Comet assay may predict assisted reproductive techniques pregnancy success

San Francisco-A familiar laboratory assay is emerging as a reliable indicator of the likelihood of success in assisted reproductive techniques (ART). Researchers from the United States and Egypt have established a cutoff value for sperm DNA damage based on a simple comet (single cell gel electrophoresis) assay.

"We can use comet values to predict which couples have a good chance of getting pregnant," Hussein Abdelrazik, MD, TS (ABB), andrologist and supervising clinical embryologist in Cairo, and consultant at the Center for Reproductive Medicine, Cleveland Clinic, told Urology Times at the American Society for Reproductive Medicine annual meeting. "There is a significant correlation between comet assay results and the likelihood of pregnancy using ART."

The comet assay classifies sperm DNA damage in a given semen sample into mild, moderate, and severe strata, Dr. Abdelrazik said. Sperm showing 28% or greater severe DNA damage are not likely to result in pregnancy for couples undergoing intracytoplasmic sperm injection (ICSI). Sperm with less than 28% severe DNA damage are likely to produce pregnancy.

The comet assay is commonly used to assess DNA damage to single cells in research and clinical settings. The technique was first described in 1984, and is named for the appearance of damaged DNA that is displaced during electrophoresis from the cell like a comet's streaming tail. Both tail length and tail moment, the fraction of cellular DNA that has been fragmented and pulled into the tail during electrophoresis, are used to assess DNA damage.

Dr. Abdelrazik said that the comet assay correlates well with both TUNEL and assays of DNA fragmentation, but comet is simpler and easier to perform. Until now, there have been no data to show a cutoff value for sperm DNA damage and pregnancy outcomes in any form of ART using the comet assay.

Tail moment is key indicator

Investigators analyzed sperm samples from 48 couples who requested in vitro fertilization. The project was a collaboration between Kasr El-eni Hospital-Cairo University, the Nile Badwri Hospital in Cairo, and Cleveland Clinic. All of the couples had used ICSI in an attempt to achieve pregnancy.

Semen samples were given conventional and computerized analyses. A portion of each sample was then subjected to comet assay. Samples were divided into two fractions for duplicate analysis and 100 sperm cells from each fraction, or 200 cells from each man, were examined and scored for DNA fragmentation.

Sperm DNA fragmentation was classified as mild, moderate, or severe. The comet score is based on tail moment, which reflects the portion of sperm DNA that has been damaged and has migrated from within the cell during electrophoresis. A tail moment of greater than 40 was considered to indicate severe sperm DNA fragmentation.

Couples whose ICSI resulted in pregnancy had a higher percentage of mild sperm DNA fragmentation (p=.023) and a lower percentage of severe sperm DNA fragmentation (p=.01) than did couples who did not achieve pregnancy. There was no predictive value for moderate sperm DNA fragmentation.

Among couples with less than 28% severe sperm DNA damage, 51.6% of the women became pregnant, while none of the women in the couples with 28% or more severe sperm DNA damage became pregnant. The 28% cutoff (p<.017) showed 100% sensitivity and 53.1% specificity, Dr. Abdelrazik reported.

Additional data are needed to confirm the results, he said, but comet appears to provide a reliable, affordable, easy-to-perform predictor for pregnancy in ICSI.

"If the score is 27% or lower, the chances of getting pregnant are good," he concluded. "If the score is 28% or above, the chances of pregnancy are not good at all."