Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection*

Abstract

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.

Cumulus cell apoptosis changes with exposure to spermatozoa and pathologies involved in infertility

“…Similar results were reported by Nakahara et al A lower incidence of apoptosis in cumulus cells from fertilized oocytes compared with nonfertilized oocytes had been reported by several investigators (18,26,39). In contrast, Raman et al (29) showed that the fertilizing ability of oocytes (after ICSI) was positively correlated with the cumulus cell DNA status (Comet assay). In a recent report, Corn et al (31) did not find any correlation between apoptosis in cumulus cells and fertilization rate.…”

“…Moreover, our findings and previous work (29) suggest an indirect relationship between the fertilizing ability of the oocyte and the cumulus DNA status. Hakuno et al (41) also agreed that ''the increase in cumulus cell apoptosis may produce a fall away of cumulus cells from the oocyte, and would be the signal that the oocyte has attained competence for fertilization.''…”

“…Other results were controversial and showed a positive relationship between cumulus cell DNA fragmentation and fertilization by ICSI (29), whereas a lack of correlation was found between the incidence of apoptotic cumulus cells and the outcome of ICSI when analyzing only fertilization and embryo quality (30). A negative correlation was reported by Saito et al (24) and Corn et al (31) between embryo quality and apoptotic rates of cumulus cells, with no influence on pregnancy and implantation.…”

Evaluation of the immature human oocytes from unstimulated cycles in polycystic ovary syndrome patients using a novel scoring system

“…A relationship between the DNA fragmentation of the cumulus cells and nuclear maturity has been proposed (31). Others have proposed the content of inhibin A and B and activin A in the follicular fluid as biochemical markers for oocyte quality and fertilizing capacity (32).…”

Clinical value of DNA fragmentation evaluation tests under ART treatments

“…Comet assay uses single cell gel electrophoresis (SCGE) to analyze DNA fragmentation in individual cells, was first introduced in 1984 by Ostling and Johanson (16) who used neutral buffer conditions to study double-stranded DNA breaks (17). This assay is extensively used in somatic cells to measure genotoxic damage, especially single and double strands breaks and was originally applied to sperm by Singh (18).…”