Mitochondrial DNA Deletion in Human Oocytes

Abstract

To compare the incidence of mitochondrial DNA (mtDNA) deletion in normal and unfertilized oocytes. Laboratory study. Metaphase II oocytes were obtained during IVF cycles. Normal oocytes were obtained from women undergoing IVF treatments because of non-obstructive azoospemia but no sperm could be retrieved from testicular biopsy. The other group of oocytes came from patients undergoing IVF treatment but the oocytes were unfertilized. The presence of mtDNA in single human oocyte was confirmed by standard PCR with a set of primers (forward primer L467 and reverse primer H725) for amplifying a fragment outside the hotspot of deletion area. The nested PCR reaction was employed to detect the presence of mtDNA deletions, and was carried out on those oocytes with a positive band from the first PCR reaction. The two pairs of primers (outer primers L8282 + H13650 and inner primers L8334 + H13578) flanked the region of the K5S mtDNA deletion. If the K5S deletion was present, the nested PCR product was 256 bp long. In the absence of the deletion, there was no DNA amplication. All experiments included water-only negative controls and a positive control. The PCR products were separated on 2% agarose gel stained with ethidium bromide. PCR products were confirmed by DNA sequencing. Thirty normal and 155 unfertilized MII oocytes were obtained from 4 and 62 donors respectively. The incidence of K5S mtDNA deletion in the normal oocytes was 23.3% which was statistically not different from the 34.6% in the unfertilized oocytes (p = 0.228). When the comparison was confined to donors of unfertilized oocytes < 35 years of age (n = 97), the incidence of K5S mtDNA deletion remained comparable (p = 0.626) between the two groups. When only the unfertilized oocytes were studied, women ≥= 35 years of age had a significantly higher incidence of K5S mtDNA deletion (46.6%) when compared with younger women (27.8%) (p = 0.018). The incidence of mtDNA deletion in unfertilized oocytes was similar to that of the normal oocytes, but was higher in women with advanced age. These findings were in concordance with the observation that the rate of accumulation of somatic mtDNA mutations increased during the aging process and hence the incidence of mtDNA deletion can reflect ovarian aging.