title: Development and evaluation of a qualitative reverse-transcriptase nested polymerase chain reaction protocol for same-day viral validation of human immunodeficiency virus type 1 ribonucleic acid in processed semen.
abstract: OBJECTIVE
  To develop a method for same-day validation of processed semen in the setting of assisted reproductive techniques (ART) with patients who are seropositive for human immunodeficiency virus, type 1 (HIV-1).
  
  
  METHODS
  Laboratory experiments.
  
  
  METHODS
  University hospital.
  
  
  METHODS
  Volunteers who are HIV-1 seronegative and seropositive.
  
  
  METHODS
  Evaluation of the sensitivity of a reverse-transcriptase (RT)-nested polymerase chain reaction (PCR) in HIV-1 RNA-positive blood plasma, in artificially infected blood plasma and semen, and in 85 semen samples of 29 HIV-1-seropositive volunteers. Semen was submitted to gradient separation, followed by swim-up.
  
  
  METHODS
  Qualitative detection of HIV-1 RNA in blood plasma and in different parts of semen preparation by using RT-nested PCR, PCR inhibition control by dilution of samples, and an internal control.
  
  
  RESULTS
  The detection limit of our PCR was 20 HIV-1 RNA copies per milliliter. Among seropositive patients, RNA was detected in 25% of fresh semen, 36.5% of seminal plasma, 27.5% of gradient supernatants, and 7.1% of final preparations before the migration-sedimentation stage. Positive final preparations were observed in patients who had blood viral loads of >/=20,000 HIV-1 RNA copies per milliliter. Inhibition was present in 17.6% of seminal plasma and in 20% gradient supernatants and in 2 final preparations among 69 tested. Among 25 preparations tested after the migration-sedimentation stage, 2 were positive (1 patient; 70,000 HIV-1 RNA copies per milliliter).
  
  
  CONCLUSIONS
  The RT-nested PCR detects low viral load and allows the validation of semen preparations of HIV-1-seropositive patients for ART on the day of sampling. For this purpose, the validation is performed on spermatozoa that are obtained after gradient separation before swim-up. Inhibition of the PCR must be controlled by using an internal control that is well-designed to explore the detection limit of the method.
authors.affiliation: Fertility Clinic, Department of Obstetrics and Gynaecology and Laboratory for Research on Human Reproduction, Campus Erasme, Universite Libre de Bruxelles (ULB), Brussels, Belgium.
authors.forename: Benoît
authors.initials: B
authors.lastname: Lesage
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Vannin
authors.forename: Serena
authors.initials: S
authors.lastname: Emiliani
authors.forename: Laurent
authors.initials: L
authors.lastname: Debaisieux
authors.forename: Yvon
authors.initials: Y
authors.lastname: Englert
authors.forename: Corinne
authors.initials: C
authors.lastname: Liesnard
mesh_terms: D002478:Cells, Cultured; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D027724:Reproductive Techniques, Assisted; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012661:Semen / Q000821:virology*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
chemical_list: D012367:RNA, Viral