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Factors From Damaged Sperm Affect Its DNA Integrity and Its Ability to Promote Embryo Implantation in Mice

From the Departamento de Reproducción Animal y Conservación de Recursos Zoogenéticos, Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria, Carretera de la Coruña, Madrid, Spain.

Correspondence to: Dr Alfonso Gutiérrez-Adán, Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria, Departamento de Reproducción Animal, Carretera de la Coruña km 5, 9, 28040 Madrid, Spain (e-mail: agutierr{at}inia.es).

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P < .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.

     Key words: Sperm endonucleases, DNA integrity, ICSI, embryo quality

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