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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
- Source :
- Fertility and Sterility. 86:121-128
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
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). Design Laboratory experiments. Setting University hospital. Patient(s) Volunteers who are HIV-1 seronegative and seropositive. Intervention(s) 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. Main Outcome Measure(s) 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. Result(s) 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). Conclusion(s) 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.
- Subjects :
- Male
Reproductive Techniques, Assisted
Reverse Transcriptase Polymerase Chain Reaction
Sperm washing
HIV
Reproducibility of Results
Obstetrics and Gynecology
Semen
Viral Load
Biology
Sensitivity and Specificity
Virology
Reverse transcriptase
law.invention
Reverse transcription polymerase chain reaction
Reproductive Medicine
law
Blood plasma
Humans
RNA, Viral
Nested polymerase chain reaction
Viral load
Cells, Cultured
Polymerase chain reaction
Subjects
Details
- ISSN :
- 00150282
- Volume :
- 86
- Database :
- OpenAIRE
- Journal :
- Fertility and Sterility
- Accession number :
- edsair.doi.dedup.....3f5ae948664350cdce8d7d15b8ae62af