insemination with semen from hiv+ men: technical considerations
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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. Composition of Semen. seminal plasma - PowerPoint PPT PresentationTRANSCRIPT
Insemination with Semen from HIV+ Men:Technical Considerations
Deborah J. Anderson, Ph.D.
Dept of Medicine and Center for AIDS ResearchHarvard Medical School and
Department of Obstetrics & Gynecology Boston University School of Medicine
Boston, MA
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)
HIV-1 in Semen
HIV-1 is found:
• in cell-free seminal plasma
• in association with seminal white blood cells (macrophages and CD4+ lymphocytes)
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.
Mermin et al. (1991) 23 Ficoll 0
Baccetti et al. (1993) 2 Ficoll & Swim-up 0
Scofield et al. (1994) 3 Percoll 100
Quayle et al. (1997) 8
13
Swim-up & IM Beads
Swim-up & Percoll
0
0
Lasheeb et al. (1997) 6 Percoll & Swim-up 0
Marina et al. (1998, 2001)1 607 Percoll or PureSperm & Swim-up 2.5
Tachet et al. (1999) 26 Percoll 42
Kim et al. (1999) 11 Medicol & Swim-up 0
Hanabusa et al. (2000) 12 Percoll & Swim-up 0
Baccetti et al. (1993) 2 Ficoll & Swim-up 100
Lasheeb et al. (1997) 6 Percoll & Swim-up 0
Chrystie et al. (1998) 20 Percoll & Swim-up 20
Tachet et al. (1999) 41 Percoll 15
Kim et al. (1999) 10 Medicol & Swim-up 0
Hanabusa et al. (2000) 12 Percoll & Swim-up 0
HIV-1 RNA and/or DNA
Pasquier et al. (2000) 40 PureSperm & Swim-up 0
HIV-1 DNA
HIV-1 RNA
Author n Separation Technique % Positive
A. Published Papers
Quayle et al. (1997)
1Represents results from published article and updated in abstract
2Round cell contamination reported
Summary of Molecular Studies on HIV-1 Association with Sperm: Published Papers
Gradient/Swim-up Method
Sw
im-u
p
Wash Pellet
Seminal Plasma
47% Separation Medium
90% Separation Medium
Motile Sperm
Motile Sperm for Insemination
Sperm Wash Medium
Nonmotile Sperm,Immature Germ CellsWBCs
DiscontinuousDensity Gradient
Swim-up
Double Tube
47% SeparationMedium
90% SeparationMedium
Outer Tube Inner Tube
Adhesive Seal
Semen
Double Tube
47% SeparationMedium
90% SeparationMedium
Outer Tube Inner Tube
Seminal Plasma
Nonmotile SpermImmature Germ Cells
WBCs
Motile Spermfor Insemination
Adhesive Seal
Double Tube
90% SeparationMedium
Outer Tube
Motile Spermfor Insemination
Lab Prototype of Double Tube
Parafilm seal
5 cc syringe
15 mL Falcon Tube
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
• 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
Design
• Compare:
• Double Tube
• Swim-up
• Gradient/Swim-up
• Single Tube Gradient
• Endpoints:
• Exclusion of HIV-1 from motile sperm fraction
• Sperm yield
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
% o
f R
NA
Cop
ies
Sp
iked
Single TubeGradient
Swim-up Gradient/Swim-up
1.0
P<0.01
P<0.01
Compared to Swim-up
n=23n=5 n=10
Results
Separation of HIV-1 from Motile Sperm Fraction by Various Methods (ISolate)
HIV
RN
A (
Cop
y#)
Single TubeGradient
Gradient/Swim-up Double Tube
n=6
n=6
n=6
P<0.01
P<0.01
Compared to Double Tube
0
500
1000
1500
2000
2500
3000
3500
4000
n=6
Removal of Infectious HIV-1 from Motile Sperm Fraction by Various Methods:
Results with ISolate
Am
oun
t H
IV R
emov
ed (
TC
ID50
)
Single TubeGradient
Gradient/Swim-up Double Tube
100000
10000
1000
100
Comparison of Sperm Yield from Different Sperm Separation Techniques:
ISolate
0
10
20
30
Sp
erm
Yie
ld (
%T
otal
Mot
ile S
per
m)
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
Comparison of Sperm Yield from Different Sperm Separation Techniques:
Percoll
0
10
20
30
Sp
erm
Yie
ld (
%T
otal
Mot
ile S
per
m)
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=12 n=6
Conclusions
Sperm processing techniques reduce levels of HIV-1 in semen
• Single gradient: 1,000X
• Gradient/swim-up: 10,000X
• Double tube 100,000X
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
Location of Pathogens in Semen
Seminal Plasma
WBC/
Epithelial Cells
Sperm
HIV-1 Yes Yes ?/No
CMV Yes Yes No
HPV Yes Yes ?/No
HBV Yes Yes No
HCV Yes Yes ?/No
HSV-2 Yes Yes No
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