ASSISTED CONCEPTION IN HIV-DISCORDANT COUPLES

ASSISTED CONCEPTION IN ASSISTED CONCEPTION IN HIV-DISCORDANT COUPLESHIV-DISCORDANT COUPLES

Augusto E Semprini, MDAugusto E Semprini, MD

University of Milan Medical SchoolUniversity of Milan Medical School

University College of LondonUniversity College of London

Chelsea & Westminster Hospital, LondonChelsea & Westminster Hospital, London

Alessandra Vucetich, MD Alessandra Vucetich, MD

Simona Fiore, MD Simona Fiore, MD

Valeria Savasi, MD Valeria Savasi, MD

Claudio Castagna, MDClaudio Castagna, MD

Simonetta Giuntelli, MDSimonetta Giuntelli, MD

Monica Oneta, BiologistMonica Oneta, Biologist

Tiziana Persico, BiologistTiziana Persico, Biologist

REMOVAL OF p18 IMMUNOREACTIVE CELLS FROM THE REMOVAL OF p18 IMMUNOREACTIVE CELLS FROM THE SEMEN HTLV-III/LAV SEROPOSITIVE MENSEMEN HTLV-III/LAV SEROPOSITIVE MENAugusto E Semprini, A Vucetich, E Morandi, CL Parravicini, G Pardi Augusto E Semprini, A Vucetich, E Morandi, CL Parravicini, G Pardi and AE Beer. Colloque INSERM, Vol. 154, 1987, pp 462and AE Beer. Colloque INSERM, Vol. 154, 1987, pp 462

A cytospin preparation of washed spermatozoa, supernatant and A cytospin preparation of washed spermatozoa, supernatant and

the second fraction of the ejaculate were tested against a the second fraction of the ejaculate were tested against a

monoclonal anti-p18 antibody by immunoperoxidase technique. monoclonal anti-p18 antibody by immunoperoxidase technique.

Washed sperm of seropositive and seronegative men were non-Washed sperm of seropositive and seronegative men were non-

reactive, while many mononuclear cells and those in the second reactive, while many mononuclear cells and those in the second

fraction of seropositive males were strongly reactive. fraction of seropositive males were strongly reactive. Experiments Experiments

are under way to test the possibility of safe intrauterine insemination are under way to test the possibility of safe intrauterine insemination

with processed semen of HIV-positive men desiring a child.with processed semen of HIV-positive men desiring a child.

TRANSMISSION OF TRANSMISSION OF

BLOOBORNE VIRUSES BLOOBORNE VIRUSES

THROUGH ART IS NOT A THROUGH ART IS NOT A

NEGLIGIBLE PROBLEMNEGLIGIBLE PROBLEM

PREVALENCE (%) OF VIRUSES PREVALENCE (%) OF VIRUSES TRANSMISSIBLE WITH ARTTRANSMISSIBLE WITH ART

•CMV 50 % CMV 50 % •HIV 0.1 - 1 %HIV 0.1 - 1 %•HBV 2 %HBV 2 %•HHV8 10- 50 %HHV8 10- 50 %•TTV 2 - 90 %TTV 2 - 90 %•HGV 1.5 %HGV 1.5 %•HCV 2 %HCV 2 %

THREE MAJOR QUESTIONSTHREE MAJOR QUESTIONS

1. SHOULD IT BE DONE ?1. SHOULD IT BE DONE ?

2. HOW SAFE IT IS ?2. HOW SAFE IT IS ?

3. HOW SHOULD WE DO IT ?3. HOW SHOULD WE DO IT ?

AIDS casesin 20-34 years old

by sex and by year of diagnosis

0

2000

4000

6000

8000

10000

12000

1996 1997 1998 1999 giu-00

TOTMaleFemale

HIV/AIDS surveillance in Europe-Mid Year report 2000, n 63

WOMEN AT THE HIGHEST RISK

OF ACQUIRING HIV THROUGH

INFECTED SEMEN ARE THE

HABITUAL PARTNERS OF HIV-

POSITIVE MEN

EPIDEMIOLOGY OF HIV EPIDEMIOLOGY OF HIV INFECTION IN ITALYINFECTION IN ITALY

•HETEROSEXUAL TRANSMISSION HAS HETEROSEXUAL TRANSMISSION HAS BECOME THE SECOND LEADING BECOME THE SECOND LEADING CAUSE OF HIV ACQUISITON AFTER CAUSE OF HIV ACQUISITON AFTER USE OF INJECTING DRUGSUSE OF INJECTING DRUGS

•THREE OUT OF FOUR NEW CASES OF THREE OUT OF FOUR NEW CASES OF SEXUAL INFECTION ARE FEMALESSEXUAL INFECTION ARE FEMALES

Life expectancy of individuals Life expectancy of individuals

infected with HIV under infected with HIV under

medication exceeds now 30 medication exceeds now 30

years from seroconversionyears from seroconversion

Natural conception in HIV-negative women with Natural conception in HIV-negative women with HIV-infected partnersHIV-infected partnersL Mandelbrot, I Heard, E Henrion-Geant, R Henrion (Lancet 1997; 349: 850)

We followed 104 consecutive pregnancies in 92 HIV-negative women with HIV-positive partners. Couples were advised to pinpoint ovulation in order to reduce possible exposure. Seroconversion was observed in two women at 7 months of pregnancy and in two others post partum. Some authors advocate intrauterine insemination with semen from the HIV-infected males, but the risk of this must be measured against the low background risk of natural conception. Stringent standard of safety must be required before inseminating potentially infected semen.

100 Couples where the male is infected after two years (10.000

episodes)

• IUI 384-641 $ (4-8%) • hMG IUI 1428-2380 (9-14%)

Costs for 100 ATTEMPTS• IUI 500= 50 000 PREGNANCIES • hMG IUI 1900= 190 000 PREGNANCIES

Benefits• 3 to 100 adult HIV- infections (300 000 $ for an

HIV infected adult) $ 900-000 to 30 000 000 • Cost for an infected child 175 000 $

NO REPORT OF FEMALE OR CONGENITAL HIV INFECTION

(up to September 2001)

2.500 IUI (Europe 3.100)

200 IVF (Europe 400)

100 ICSI (Europe 200)

SPECIAL CONSIDERATIONS REARDING HIV AND SPECIAL CONSIDERATIONS REARDING HIV AND

ASSISTED REPRODUCTIVE TECHNOLOGIESASSISTED REPRODUCTIVE TECHNOLOGIESAmericanAmerican Society for Reproductive MedicineSociety for Reproductive Medicine

Fertility and Sterility Vol. 62, No. 5, November 1994Fertility and Sterility Vol. 62, No. 5, November 1994

USE OF SEMEN FROM HIV-POSITIVE PARTNERS FOR USE OF SEMEN FROM HIV-POSITIVE PARTNERS FOR

INSEMINATION OF SERONEGATIVE WOMEN PARTNERSINSEMINATION OF SERONEGATIVE WOMEN PARTNERS

“The Committee recommends that the physician counsel “The Committee recommends that the physician counsel

the couple regarding the risks to the woman and the couple regarding the risks to the woman and

offspring through homologous insemination by any offspring through homologous insemination by any

means and that the couple consider the options of donor means and that the couple consider the options of donor

insemination, adoption, or child-free living.”insemination, adoption, or child-free living.”

RISK OF VIRAL INFECTION WITH ART FOR HEALTH CARE PROVIDERS

THERE IS NO REPORT OF THERE IS NO REPORT OF

ACQUISITION OF SEXUALLY ACQUISITION OF SEXUALLY

TRANSMISSIBLE OR BLOODBORNE TRANSMISSIBLE OR BLOODBORNE

VIRUSES BY HEALTH CARE VIRUSES BY HEALTH CARE

PROVIDERS DURING ART PROVIDERS DURING ART

PROCEDURESPROCEDURES

INSEMINATION OF HIV-NEGATIVE WOMEN WITH INSEMINATION OF HIV-NEGATIVE WOMEN WITH PROCESSED SEMEN OF HIV-POSITIVE PARTNERSPROCESSED SEMEN OF HIV-POSITIVE PARTNERS

85 HIV-discordant couples were screened for fertility;

29 women were found suitable for a timed

insemination course with the processed semen of

their HIV-positive partner. None of the inseminated

women seroconverted and 17 pregnancies were

achieved in 15 women. All 10 infants born to these

mothers remain HIV seronegative. The eldest child is

now three years old, healthy and uninfected. (Semprini et al. - Lancet 1992; 340: 1317-19)

COMBINED PROCESSING METHOD TO REDUCE COMBINED PROCESSING METHOD TO REDUCE SEMINAL HIV DNA AND RNA VIRAL CONTENTSEMINAL HIV DNA AND RNA VIRAL CONTENT

• Liquified semen is diluted in culture media

• It is centrifuged against gradient to separate mononuclear cells (round cells, seminal leukocytes, non-motile sperm)

• The pellet is re-suspended and washed

• The pellet is overlaid with nutrient medium and kept for 1 hr at 5% CO

• Motile spermatozoa are collected by pipetting

• A fraction of the final aliquot is tested for HIV RNA by nuclear acid sequence-based amplification (NASBA) method with a final sensitivity of 250 viral copies/ml

CRITICAL STEPS FOR DETECTION OF CRITICAL STEPS FOR DETECTION OF VIRAL NUCLEIC ACIDS IN SEMENVIRAL NUCLEIC ACIDS IN SEMEN

• Concentration of viral RNA and DNA• Sensitivity of detecting methods• Presence of inhibitors of detecting

methods• Separation of seminal fractions• Presence of nucleases• Presence of non-specific viral inhibitors• Presence of antiviral drugs

AMOUNT OF HIV-1 IN SPERM FRACTION AFTER SPERM PROCESSING TECHNIQUES (normal semen spiked with 106 pg as by Abbot HIV-Ag ELISA)

Anderson and Semprini, Fertil Steril 1993, abstract

TECHNIQUE AMOUNT REDUCTION

Simple cell wash 80 pg 10.000-fold

Gradient centrifugation Undetectable >100.000-fold

Swim-up Undetectable >100.000-fold

HIV-DNA AND RNA BY PCRHIV-DNA AND RNA BY PCR (LDL 240 copies/ml)(LDL 240 copies/ml)

Seminal fraction HIV-DNA HIV-RNA

Unprocessed semen 127/254 180/240

Washed spermatozoa 0/254 0/540

HIV-1 RNA IN SEMEN AND BLOOD PLASMA HIV-1 RNA IN SEMEN AND BLOOD PLASMA (LOWER DETECTION LIMIT 100 COPIES/ML)(LOWER DETECTION LIMIT 100 COPIES/ML)

Plasma(blood)

N 50

WholeSemen

N 48

SeminalPlasma

N 46

NSCs

N 38

Spermatozoabefore

migration

N 38

FinalSpermatozoa

fraction

N 46

21(42%)

0 4(8.7%)

1(2.6%)

0 0

HIV-1 DNA IN SEMEN AND PBMC BY HIV-1 DNA IN SEMEN AND PBMC BY PCR ASSAYPCR ASSAY

(LOWER DETECTION LIMIT 50 COPIES/ML)(LOWER DETECTION LIMIT 50 COPIES/ML)

PBMC

n 50

Wholesemen

n 49

NSCs

n 48

Spermatozoabefore

migration

n 46

Finalspermatozoa

fraction

n 40

50 0 7(14.6%)

0 0

DETECTION OF HIV-1 DNA IN DETECTION OF HIV-1 DNA IN SPERMATOZOA PELLET OF A SPERMATOZOA PELLET OF A

SEROPOSITIVE MAN BY IS- PCRSEROPOSITIVE MAN BY IS- PCR

DETECTION OF HIV-1 DNA IN DETECTION OF HIV-1 DNA IN SPERMATOZOA PELLET OF A SPERMATOZOA PELLET OF A

SERONEGATIVE MAN BY IS- PCRSERONEGATIVE MAN BY IS- PCR

ABSENCE OF HEPATITIS C VIRUS AND DETECTION OF HEPATITIS G ABSENCE OF HEPATITIS C VIRUS AND DETECTION OF HEPATITIS G VIRUS/GB VIRUS C RNA SEQUENCES IN THE SEMEN OF INFECTED MENVIRUS/GB VIRUS C RNA SEQUENCES IN THE SEMEN OF INFECTED MENAE Semprini, T Persico, V Thiers, M Oneta, R Tuveri, P Serafini,A Boschini, S Giuntelli, G Pardi and C Brechot. J Infect Dis 1998; 177(4): 848-54

Serum and semen from 90 anti-HCV-positive drug users were tested (27

infected with HIV) for HCV and HGV/GBV-C RNAs by polymerase chain

reaction (PCR) assay, hybridisation, and Sequence analysis. Semen was

processed into round cells, seminal plasma and spermatozoa.

Fifty-six patients were HCV-viraemic, but HCV-RNA was not identify in their

seminal fractions. However, PCR inhibitors were found in the semen of 34

of these men. Twenty-eight patients had HGV/GBV-C RNA in their blood and

for 24 of them, ejaculates were available for analysis. HGV/GBV-C RNA was

found in the seminal plasma of 6 of 12 samples free from PCR inhibitors.

These results agree with the low risk of sexual transfer of HCV and provide

preliminary evidence for the presence of HGV/GBV-C in semen.

Detection of viral nucleic acids in spermDetection of viral nucleic acids in sperm

VIRAL NUCLEICACID

NON-SPERMATOZOACELLS

SEMINALPLASMA

SPERMATOZOA

HCV-RNA NO NO/YES NO

HGV-RNA NO YES NO

HIV-RNA YES YES NO

HIV-DNA YES NO NO

TTV-DNA YES NO NO

In HIV-discordant couples ART has two different scopes

• Protection for uninfected partner

• Overcoming an infertility problem

LIST OF TESTS FOR THE MANLIST OF TESTS FOR THE MAN

• Urethral swab for pathogenic bacteria, Chlamydia t. and Mycoplasma h. and semen bacteriological colture

• ELISA and Western-blot for HIV

• HIV viraemia (quantitative PCR assay)

• CD4 and CD8 assessment

• Haemocytometric analysis, platelet and white cells counts

• HBsAg, anti-HBs, anti-HBc, anti-HCV

• HCV viraemia, anti-HGV and HGV PCR (if anti-HCV positive)

• AST and ALT

• VDRL-TPHA

• Prolactin , LH, FSH, TSH, Testosterone

• Anti-CMV IgM and IgG antibody determination

• Semen analysis

LIST OF TESTS FOR THE WOMANLIST OF TESTS FOR THE WOMAN• Cervical swab for pathogenic bacteria, Chlamydia t. and

Mycoplasma h.

• Hysterosalpingogram or explorative laparoscopy

• ELISA and Western-blot tests for HIV

• HIV-p24 antigen titre or HIV DNA PCR testing

• Haemocytometric analysis, platelet and white cells counts

• HBsAg, anti-HBs, anti-HBc, anti -HCV

• HGV RNA and anti-E

• AST and ALT

• VDRL-TPHA

• LH, FSH, TSH, between the 3rd and the 5th day of the cycle

• Progesterone and Prolactin on the 22nd and 24th day of the cycle

• Anti-CMV (IgG and IgM)

• Cervical cytopathological smear (Pap test).

FREQUENT INFERTILITY PROBLEMS FREQUENT INFERTILITY PROBLEMS IN COUPLES WITH HIVIN COUPLES WITH HIV

• Chronic genital tract infections

• Tubal damage up to bilateral obstruction

• Poor spermatozoa recovery after washing

• Complete washing procedure unfeasible

(necrospermia, severe asthenospermia)

• No conception after repeated spontaneous

or IUI attempts

WHICH ART FOR HIV-DISCORDANT COUPLES ?

IUI TIMED ON SPONTANEOUS OVULATION • absence of infertility factors

• woman with < 35 y.o.• normal hormonal profile

• >1 million spermatozoa after washing

IUI WITH MULTIPLE FOLLICULAR INDUCTION• clinical indication for the use of fertility drugs

• woman with > 35 y.o.• failure to conceive after 3 timed IUI attempts

• >1 million spermatozoa after washing

WHICH ART FOR HIV-DISCORDANT COUPLES ?

IVF-ET

• severe pelvic infertility factor • < 1 million spermatozoa after washing

• no pregnancy after repeated IUI attempts

ICSI• < 0.5 million spermatozoa after washing• severe asthenospermia or necrospermia

(incompatible with complete washing processing)

ALL THEY NEED IS SEMEN WASHING?ALL THEY NEED IS SEMEN WASHING?

•semen washing and timed IUI does not requires

intensive follicular monitoring, carries no risk of

multifetality but has a 10% pregnancy rate per

attempt

•semen washing and IUI with induced multiple

follicular maturation, requires follicular monitoring

and expensive drugs, carries a 20% risk of

multifetality but has a 15-20% pregnancy rate per

attempt

ALL THEY NEED IS SEMEN WASHING?ALL THEY NEED IS SEMEN WASHING?

•semen washing and IVF requires all the above,

plus egg retrieval under sedation, costly

laboratory procedures, carries a 20-30% risk of

multifetality and has a 25-40% pregnancy rate per

cycle

•semen washing and ICSI involves all the above

plus additional laboratory costs, carries a 20-30

risk of multifetality and has a 30-60% pregnancy

rate

FACTORS TO BE CONSIDERED IN SELECTING FACTORS TO BE CONSIDERED IN SELECTING ART FOR HIV-DISCORDANT COUPLESART FOR HIV-DISCORDANT COUPLES

•FERTILITY OF THE COUPLE

•EXPERIENCE OF THE CENTER

•EXPECTED PREGNANCY RATE PER ART METHOD

•TIMING OF PCR RESULTS

•COSTS OF DIFFERENT ART PROCEDURES

• ACCEPTANCE OF MULTIFETAL OUTCOME

• LOGISTICS OF THE COUPLE

• NUMBER OF PREVIOUS ART ATTEMPTS

PROBLEMS FOR HIV-DISCORDANT PROBLEMS FOR HIV-DISCORDANT COUPLES ACCESSING TO ARTCOUPLES ACCESSING TO ART

•CONFLICTING COUNSELLING FROM DIFFERENT PROVIDERS

OF CARE

•ANXIETY OVER THE POSSIBILITY OF INFECTION

•DIFFICULTIES IN COMPLETING THE PRE-INSEMINATION

SCREENING (COST, CONFIDENTIALITY, LOGISTICS)

•DIFFICULTIES IN REACHING THE CENTER

•LONG WAITING LIST LEADING TO SPONTANEOUS

ATTEMPTS AT CONCEPTION

•POSSIBILITY OF CYCLE CANCELLATION DUE TO POOR

OVARIAN RESPONSE OR HIV PCR TECHNICAL PROBLEMS

LIFE

IS A FATAL

SEXUALLY TRANSMITTED

DISEASE

(R V

SHORT)

ASSISTED CONCEPTION IN HIV-DISCORDANT COUPLES

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