title: DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction.
abstract: OBJECTIVE
  To determine whether ligation-mediated real-time polymerase chain reaction (LM-RT-PCR), which combines LM-PCR, and RT-PCR, can detect sperm DNA fragmentation (DF) in human semen samples.
  
  
  METHODS
  Three-way comparison of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD), and LM-RT-PCR for detecting sperm DNA fragmentation.
  
  
  METHODS
  University hospital-based research laboratory.
  
  
  METHODS
  Twenty-five men presenting at an infertility clinic.
  
  
  METHODS
  Basic analysis of sperm concentration, motility, vitality, and morphology, with each semen sample equally divided into three aliquots that were evaluated for fragmentation using TUNEL, SCD, and LM-RT-PCR assays.
  
  
  METHODS
  In TUNEL and SCD assays, counts of the number of sperm with tetramethylrhodamine (TMR) red signals or no halo; in LM-RT-PCR results, evaluation of the threshold cycles (Ct) and relative fluorescence unit (RFU) values.
  
  
  RESULTS
  The median percentage of sperm with positive results for fragmentation in the TUNEL and SCD assays were 20.5% and 20.7%, respectively. To compare the accuracy of the TUNEL, SCD, and LM-RT-PCR assays, we divided the semen samples into two groups according to the TUNEL results: low and high percentage of sperm fragmentation. In the LM-RT-PCR results, the values of the cycles of threshold (Ct) and relative fluorescence unit (RFU) statistically significantly differed between the low and high percentage of sperm fragmentation groups. Comparisons among the TUNEL, SCD, and LM-RT-PCR assays revealed that the correlation patterns according to DNA fragmentation were similar in both the groups with high and low percentage of DNA fragmentation. Our morphologic analysis indicated that the fragmentation of sperm DNA did not appear to influence sperm morphology.
  
  
  CONCLUSIONS
  These results indicate that the LM-RT-PCR technique is another useful tool for detecting DNA fragmentation, a parameter of sperm quality in human semen alone or combined with TUNEL or SCD assays.
authors.affiliation: Fertility Center of CHA Gangnam Medical Center, College of Medicine, CHA University, Seoul, South Korea.
authors.forename: Jung Jin
authors.initials: JJ
authors.lastname: Lim
authors.forename: Jin Il
authors.initials: JI
authors.lastname: Lee
authors.forename: Dong Hwan
authors.initials: DH
authors.lastname: Kim
authors.forename: Seung-Hun
authors.initials: SH
authors.lastname: Song
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Kim
authors.forename: Woo Sik
authors.initials: WS
authors.lastname: Lee
authors.forename: Dong Ryul
authors.initials: DR
authors.lastname: Lee
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000235:genetics*; D053938:DNA Fragmentation; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D055101:Semen Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D013094:Spermatozoa / Q000502:physiology*
chemical_list: D004247:DNA
keywords: DNA fragmentation; LM-RT-PCR; SCD assay; TUNEL assay; human sperm