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Insemination with Semen from HIV+ Men: Technical Considerations
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Insemination with Semen from HIV+ Men: Technical Considerations

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  1. Insemination with Semen from HIV+ Men: Technical Considerations Deborah J. Anderson, Ph.D. Dept of Medicine and Center for AIDS Research Harvard Medical School and Department of Obstetrics & Gynecology Boston University School of Medicine Boston, MA

  2. Composition of Semen • seminal plasma • spermatozoa (0-250 X 106) • immature germ cells • PMNs (0.1-10 X 106) • macrophages (0-2 X 106) • T lymphocytes (0-1 X 106)

  3. HIV-1 in Semen HIV-1 is found: • in cell-free seminal plasma • in association with seminal white blood cells (macrophages and CD4+ lymphocytes)

  4. HIV-1 in Semen cont’d Electron microscopy and early in situ PCR studies showed possible HIV-attachment/ infection of sperm. Quantitative molecular studies do not indicate a significant association of HIV-1 with viable, motile sperm.

  5. Summary of Molecular Studies on HIV-1 Association with Sperm: Published Papers

  6. Gradient/Swim-up Method Discontinuous Density Gradient Swim-up Seminal Plasma 47% Separation Medium Nonmotile Sperm, Immature Germ Cells WBCs Motile Sperm for Insemination 90% Separation Medium Swim-up Sperm Wash Medium Wash Pellet Motile Sperm

  7. Adhesive Seal Outer Tube Inner Tube Semen 47% Separation Medium 90% Separation Medium Double Tube

  8. Adhesive Seal Seminal Plasma Nonmotile Sperm Immature Germ Cells WBCs Motile Sperm for Insemination Double Tube Outer Tube Inner Tube 47% Separation Medium 90% Separation Medium

  9. Double Tube Outer Tube 90% Separation Medium Motile Sperm for Insemination

  10. Lab Prototype of Double Tube Parafilm seal 5 cc syringe 15 mL Falcon Tube

  11. General Methods • Semen samples from HIV-1 seronegative donors • Semen spiked with high concentrations of HIV-1 • MN strain propagated in H9 cell cultures • 1-105 TCID50 • 47%/90% ISolate or Percoll Gradient • Centrifuge 400 x g for 20 min • Swim-up for 1 hour

  12. General Methods cont’d • Motile sperm fraction analyzed for HIV-1 RNA by RT-PCR • Motile sperm fraction analyzed for infectious HIV-1 by quantitative culture on PBMC or H9 target cells for 28 days

  13. Design • Compare: • Double Tube • Swim-up • Gradient/Swim-up • Single Tube Gradient • Endpoints: • Exclusion of HIV-1 from motile sperm fraction • Sperm yield

  14. Compared to Swim-up Results Separation of HIV-1 from Motile Sperm Fraction by Various Methods (ISolate) 1.0 0.9 0.8 0.7 0.6 % of RNA Copies Spiked 0.5 0.4 0.3 P<0.01 0.2 0.1 P<0.01 0 Single Tube Gradient Swim-up Gradient/Swim-up n=5 n=10 n=23

  15. Separation of HIV-1 from Motile Sperm Fraction by Various Methods (ISolate) 4000 Compared to Double Tube P<0.01 3500 3000 2500 P<0.01 HIV RNA (Copy#) 2000 n=6 n=6 1500 n=6 1000 500 n=6 0 Single Tube Gradient Gradient/Swim-up Double Tube

  16. Removal of Infectious HIV-1 from Motile Sperm Fraction by Various Methods:Results with ISolate Single Tube Gradient Gradient/Swim-up Double Tube 100 1000 Amount HIV Removed (TCID50) 10000 100000

  17. Comparison of Sperm Yield from Different Sperm Separation Techniques:ISolate 30 Compared to Gradient/Swim-up P<0.0001 * Compared to Single Tube Gradient * P<0.05 20 P<0.05 Sperm Yield (%Total Motile Sperm) 10 n=15 n=9 n=9 0 Single Tube Gradient Double Tube Gradient/Swim-up

  18. Comparison of Sperm Yield from Different Sperm Separation Techniques:Percoll 30 Compared to Gradient/Swim-up P<0.0001 * Compared to Single Tube Gradient * P<0.05 20 P<0.05 Sperm Yield (%Total Motile Sperm) 10 n=15 n=9 n=12 n=6 n=6 0 Single Tube Gradient Double Tube Gradient/Swim-up

  19. Conclusions Sperm processing techniques reduce levels of HIV-1 in semen • Single gradient: 1,000X • Gradient/swim-up: 10,000X • Double tube 100,000X

  20. Bacteria Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma hominis Ureaplasma urealyticum Mycoplasma genitalium Treponema pallidum Haemophilus ducreyi Viruses Human immunodeficiency virus 1, 2 HTLV-1, 2 Herpes simplex virus 1, 2 Epstein-Barr virus Human Herpesvirus 6, 8 Human papillomavirus Hepatitis A, B, C, G virus Cytomegalovirus Sexually Transmissable Pathogens • Other • Trichomonas vaginalis • Candida albicans • Trepanema pallidum

  21. Location of Pathogens in Semen

  22. Technical considerations when working with semen • seminal plasma is immunosuppressive/toxic to T cells • semen contains more variable numbers and a higher concentration of nucleated cells than peripheral blood. • sperm DNA is tightly condensed in histones and is not efficiently extracted without the use of DTT. • macrophages pellet with sperm in Ficoll gradients. • Percoll inhibits RT-PCR detection of HIV-1