2. 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

3. Composition of Semen seminal plasma spermatozoa (0-250 X 10 6 ) immature germ cells PMNs (0.1-10 X 10 6 ) macrophages (0-2 X 10 6 ) T lymphocytes (0-1 X 10 6 )

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

5. 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.

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

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

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

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

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

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

12. General Methods Semen samples from HIV-1 seronegative donors Semen spiked with high concentrations of HIV-1 MN strain propagated in H9 cell cultures 1-10 5 TCID 50 47%/90% ISolate or Percoll Gradient Centrifuge 400 x g for 20 min Swim-up for 1 hour

13. 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 General Methods cont’d

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

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

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

17. Removal of Infectious HIV-1 from Motile Sperm Fraction by Various Methods: Results with ISolate Amount HIV Removed (TCID 50 ) Single Tube Gradient Gradient/Swim-up Double Tube 100000 10000 1000 100

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

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

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

21. Sexually Transmissable Pathogens 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 Other – Trichomonas vaginalis – Candida albicans – Trepanema pallidum

22. Location of Pathogens in Semen Seminal Plasma WBC/ Epithelial Cells Sperm HIV-1Yes ?/No CMVYes No HPVYes ?/No HBVYes No HCVYes ?/No HSV-2Yes No

23. 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