RCD pathways, assessing the impact of ferroptosis inhibition on in vivo fertility and embryonic development, and deter- mining the cost–benefit analysis for commercial implementation.

  Ferroptosis contributes to boar sperm deterioration during liquid semen storage · 2026 · DOI

• The study integrated rumen and plasma metabolomic data to propose the rumen–plasma–sperm axis, but did not directly measure metabolite concentrations in seminal plasma or sperm cells themselves. Targeted lipidomic and metabolomic profiling of seminal plasma and isolated sperm would confirm whether the identified bioactive lipids and energy metabolites (N-decanoyl-L-homoserine lactone, glycerophospholipids, arginine) are actually bioavailable at the sperm compartment.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• The optimal NAC dosage was identified at 0.3% in goats, but the dose-response relationship was not systematically characterized across a wider range of concentrations. Testing additional NAC concentrations (e.g., 0.1%, 0.2%, 0.4%, 0.5%) would establish whether the 0.3% optimum represents a true peak or represents an inflection point within a broader dose-response curve affecting the rumen microbiome composition and sperm motility parameters.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• NAC supplementation enriched multiple lipid-related pathways including biosynthesis of unsaturated fatty acids, glycerophospholipid metabolism, and steroid hormone biosynthesis in the rumen microbiome, but the specific contribution of each pathway to systemic sperm motility enhancement was not quantitatively delineated. Pathway-selective inhibition studies would be required to determine which lipid metabolic alterations are necessary and sufficient for the observed phenotype.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• The study identified elevated plasma levels of taurine-conjugated bile acids positively correlated with sperm motility, but the mechanistic pathway by which microbiota-modulated bile acid composition enhances membrane fluidity and sperm physiological function remains incompletely characterized. Direct mechanistic studies examining how taurine-conjugated versus glycine-conjugated bile acids affect sperm mitochondrial membrane potential and ATP synthesis are needed.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• The study demonstrates correlations between specific microbial taxa (Bacteroides acidifaciens, Romboutsia) and energy/lipid metabolism pathways, but does not establish causality. Microbial transplantation experiments or controlled probiotics administration targeting these specific bacterial taxa are needed to confirm their causal role in NAC-mediated enhancement of sperm motility in goats.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• The intragastric administration of N-methyl-L-asparagine in mice showed enhanced sperm motility, but the authors acknowledge that interspecies differences between mice and goats limit the direct applicability of these findings. Future work must validate the species-specific roles of N-methyl-L-asparagine and D-glutamine in regulating sperm function through goat-based in vitro and in vivo studies before confirming their functional contribution to the rumen–plasma–sperm axis in ruminants.

  Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · 2026 · DOI

• Standardized protocols for biomarker evaluation in seminal plasma and serum—including sample collection, processing, and analysis procedures—are necessary to convert research findings on male fertility markers (SGOT, LDH, cholesterol, copper, glucose) into clinical practice and enable integration into standard clinical procedures.

  Biochemical Markers In Seminal Plasma And Serum: A Comparative Assessment In Fertile And Infertile Men · 2026 · DOI

• Clinical studies directly correlating seminal plasma biomarkers with assisted reproductive technology outcomes are limited. Future longitudinal cohort studies must monitor how SP biomarkers for male fertility (Cu, SGOT, LDH, cholesterol) evolve over time and in response to different therapeutic drugs.

  Biochemical Markers In Seminal Plasma And Serum: A Comparative Assessment In Fertile And Infertile Men · 2026 · DOI

Most-cited papers in Sperm and Testicular Function

• The evaluation of morphological characteristics of human spermatozoa according to stricter criteria · Human Reproduction · 1990 · 533 citations
• Spermatogenesis · Human Reproduction · 1998 · 413 citations
• The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development · Human Reproduction · 2002 · 395 citations
• Physiological consequences of testicular sperm extraction · Human Reproduction · 1997 · 363 citations
• Regional differences in semen quality in Europe · Human Reproduction · 2001 · 361 citations
• A survey of small RNAs in human sperm · Human Reproduction · 2011 · 345 citations
• A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys · Human Reproduction · 2015 · 305 citations
• Diagnosis and treatment of infertility in men: AUA/ASRM guideline part I · Fertility and Sterility · 2020 · 284 citations
• The relationship between male BMI and waist circumference on semen quality: data from the LIFE study · Human Reproduction · 2013 · 265 citations
• Reduced amounts and abnormal forms of phospholipase C zeta (PLC ) in spermatozoa from infertile men · Human Reproduction · 2009 · 262 citations

Most recent work

• Differential microRNAs in human serum and sperm after childhood trauma with potential implications for offspring health · medRxiv · 2026
• Structure and inhibition of the sperm TMEM95-FIMP complex in mammalian fertilization · bioRxiv · 2026
• Deep learning-enabled morphology analysis of bovine sperm for label-free imaging flow cytometry · Frontiers in Veterinary Science · 2026
• Mouse ICSI impairs fertility of male offspring by disruption of fetomaternal tolerance · Development · 2026
• Integrating multi-omics and network toxicology to identify FIS1 as a key target of environmental pollutants in male infertility · Frontiers in Cell and Developmental Biology · 2026
• Single-cell transcriptomics reveals a spermatogonial stem cell-centered spermatogenic microenvironment: Pathophysiological mechanisms and therapeutic targets in male infertility · World Journal of Stem Cells · 2026
• Male testosterone synthesis disorders: oxidative stress pathways, regulatory mechanisms, potential of antioxidant therapy, and botanical medicine prospects · Asian Journal of Andrology · 2026
• miRNA Sequencing and Differential Analysis of Testes from 1-Year-Old and 3-Year-Old Kazakh Horses · Biology · 2026
• Biochemical Markers In Seminal Plasma And Serum: A Comparative Assessment In Fertile And Infertile Men · International Journal of Drug Delivery Technology · 2026
• Dietary N-acetylcysteine enhances sperm motility by remodeling the rumen microbiome and its metabolic axis in goats · Journal of Animal Science and Biotechnology · 2026

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