title: HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality.
abstract: OBJECTIVE
  Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay?
  
  
  CONCLUSIONS
  We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic.
  
  
  BACKGROUND
  The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency.
  
  
  METHODS
  The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses ( ITALIC! n = 3-5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia.
  
  
  METHODS
  Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles.
  
  
  RESULTS
  We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi-manual analysis software. Using this method, a cross-sectional study on 123 men showed no significant correlation between sperm concentration and sperm DNA damage, confirming the existence of hidden chromatin damage in men with apparently normal semen characteristics, and a significant correlation between percentage DNA in the tail and percentage of progressively motile spermatozoa. Finally, the use of DNA damage profiles helped to distinguish subjects between and within sperm concentration categories, and allowed a determination of the proportion of highly damaged cells.
  
  
  CONCLUSIONS
  The main limitations of the HT-COMET are the high, yet indispensable, investment in an automated liquid handling system and heating block to ensure accuracy, and the availability of an automated plate reading microscope and analysis software.
  
  
  CONCLUSIONS
  This standardized HT-COMET assay offers many advantages, including higher accuracy and evenness due to automation of sensitive steps, a 14.4-fold increase in sample analysis capacity, and an imaging and scoring time of 1 min/well. Overall, HT-COMET offers a decrease in total experimental time of more than 90%. Hence, this assay constitutes a more efficient option to assess sperm chromatin quality, paves the way to using this assay to screen large cohorts, and holds prognostic value for infertile patients.
  
  
  BACKGROUND
  Funded by the CIHR Institute of Human Development, Child and Youth Health (IHDCYH; RHF 100625). O.A. is a fellow supported by the Fonds de la Recherche du Québec - Santé (FRQS) and the CIHR Training Program in Reproduction, Early Development, and the Impact on Health (REDIH). B.R. is a James McGill Professor. The authors declare no conflicts of interest.
authors.affiliation: Department of Pharmacology &Therapeutics, McGill University, Montreal, QC, H3G 1Y6, Canada.
authors.forename: Océane
authors.initials: O
authors.lastname: Albert
authors.affiliation: Department of Pharmacology & Therapeutics, McGill University, Green Chemistry CFI Platform, Montreal, QC, H3G 1Y6, Canada.
authors.forename: Wolfgang E
authors.initials: WE
authors.lastname: Reintsch
authors.affiliation: Department of Urology, McGill University Health Centre, Montreal, QC, H4A 3J1, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Chan
authors.affiliation: Department of Pharmacology &Therapeutics, McGill University, Montreal, QC, H3G 1Y6, Canada Department of Obstetrics & Gynecology, McGill University, Montreal, QC, H4A 3J1, Canada bernard.robaire@mcgill.ca.
authors.forename: Bernard
authors.initials: B
authors.lastname: Robaire
mesh_terms: D002843:Chromatin; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007248:Infertility, Male / Q000235:genetics; D008297:Male; D011237:Predictive Value of Tests; D055101:Semen Analysis / Q000379:methods*; D012984:Software; D013094:Spermatozoa / Q000166:cytology* / Q000648:ultrastructure
chemical_list: D002843:Chromatin
keywords: DNA damage profiling; fertility; fertility prognosis; high throughput comet assay; sperm DNA damage; sperm chromatin quality