Assessment of Somatic DNA Quality in the Eighth Generation of Mice Produced by ICSI

Assessment of somatic DNA Quality in the Eighth Generation of Mice Produced by ICSI

R. Fernández-González¹, M. Maroto¹, C. García-Merino¹, P. Navarrete-López¹, A. Gutiérrez-Adán¹

 1. Department of Animal Reproduction, INIA-CSIC, Madrid, Spain

 

Abstract Text:

Although the intracytoplasmic sperm injection (ICSI) technique has been widely used as a treatment for infertility, there are still concerns about its safety and potential consequences for the fertility of individuals produced by ICSI. In this study, we produced eight generations of mice through ICSI to analyze whether repeated use of the technique affects fertility and the stability of somatic tissue DNA in the animals. Ten hybrid B6D2F1/J mice were used to generate the F1 generation through ICSI using sperm from vas deferens squeezing and cauda epididymis dissection. Spermatozoa were allowed to swim-out for 30 min at 37ºC in a 500mL droplet of HTF medium, and sperm from the surface were collected for ICSI. Twenty males were produced and mated to assess their fertility. Subsequently, between 5 and 10 fertile males were selected to produce the next generation, and the fertility of 20 males from the new generation was analyzed again. This protocol was repeated for eight generations. Finally, DNA was sequenced from samples obtained from fertile 5-month-old males of generations G0 (n=5), G1 (n=8), and G8 (n=8).

The results indicate that, regardless of generation, only 50% of the males born were fertile, showing testicular size (represented as gonadosomatic index) and sperm quality (motility, sperm count, and morphology) similar to the control group. However, infertile animals exhibited smaller testicular size, lower sperm count, reduced progressive motility, and a higher incidence of morphological abnormalities. Regarding DNA quality, we extracted DNA from mice tails and we analyzed Single Nucleotide Polymorphisms (SNPs), Small insertions or deletions (InDels), Structural Variants (SVs), which are genomic alterations of relatively larger size ( >50 bp), and Copy Number Variations (CNVs). The frequency of SNPs decreased in both G1 and G8, with no differences between these two generations. For InDels, an increase in both deletions and insertions was observed in G1 and G8, with no differences between the two generations. In relation to SVs, a progressive increase in inter-chromosomal translocation mutations was observed in G1 and G8, along with increases in deletions, inversions, and intra-chromosomal translocations in G1, which persisted in G8, however the differences were not significant. Finally, regarding CNVs, there was an increase in the proportion of CNVs, both in deletions and duplications, in G1 and G8. There were no significant differences in deletions between G1 and G8, but duplications increased in G8 compared to G1.

The findings suggest that while repeated ICSI over multiple generations does not reduce the proportion of fertile males, it leads to significant genomic alterations, including increases in structural variants and copy number variations. These changes, observed from the first generation and persisting through the eighth, indicate that repeated use of ICSI may contribute to genomic instability, highlighting the importance of continued research on its long-term safety and effects on reproductive health.