To determine whether oocytes from women harbor deletions in mitochondrial DNA (mtDNA) and whether deleted mtDNA is more common in oocytes from older women than oocytes from younger women.
A polymerase chain reaction (PCR)-based strategy, which depends on deletions approximating otherwise widely separated primers to demonstrate mtDNA deletions in individual oocytes, was used.
Yale In Vitro Fertilization Clinic and Laboratory at Yale University School of Medicine.
Main Outcome Measures
Primers flanked a region of the mitochondrial genome in which long direct repeated sequence predispose to deletions. The primers identified the 0.5-kb “common” deletion. Deleted mtDNA was represented by a 0.5-kb band when primers separated by 5 kb were used. Control reactions used primers that amplify mtDNA outside the deletion hotspot. Positive controls included brain and/or muscle from aged individuals, and negative controls included fetal tissue and DNA-free blanks. Nested primers confirmed the specificity of the deleted product.
Unfertilized oocytes, muscle, and brain tissue contained PCR products consistent with deleted mtDNA. Fetal tissue lacked the mtDNA deletion product. Deleted mtDNA was detected in single oocytes. Oocytes from older women were more likely to contain deleted mtDNA then oocytes from younger women.
Deleted mtDNA in unfertilized oocytes may serve as a marker of oocyte senescence.