THE IMPACT OF DOUBLE-STRANDED SPERM DNA BREAKS ON ICSI OUTCOME

Abstract

We sought to evaluate specific sperm DNA damage, double-stranded DNA breaks (dsDNA), and its effect on embryo development and implantation. Over a year-long period, a prospective pilot study was carried out on sperm samples to evaluate the proportion of dsDNA on samples screened by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) to assess total DNA fragmentation. Once a correlation was established, we extrapolated dsDNA values retrospectively onto patients who had their ejaculates screened by TUNEL to evaluate clinical outcome. Samples from consenting couples were screened for dsDNA rates by neutral Comet assay using an in-house protocol; 200 spermatozoa were assessed per patient. These samples were also assessed by TUNEL using a commercially available kit, analyzing at least 500 spermatozoa per patient. ICSI was performed in the standard fashion. The pilot study reported an average total DNA fragmentation of 11.3±6% by TUNEL and an average dsDNA of 2.2±3% by neutral Comet. The results showed a linear relationship between the overall SCF and dsDNA rates (R2=0.96). This equation was applied to extrapolate the dsDNA levels from 573 normozoospermic men (volume of 2.6±1 mL, concentration of 42.3±33x106/mL, 43.1±10% motility, and 4.2±1% normal morphology) with an average SCF of 14.2±8%. Therefore, on the basis of this preliminary test, we established a dsDNA threshold of 3%. A total of 417 couples (maternal age, 37.0±4 yrs; paternal age, 38.6±5 yrs) underwent 777 ICSI cycles and presented with dsDNA levels of 1.8±0.6%. These cycles had a 73.0% fertilization rate, a 12.7% (123/966) implantation rate, and a 23.2% (102/440) clinical pregnancy rate (CPR), of which 15 were lost (14.7%), leaving a 19.7% (87/440) ongoing/delivery rate. There were 155 couples with dsDNA levels of 4.2±1%. These couples underwent 268 ICSI cycles with a comparable maternal age of 37.3±5 years, but with older male partners at 41.3±8 years of age (P<0.001). These cycles had a comparable fertilization rate of 71.0%, implantation rate of 11.4% (53/464), and clinical pregnancy rate of 24.3% (44/181). However, these couples were much more likely to lose their pregnancy at 29.5% (13/44; P = 0.03), leaving an ongoing/delivery rate of 17.1% (31/181). These findings provide further evidence that dsDNA has an important role in the success of a pregnancy generated by ICSI. dsDNA damage has been linked to aneuploidy and consequent pregnancy loss. The use of an assay in a laboratory setting to screen exclusively for dsDNA would help to identify paternally linked aneuploidy that traditional screening would only report as total SCF.