ASSISTED CONCEPTION IN HIV-DISCORDANT COUPLES

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

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. 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 $ 100 Couples where the male is infected after two years (10.000 episodes)

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 1994USE OF SEMEN FROM HIV-POSITIVE PARTNERS FOR INSEMINATION OF SERONEGATIVE WOMEN PARTNERS“The 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 < 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

38. 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)

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)