title: Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment.
abstract: OBJECTIVE
  Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)?
  
  
  CONCLUSIONS
  Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study.
  
  
  BACKGROUND
  Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown.
  
  
  METHODS
  Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013.
  
  
  METHODS
  Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated.
  
  
  RESULTS
  Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05).
  
  
  CONCLUSIONS
  A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage.
  
  
  CONCLUSIONS
  Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE.
  
  
  BACKGROUND
  No personal or direct financial support has been received for any of this work. The authors declare no competing interests.
  
  
  BACKGROUND
  N/A.
authors.affiliation: Andrology and IVF Laboratory, Department of Surgery (Urology), University of Utah, Salt Lake City, UT 84108, USA.
authors.forename: L
authors.initials: L
authors.lastname: Simon
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: K
authors.initials: K
authors.lastname: Murphy
authors.forename: S
authors.initials: S
authors.lastname: Ge
authors.forename: J
authors.initials: J
authors.lastname: Hotaling
authors.forename: K I
authors.initials: KI
authors.lastname: Aston
authors.forename: B
authors.initials: B
authors.lastname: Emery
authors.forename: D T
authors.initials: DT
authors.lastname: Carrell
mesh_terms: D002843:Chromatin / Q000378:metabolism; D020552:Comet Assay / Q000379:methods*; D003430:Cross-Sectional Studies; D004249:DNA Damage; D053938:DNA Fragmentation; D005260:Female; D005307:Fertilization in Vitro; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D007248:Infertility, Male / Q000235:genetics*; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D055101:Semen Analysis / Q000379:methods*; D020554:Sperm Injections, Intracytoplasmic
chemical_list: D002843:Chromatin
keywords: Comet assay; TUNEL assay; assisted reproduction treatment outcomes; histone retention; sperm DNA damage