M O'Connell1, N McClure, S E M Lewis. 1. School of Medicine, Obstetrics & Gynaecology, Queen's University Belfast, Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, UK.
Abstract
BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.
BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.
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