The global fertility crisis is increasingly attributed to a steady decline in human semen quality, with conditions such as asthenozoospermia (reduced sperm motility) and teratozoospermia (abnormal sperm morphology) accounting for more than half of male subfertility cases.

While small noncoding RNAs (sncRNAs) are known to be abundant in mature sperm and essential for regulating spermatogenesis, traditional sequencing methods have predominantly focused on miRNAs, which represent less than 1% of the total sncRNA population in sperm, whereas transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) comprise the majority of the sperm sncRNA profile. These sncRNAs frequently possess chemical modifications and non-canonical terminal structures that hinder adapter ligation and reverse transcription during standard library preparation, thereby making their detection challenging with conventional methods.

In a prospective cohort study published in Genes & Diseases, researchers from Tongji University, Shanghai Institute for Biomedical and Pharmaceutical Technologies (SIBPT), and Chinese Academy of Sciences utilized PANDORA-seq—a panoramic RNA display strategy that employs a two-step enzymatic treatment with T4 polynucleotide kinase (T4PNK) and α-ketoglutarate-dependent dioxygenase (AlkB)—to remove these inhibitory modifications. By applying this method to a cohort of 25 participants categorized into normozoospermia (NZS), asthenozoospermia (AZS), and teratozoospermia (TZS) groups, the researchers generated one of the most comprehensive landscapes of human sperm sncRNAs to date.

The study revealed that tsRNAs and rsRNAs are not only the dominant species, constituting over 97% of the total small RNA population, but are also strongly correlated with clinical indicators of sperm quality, such as motility and morphology. Among rsRNAs, the majority are derived from cytoplasmic 28S and 18S subunits, with 28S-derived sequences alone accounting for over 74% of the rsRNA population. tsRNAs also exhibit distinct patterns, with nuclear-encoded species primarily originating from the 5' end of tRNAs, whereas mitochondrial-encoded tsRNAs are skewed toward internal cleavage sites.

Functional analysis identified robust linear correlations between these specific molecular species and clinical indicators: nuclear-encoded tsRNA-Phe and tsRNA-Lys are positively correlated with progressive motility (PR), whereas rsRNA-28S exhibits a significant negative correlation with motility parameters. Furthermore, rsRNA-5.8S showed a notable negative correlation with both the head shape index (TZI) and the percentage of intact sperm heads, suggesting a potential mechanistic role in regulating sperm morphology.

Conversely, tsRNA species such as tsRNA-iMet, tsRNA-Val, and various 28S-derived rsRNAs were negatively correlated with motility, indicating their association with subfertile states. While correlations between sncRNAs and morphology were generally less pronounced, rsRNA-5.8S remained negatively associated with intact head and head shape indices, and tsRNA-Val showed a positive association with abnormal morphological indices.

To translate these molecular findings into clinical utility, the researchers employed machine learning and LASSO regression based on sperm rsRNA and tsRNA profiles, establishing the male subfertility sncRNA signature (MSsncSig), the AZS-related signature (AZSsncSig), and the TZS-related signature (TZSsncSig). These models demonstrated exceptional diagnostic power, achieving area under the curve (AUC) scores of 0.83 or higher. This predictive capability represents a substantial improvement over traditional WHO semen quality assessments, providing a novel molecular framework for diagnosing male infertility.

In conclusion, PANDORA-seq provides critical insights into the landscape of the human sperm sncRNA repertoire, identifying tsRNAs and rsRNAs as pivotal markers of reproductive health. By establishing correlations between these modified RNAs and sperm fitness, this research offers a robust framework for assessing sperm quality and understanding the molecular mechanisms underlying male subfertility and its potential intergenerational impacts.

Reference

Title of the original paper: PANDORA-seq reveals human sperm sncRNA signature endowed with sperm quality assessment

Journal: Genes & Diseases
Genes & Diseases is a journal for molecular and translational medicine. The journal primarily focuses on publishing investigations on the molecular bases and experimental therapeutics of human diseases. Publication formats include full length research article, review article, short communication, correspondence, perspectives, commentary, views on news, and research watch.

DOI: https://doi.org/10.1016/j.gendis.2025.101807

Funding Information:
National Key Research and Development Program of China (No. 2019YFA0802600, 2022YFA1103200)
National Natural Science Foundation of China (No. 82371727, 82022029, 81971460, 32170553, 32370596)
Science and Technology Commission of Shanghai Municipality, China (No. 23JC1403802)
Innovation Promotion Program of NHC and Shanghai Key Labs, SIBPT (China) (No. RC2024-04)
Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA0460302)
Fundamental Research Funds for the Central Universities of China (No. 22120240435)
National Key Research and Development Program of China provided the funding for the open access charge.

Genes & Diseases publishes rigorously peer-reviewed and high quality original articles and authoritative reviews that focus on the molecular bases of human diseases. Emphasis is placed on hypothesis-driven, mechanistic studies relevant to pathogenesis and/or experimental therapeutics of human diseases. The journal has worldwide authorship, and a broad scope in basic and translational biomedical research of molecular biology, molecular genetics, and cell biology, including but not limited to cell proliferation and apoptosis, signal transduction, stem cell biology, developmental biology, gene regulation and epigenetics, cancer biology, immunity and infection, neuroscience, disease-specific animal models, gene and cell-based therapies, and regenerative medicine.
Scopus CiteScore: 8.4 | Impact Factor: 9.4

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