Diapositiva 1

1
(No Transcript)
2
(No Transcript)
3
ASSISTED CONCEPTION IN HIV-DISCORDANT COUPLES

• Augusto E Semprini, MD
• University of Milan Medical School
• University College of London
• Chelsea Westminster Hospital, London

4
Alessandra Vucetich, MD Simona Fiore,
MDValeria Savasi, MD Claudio Castagna,
MDSimonetta Giuntelli, MDMonica Oneta,
BiologistTiziana Persico, Biologist
5
REMOVAL OF p18 IMMUNOREACTIVE CELLS FROM THE
SEMEN HTLV-III/LAV SEROPOSITIVE MENAugusto E
Semprini, A Vucetich, E Morandi, CL Parravicini,
G Pardi and AE Beer. Colloque INSERM, Vol. 154,
1987, pp 462

• A cytospin preparation of washed spermatozoa,
  supernatant and the second fraction of the
  ejaculate were tested against a monoclonal
  anti-p18 antibody by immunoperoxidase technique.
  Washed sperm of seropositive and seronegative men
  were non-reactive, while many mononuclear cells
  and those in the second fraction of seropositive
  males were strongly reactive. Experiments are
  under way to test the possibility of safe
  intrauterine insemination with processed semen of
  HIV-positive men desiring a child.

6
TRANSMISSION OF BLOOBORNE VIRUSES THROUGH ART IS
NOT A NEGLIGIBLE PROBLEM
7
PREVALENCE () OF VIRUSES TRANSMISSIBLE WITH ART

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

8
THREE MAJOR QUESTIONS

• 1. SHOULD IT BE DONE ?
• 2. HOW SAFE IT IS ?
• 3. HOW SHOULD WE DO IT ?

9
1. SHOULD IT BE DONE ?
10
AIDS casesin 20-34 years old by sex and by year
of diagnosis
HIV/AIDS surveillance in Europe-Mid Year report
2000, n 63
11
(No Transcript)
12
WOMEN AT THE HIGHEST RISK OF ACQUIRING HIV
THROUGH INFECTED SEMEN ARE THE HABITUAL PARTNERS
OF HIV-POSITIVE MEN
13
EPIDEMIOLOGY OF HIV INFECTION IN ITALY

• HETEROSEXUAL TRANSMISSION HAS BECOME THE SECOND
  LEADING CAUSE OF HIV ACQUISITON AFTER USE OF
  INJECTING DRUGS
• THREE OUT OF FOUR NEW CASES OF SEXUAL INFECTION
  ARE FEMALES

14
Life expectancy of individuals infected with HIV
under medication exceeds now 30 years from
seroconversion
15
Natural conception in HIV-negative women with
HIV-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.

16
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

17
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)

18
SPECIAL CONSIDERATIONS REARDING HIV AND ASSISTED
REPRODUCTIVE TECHNOLOGIESAmerican Society for
Reproductive MedicineFertility and Sterility
Vol. 62, No. 5, November 1994 USE OF SEMEN FROM
HIV-POSITIVE PARTNERS FOR INSEMINATION OF
SERONEGATIVE WOMEN PARTNERSThe Committee
recommends that the physician counsel the couple
regarding the risks to the woman and offspring
through homologous insemination by any means and
that the couple consider the options of donor
insemination, adoption, or child-free living.
19
2. HOW SAFE IT IS ?
20
RISK OF VIRAL INFECTION WITH ART FOR HEALTH CARE
PROVIDERS

• THERE IS NO REPORT OF ACQUISITION OF SEXUALLY
  TRANSMISSIBLE OR BLOODBORNE VIRUSES BY HEALTH
  CARE PROVIDERS DURING ART PROCEDURES

21
INSEMINATION OF HIV-NEGATIVE WOMEN WITH PROCESSED
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)

22
COMBINED PROCESSING METHOD TO REDUCE SEMINAL 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

23
CRITICAL STEPS FOR DETECTION OF VIRAL 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

24
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
25
HIV-DNA AND RNA BY PCR (LDL 240 copies/ml)

• Seminal fraction HIV-DNA HIV-RNA
• Unprocessed semen 127/254 180/240
• Washed spermatozoa 0/254 0/540

26
HIV-1 RNA IN SEMEN AND BLOOD PLASMA (LOWER
DETECTION LIMIT 100 COPIES/ML)
27
HIV-1 DNA IN SEMEN AND PBMC BY PCR ASSAY(LOWER
DETECTION LIMIT 50 COPIES/ML)
28
DETECTION OF HIV-1 DNA IN SPERMATOZOA PELLET OF A
SEROPOSITIVE MAN BY IS- PCR
29
DETECTION OF HIV-1 DNA IN SPERMATOZOA PELLET OF A
SERONEGATIVE MAN BY IS- PCR
30
ABSENCE OF HEPATITIS C VIRUS AND DETECTION OF
HEPATITIS G VIRUS/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.

31
Detection of viral nucleic acids in sperm
32
3. HOW SHOULD WE DO IT ?
33
In HIV-discordant couples ART has two different
scopes

• Protection for uninfected partner
• Overcoming an infertility problem

34
LIST 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

35
LIST 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).

36
FREQUENT INFERTILITY PROBLEMS IN 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

37
WHICH ART FOR HIV-DISCORDANT COUPLES ?

• IUI TIMED ON SPONTANEOUS OVULATION
• absence of infertility factors
• woman with lt 35 y.o.
• normal hormonal profile
• gt1 million spermatozoa after washing
• IUI WITH MULTIPLE FOLLICULAR INDUCTION
• clinical indication for the use of fertility
  drugs
• woman with gt 35 y.o.
• failure to conceive after 3 timed IUI attempts
• gt1 million spermatozoa after washing

38
WHICH ART FOR HIV-DISCORDANT COUPLES ?

• IVF-ET
• severe pelvic infertility factor
• lt 1 million spermatozoa after washing
• no pregnancy after repeated IUI attempts
• ICSI
• lt 0.5 million spermatozoa after washing
• severe asthenospermia or necrospermia
• (incompatible with complete washing
  processing)

39
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

40
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

41
FACTORS TO BE CONSIDERED IN SELECTING ART 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

42
PROBLEMS FOR HIV-DISCORDANT COUPLES 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

43
1. SHOULD IT BE DONE ?2. HOW SAFE IT IS ?3. HOW
SHOULD WE DO IT ?
44
LIFEIS A FATAL SEXUALLY TRANSMITTED
DISEASE (R V SHORT)
45
(No Transcript)