Download Technical issues for semen processing and assisted reproductive

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
• 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.
Summary of Molecular Studies on HIV-1 Association with Sperm: Published Papers
Author
n
Separation Technique
% Pos itive
A. Publis hed Papers
HIV-1 DNA
Mermin et al. (1991)
23
Ficoll
0
0
Baccetti et al. (1993)
2
Ficoll & Swim-up
Scofield et al. (1994)
3
Perco ll
Quayle et al. (1997)
8
Swim-up & IM Beads
0
Quayle et al. (1997)
13
Swim-up & Percoll
0
6
Perco ll & Swim-up
0
Perco ll o r PureSperm & Swim-up
2.5
Lash eeb et al. (1997)
Marina et al. (1998, 2001)
1
607
100
Tachet et al. (1999)
26
Perco ll
42
Kim et al. (1999)
11
Medicol & Swim-up
0
Han abu sa et al. (2000)
12
Perco ll & Swim-up
0
HIV-1 RNA
Baccetti et al. (1993)
2
Ficoll & Swim-up
100
Lash eeb et al. (1997)
6
Perco ll & Swim-up
0
Chry stie et al. (1998)
20
Perco ll & Swim-up
20
Tachet et al. (1999)
41
Perco ll
15
Kim et al. (1999)
10
Medicol & Swim-up
0
Han abu sa et al. (2000)
12
Perco ll & Swim-up
0
40
Pu reSp erm & Swim-up
0
HIV-1 RNA and/or DNA
Pasq uier et al. (2000)
1Repres en ts resu lts from pub lis hed article and u pda ted in abs tract
2Rou nd cell contamination reported
Gradient/Swim-up Method
Discontinuous
Density Gradient
Swim-up
Seminal
Plasma
Nonmotile Sperm,
Immature Germ Cells
WBCs
Swim-up
47% Separation
Medium
90% Separation
Medium
Wash Pellet
Motile Sperm
Motile Sperm
for Insemination
Sperm Wash
Medium
Double Tube
Adhesive Seal
Outer Tube
Inner Tube
Semen
47% Separation
Medium
90% Separation
Medium
Double Tube
Adhesive Seal
Outer Tube
Inner Tube
Seminal Plasma
47% Separation
Medium
Nonmotile Sperm
Immature Germ Cells
WBCs
90% Separation
Medium
Motile Sperm
for Insemination
Double Tube
Outer Tube
90% Separation
Medium
Motile Sperm
for 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
General Methods cont’d
• Motile sperm fraction analyzed for HIV-1 RNA by RTPCR
• Motile sperm fraction analyzed for infectious HIV-1 by
quantitative culture on PBMC or H9 target cells for 28
days
Design
• Compare:
• Double Tube
• Swim-up
• Gradient/Swim-up
• Single Tube Gradient
• Endpoints:
• Exclusion of HIV-1 from motile sperm fraction
• Sperm yield
Results
Separation of HIV-1 from Motile Sperm Fraction
by Various Methods (ISolate)
% of RNA Copies Spiked
1.0
Compared to Swim-up
0.9
0.8
0.7
0.6
0.5
0.4
0.3
P<0.01
0.2
0.1
P<0.01
0
Single Tube
Gradient
n=23
Swim-up
n=5
Gradient/Swim-up
n=10
Separation of HIV-1 from Motile Sperm
Fraction by Various Methods (ISolate)
4000
P<0.01
3500
HIV RNA (Copy#)
Compared to Double Tube
3000
2500
P<0.01
2000
n=6
1500
n=6
1000
500
0
n=6
Single Tube
Gradient
Gradient/Swim-up
Double Tube
Removal of Infectious HIV-1 from Motile Sperm
Fraction by Various Methods:
Results with ISolate
Amount HIV Removed (TCID50)
100
1000
10000
100000
Single Tube
Gradient
Gradient/Swim-up
Double Tube
Sperm Yield (%Total Motile Sperm)
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
* P<0.05
20
10
n=15
0
Single Tube
Gradient
n=9
Double Tube
n=9
Gradient/Swim-up
Sperm Yield (%Total Motile Sperm)
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
* P<0.05
20
10
n=15
n=12
0
Single Tube
Gradient
n=9
n=6
Double Tube
n=6
Gradient/Swim-up
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
• Other
– Trichomonas vaginalis
– Candida albicans
– Trepanema pallidum
• 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
Location of Pathogens in Semen
Seminal
Plasma
HIV-1
Yes
WBC/
Epithelial Cells
Yes
Sperm
CMV
Yes
Yes
No
HPV
Yes
Yes
?/No
HBV
Yes
Yes
No
HCV
Yes
Yes
?/No
HSV-2
Yes
Yes
No
?/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