Integrative transcriptomics and systems genetics position AKAP4 as an evolutionarily constrained network hub gene in human sperm cryopreservation

Sperm cryopreservation is a key component of assisted reproductive technologies; however, freeze–thaw stress often compromises sperm motility and fertilization capacity. Identifying molecular determinants of cryoinjury is essential to improving fertility preservation strategies. To identify cryostress-responsive molecular networks and key regulatory determinants associated with sperm cryoinjury using an integrative systems-level approach. Human sperm transcriptomics was integrated with network modelling, regulatory prediction, population genetics, comparative genomics, and structural annotation. Transcriptomic analysis of dataset GSE225320 was performed to evaluate cryostress-responsive changes. Protein–protein interaction and co-expression analyses were used to identify central hub genes. RT-qPCR validation, independent transcriptomic and proteomic datasets, population genetic analysis, cross-species conservation assessment, and domain-level structural mapping were employed to characterize candidate genes further. Transcriptomic analysis revealed dynamic remodelling of cryostress-responsive networks. Short-term storage affected pathways related to cytoskeletal organization, phosphorylation signalling, and sperm motility, whereas prolonged storage induced proteostasis, metabolic stress adaptation, and DNA damage repair processes. Network analysis identified A-kinase anchoring protein 4 (AKAP4) as a central hub in the short-term cryostress network. RT-qPCR validation confirmed a significant reduction in AKAP4 transcript abundance following sperm cryopreservation.Independent datasets showed strong testis-specific enrichment and functional connectivity of AKAP4 with flagellar architecture, calcium signalling, and cAMP/PKA-dependent motility pathways. Population genetics and comparative analyses indicated strong purifying selection, mammalian conservation, and selective constraint at key functional domains. AKAP4 emerges as an evolutionarily constrained and cryopreservation-sensitive network hub associated with sperm motility pathways. These findings provide systems-level insight into molecular alterations associated with sperm cryopreservation and identify AKAP4 as a promising candidate biomarker for future functional investigation.