Abstract Authors: María Maroto; Cristina García-Merino; Paula Navarrete-López; Alfonso Gutiérrez-Adán; Raúl Fernández-González
Department of Animal Reproduction, INIA-CSIC, Madrid, Spain
Abstract Text: Although intracytoplasmic sperm injection (ICSI) is widely used to treat infertility, concerns persist regarding its long-term safety and potential effects on the fertility of ICSI-derived offspring. In this study, we investigated whether repeated use of ICSI across generations alters fertility and the transcriptomic landscape of sperm. We generated eight successive generations of mice via ICSI to evaluate potential cumulative effects. Ten hybrid B6D2F1/J males were used to create the F1 generation through ICSI, using sperm collected via vas deferens squeezing and cauda epididymis dissection. Spermatozoa were allowed to swim out for 30 minutes at 37 °C in a 500 μL droplet of HTF medium, and motile sperm were collected from the surface. The ICSI protocol included the use of frozen-thawed sperm that were centrifuged, frozen in liquid nitrogen, and stored up to one month. Twenty male offspring from each generation were assessed for fertility through natural mating. Between five and ten fertile males were selected from each generation to produce the next, repeating the procedure through eight generations (G1–G8). Total RNA was isolated from sperm of fertile 5-month-old males from both the G0 (n = 7) and G8 (n = 5) generations using TRIzol® (Invitrogen) following the manufacturer’s instructions. RNA was sequenced on an Illumina NovaSeq X Plus platform with PE150 reads. Raw RNA-Seq data were quality-checked, trimmed, and aligned to the reference genome, followed by gene expression quantification. Low-abundance transcripts were filtered out using DESeq2 (FDR < 0.05, log2 fold change > 1), and gene ontology enrichment was performed with clusterProfiler using the enrichGO function. A total of 234 genes were found to be differentially expressed between G8 and G0 sperm samples: 162 were upregulated and 72 downregulated in G8. Among these, 89 were long non-coding RNAs (lncRNAs), of which 52 were upregulated in G8 and 37 in G0. Of the 120 protein-coding DEGs, 102 were upregulated in G8 and only 18 in G0. Notably, the proportion of lncRNAs among the DEGs was unusually high for mature spermatozoa, which are typically transcriptionally quiescent. Gene ontology analysis revealed that upregulated genes in G8 were enriched for biological processes related to muscle and heart function, including muscle contraction, striated muscle contraction, and heart contraction. In contrast, downregulated genes were associated with immune regulation, immune cell proliferation, and neural development. Given the abundance of lncRNAs, we also performed protein–lncRNA interaction analyses, which showed strong enrichment in biological processes such as muscle organ development, cardiac muscle development, and striated muscle cell differentiation. Furthermore, several GO terms related to miRNA regulation were enriched, including regulation of miRNA metabolic process, miRNA transcription, and general miRNA processing. Terms related to cellular development and differentiation, such as stem cell differentiation, cardiomyocyte differentiation, lymphocyte differentiation, and T cell differentiation, were also enriched. Our study demonstrates that repeated application of ICSI across generations can lead to changes in the sperm transcriptome, particularly through increased expression of lncRNAs and genes involved in cardiac and muscle development. The elevated presence of regulatory non-coding RNAs suggests a potential role in epigenetic inheritance. These findings raise important questions about the long-term safety of ICSI, particularly regarding subtle molecular changes that may impact offspring development and transgenerational epigenetic regulation.
