SAT-173 Exploring Sperm-Carried IGF2 Transcript Variants: Cloning, qPCR Analysis, And Variant Profiling

Abstract Disclosure: R. Cannarella: None. A. Miller: None. S.D. Lundy: None. A.E. Calogero: None. Background. Prior research from our group identified the presence of insulin-like growth factor 2 (IGF2) gene transcript in human spermatozoa, suggesting a pivotal role in early embryo development. IGF2, a paternally imprinted gene, is inactive in oocytes, providing a unique paternal genetic contribution during embryogenesis. While five distinct IGF2 transcript variants have been identified in humans, the specific variants expressed in spermatozoa compared to granulosa cells (GCs)—a biologically significant maternal source during early development- remain uncharacterized. Clarifying these variants and their expression patterns could yield novel insights into the paternal genetic role in embryogenesis. Objective. To characterize the diversity and expression levels of IGF2 transcript variants in human spermatozoa and compare them with those in GCs. Methods. Post-gradient sperm samples were obtained from two healthy, fertile men with proven paternity. GCs were isolated and purified from follicular fluid obtained during oocyte retrieval. RNA extraction was followed by cDNA synthesis and PCR amplification in both sperm and GC samples. PCR products were cloned into the PCR4-TOPO vector and transformed into TOPO10 One-Shot electro-competent Escherichia coli. Ninety-six positive colonies (32 per sample) were identified and grown. Each clone underwent Sanger sequencing to identify nucleotide and amino acid sequence variations, followed by phylogenetic analysis to classify variants. Quantitative PCR with gene-specific primers assessed transcript levels, melting temperatures, and sequence characteristics. Results. Fourteen distinct transcript variants were identified, including deletions, insertions, and single nucleotide polymorphisms that resulted in amino acid sequence changes. Among these, two variants were common to both spermatozoa and GCs, two were unique to GCs, and ten were exclusive to spermatozoa. While some sequences matched known NCBI variants, others formed two previously unreported phylogenetically distinct clusters. Quantitative analysis revealed higher expression differences of sperm-specific variants compared to GC variants. Notably, sperm-specific variants exhibited higher expression levels, suggesting their potential functional significance during the early stages of embryo development. Conclusion. This study provides the first comprehensive characterization of IGF2 transcript diversity in human spermatozoa. The identification of ten sperm-specific variants distinct from GC-expressed variants highlights the unique paternal genetic contribution to embryogenesis. These findings pave the way for further research to investigate the functional implications of sperm-specific IGF2 transcripts and particularly their role in regulating early embryo development and their potential implications for reproductive outcomes. Presentation: Saturday, July 12, 2025