Microfluidic Sperm Selection Is an Effective Method for Improving Embryo Developmental Competence in Ivf With Older Patients

Abstract

OBJECTIVE: We sought to evaluate specific sperm DNA damage, doublestranded DNA breaks (dsDNA), and its effect on embryo development and implantation.DESIGN: 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.MATERIALS AND METHODS: 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.RESULTS: The pilot study reported an average total DNA fragmentation of 11.3AE6% by TUNEL and an average dsDNA of 2.2AE3% by neutral Comet. The results showed a linear relationship between the overall SCF and dsDNA rates (R 2 ¼0.96). This equation was applied to extrapolate the dsDNA levels from 573 normozoospermic men (volume of 2.6AE1 mL, concentration of 42.3AE33x10 6 /mL, 43.1AE10% motility, and 4.2AE1% normal morphology) with an average SCF of 14.2AE8%. Therefore, on the basis of this preliminary test, we established a dsDNA threshold of 3%.A total of 417 couples (maternal age, 37.0AE4 yrs; paternal age, 38.6AE5 yrs) underwent 777 ICSI cycles and presented with dsDNA levels of 1.8AE0.6%. 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 4.2AE1%. These couples underwent 268 ICSI cycles with a comparable maternal age of 37.3AE5 years, but with older male partners at 41.3AE8 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 ¼ 0.03), leaving an ongoing/delivery rate of 17.1% (31/181).CONCLUSIONS: 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.