Infertility is a global reproductive health issue, with male factors contributing significantly to its etiology.[1,2] Despite extensive research, the genetic underpinnings of a substantial proportion of male infertility cases remain elusive. One area under investigation is mRNA splicing, a fundamental, post-transcriptional ‘cut-and-paste’ process that generates mRNA from our genetic code. Over 95% of human genes undergo ‘alternative splicing’, which allows more than one mRNA sequence to be created from the same gene. This is essential for generating a diverse range of proteomes—the library of proteins a cell or tissue can express—from our finite number of genes.[3]

The genetic variants that influence alternative splicing are broadly categorized into two classes: canonical and non-canonical splicing variants. Canonical splicing variants refer to mutations at the donor (GT) and acceptor (AG) dinucleotides of the sites where splicing occurs; non-canonical splicing variants (NCSVs) encompass mutations in other essential cis-acting splicing regulatory elements including polypyrimidine tract, branch point, as well as exonic and intronic splicing enhancers and silencers.

Disruption of this intricate regulatory code can lead to aberrant splicing and is a significant contributor to human genetic disorders. Traditional guidelines only define canonical splicing variants as the ones leading to a potential loss of function in the resulting mRNA,[4] but genetic variants in non-canonical splicing sites can also result in abnormal mRNA splicing.

Emerging evidence highlights the clinical significance of these non-canonical splicing variants,[5,6] and a recent comprehensive review of reported splicing variants in male infertility cases implicated a total of 54 functionally validated splicing variants in 42 genes, with 22 of them located outside the canonical splice sites.[3] This offers a groundbreaking new perspective on the previously overlooked role of non-canonical splicing variants in this complex condition.

Impact of non-Canonical splicing

In the context of male infertility, while pathogenic variants have been reported, the true impact of NCSVs remains largely unexplored. A critical issue is that many variants—particularly missense mutations, which result in a different amino acid being encoded, resulting in a change of function of the encoded protein—are prioritized based on pathogenicity prediction tools that assess amino acid changes. This approach may inadvertently overlook variants whose primary pathogenic mechanism is the disruption of mRNA splicing, not the missense change itself. Consequently, a significant fraction of the genetic architecture of male infertility remains obscured.

To address this knowledge gap, a study published in Advanced Science by K. Li et al.[8] curates 2,404 reported variants associated with male infertility and, using robust in silico prediction tools, reprioritizes potential splicing variants.

Through meticulous functional validation via minigene assays, the researchers demonstrated that 58.33% (28/48) of tested potential NCSVs indeed cause abnormal splicing. This expands the contribution of functionally validated NCSVs by 53.13%, establishing that NCSVs account for 28.99% (49/169) of all infertility-associated splicing variants. Furthermore, the authors reanalyzed whole-exome sequencing data from 718 idiopathic male infertility patients. They identified 13 pathogenic NCSVs in 12 patients (1.67% of the cohort) that had been missed by conventional genetic analysis pipelines.

Concrete evidence for non-canonical splicing-related male infertility

One highlight of this study is its detailed analysis of the TMF1 gene, and the discovery of the first pathogenic NCSV in TMF1. This leads to exon skipping (where the splicing process misses out key functional parts of the mRNA) and is associated with decreased sperm motility and morphological abnormalities, providing a concrete example of how NCSVs can directly impact male fertility. The team were able to generate a TMF1 NCSV knock-in mouse model which mimicked this human phenotype: the mouse model not only demonstrated significant decreases in sperm count and motility but also revealed ultrastructural defects in sperm, strengthening the evidence for an underlying TMF1-related infertility.

While the work by Li et al. represents a significant step forward in our understanding of the genetic basis of male infertility, it also highlights current challenges in NCSV research. It creates a robust framework for uncovering the hidden role of NCSVs in a wide spectrum of human genetic disorders: the use of splicing prediction methods for prioritizing potential NCSVs, followed by functional validation through minigene assays, ensures that the identified variants are likely to have a genuine impact on mRNA splicing.

However, the study was limited to single-nucleotide variants, the potential oversight of splicing defects caused by stop-gain variants, and the technical constraints of minigene assays, which may not detect large-scale mis-splicing events due to the absence of distant regulatory sequences. The positive validation rate of 62.12%, though impressive, indicates that current splicing prediction algorithms still require refinement.

The researchers believe that the future therefore lies in developing more accurate genome-wide and tissue-specific models, potentially leveraging emerging AI models, to predict splicing and other molecular phenotypes comprehensively. Moreover, the functional impact of some of these variants on protein structure and function could be further explored through additional experimental assays.

By highlighting the role of NCSVs, their work underscores the diverse genetic architecture of male infertility and the potential for NCSVs to contribute to different phenotypes. It also compellingly argues for the integration of NCSV detection into standard genetic analyses for idiopathic male infertility, opening up new avenues for research and for the development of diagnostic tools and, eventually, therapeutic strategies.

References:

[1] A. Agarwal et al., A unique view on male infertility around the globe. Reproductive Biology and Endocrinology (2015), DOI: 10.1186/s12958-015-0032-1.

[2] M. L. Eisenberg et al., Male infertility. Nature Reviews Disease Primers (2023), DOI: 10.1038/s41572-023-00459-w.

[3] K. Li et al., Defects in mRNA splicing and implications for infertility: a comprehensive review and in silico analysis. Human Reproduction Update (2025), DOI: 10.1093/humupd/dmae037.

[4] S. Richards et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine (2015), DOI: 10.1038/gim.2015.30.

[5] S. Zhang et al., Base-specific mutational intolerance near splice sites clarifies the role of nonessential splice nucleotides. Genome Research (2018), DOI: 10.1101/gr.231902.117.

[6] J. Lord et al. Pathogenicity and selective constraint on variation near splice sites. Genome Research (2019), DOI: 10.1101/gr.238444.118.

[7] K. Li et al., Mapping the Non-Canonical Splicing Variants: Decrypting the Hidden Genetic Architecture of Idiopathic Male Infertility. Advanced Science (2025), DOI: 10.1002/advs.202515512.

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