Reproductive Technology

Long-Term Effects of Mouse Intracytoplasmic Sperm Injection with DNA-Fragmented Sperm on Health and Behavior of Adult Offspring

Abstract
Genetic and environmental factors produce different levels of DNA damage in spermatozoa. DNA-fragmented spermatozoa (DFS) are usually used in intracytoplasmic sperm injection (ICSI) treatments in human reproduction and are still a matter of concern. The purpose of the present study was to investigate the long-term consequences on development and behavior of mice generated by ICSI with DFS. Using CD1 and B6D2F1 mouse strains, oocytes were injected with fresh spermatozoa or frozen-thawed spermatozoa without cryoprotector. This treatment increased the percentage of TUNEL positive spermatozoa, tail length measured by Comet assay, and loss of telomeres measured by quantitative PCR. ICSI generated embryos were cultured 24 h in KSOM, and 2-cell embryos were transferred into CD1 females. DFS reduced the rate of both preimplantation embryo development and offspring. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed a delay of 2 h on the active demethylation of male pronucleus in the embryos produced by ICSI. Moreover, ICSI affected gene transcription and methylation of some epigenetically regulated genes like imprinting, X-linked genes, and retrotransposon genes. At 3 and 12 months of age, ICSI produced animals with DFS and in vivo fertilized controls were submitted to behavioral tests: locomotor activity (open field), exploratory/anxiety behavior (elevated plus maze, open field), and spatial memory (free-choice exploration paradigm in Y maze). Females produced by ICSI showed increased anxiety, lack of habituation pattern, deficit in short term spatial memory and age-dependent hypolocomotion in the open field test (P<0.05). Postnatal weight gain of mice produced by ICSI with fresh or frozen sperm was higher than those from their control counterparts from 16 weeks on (P<0.01). Anatomopathological analysis of animals at 16 months of age showed some large organs and an increase in pathologies (33% of CD1 females produced with DFS presented some solid tumors in lungs and dermis of back or neck). Moreover, 20% of the B6D2F1 generated with DFS died during the first 5 months of age, 25% of the surviving animals showed premature ageing symptoms, and 70% of mice died earlier than control with different kind of tumors. We propose that, depending on the level of DFS, oocytes may partially repair fragmented DNA, producing blastocysts able to implant and produce live offspring. However the incomplete repair may lead to long term pathologies. Our data indicate that, the use of DFS in ICSI can generate effects that only emerge in later life, such as, aberrant growth, premature ageing, abnormal behavior and mesenchymatical tumors.

Key words: Embryo • Assisted Reproductive Technology • Early development • DNA fragmented • ICSI

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