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