An investigation of the potential effect of vacuoles in human sperm on DNA damage using a chromosome assay and the TUNEL assay

BACKGROUND: The aims of this study were to establish whether individual differences exist in the frequency and size of vacuoles found in human sperm and to ascertain whether such vacuoles are involved in causing DNA damage. METHODS: Morphologically normal sperm were obtained from 15 IVF and 2 ICSI patients and 3 fertile donors. (i) Sperm heads were analyzed for the presence of vacuoles under a 1000x differential interference contrast microscope. (ii) In three patients and two donor samples, structural chromosomal damage was evaluated in normal sperm containing large vacuoles and selected at 1000x magnification for injection into mouse oocytes. (iii) In 10 patients and two donor samples, confocal laser microscopy detected DNA damage in sperm-exhibiting large vacuoles and stained with an in situ cell death detection kit. RESULTS: (i) Vacuoles were observed in almost all normal sperm from patient and donor ejaculates and were mainly located at the tip or middle area of the sperm heads. However, average incidence of normal sperm exhibiting large vacuoles was 4.6 and 4.2% in the patient and donor groups, respectively. (ii) Sperm chromosome assays did not reveal any differences in the incidence of structural chromosome aberrations between sperm exhibiting large vacuoles and those without them (9.1 versus 4.1%). (iii) No significant difference in frequency of TUNEL-positive cells was found between normal sperm with large vacuoles and those without them in the samples examined. Among 227 sperm exhibiting large vacuoles, only 7 cells were TUNEL positive. CONCLUSION: The results showed that large vacuoles were not responsible for DNA damage, suggesting that intra-cytoplasmic injection of morphologically selected sperm may not be required for patients who produce high-quality semen.