Review Sperm washing techniques address the fertility needs of HIV-seropositive men: a clinical review

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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 26 November 2004 Review Sperm washing techniques address the fertility needs of HIV-seropositive men: a clinical review Dr Mark Sauer Mark Sauer is a tenured professor of Obstetrics & Gynecology at Columbia University in New York. He is Vice Chairman of the Department, and Director of the Division of Reproductive Endocrinology. He is also Program and Laboratory Director of the Center for Women s Reproductive Care, the IVF unit at the University. Since fellowship, his research interests have focused on egg and embryo donation, having developed programmes at the University of California Los Angeles (UCLA) and the University of Southern California (USC) before moving to New York in His research involving women of advanced reproductive age (40 55 years) has been instrumental in redefining fertility care in older patients, while providing insight into the importance of oocyte age on successful implantation. Mark V Sauer Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, Division of Reproductive Endocrinology, New York, NY, USA Correspondence: Centre for Women s Reproductive Care at Columbia University, 1790 Broadway, 2nd Floor, New York, NY 10019, USA. Tel: ; Fax: ; mvs9@columbia.edu Abstract It is estimated that nearly 40 million people worldwide are infected with HIV. HIV/AIDS prevalence among young people is high, with youth under age 25 years accounting for approximately half of all new infections. Nearly 1 million Americans are HIV-seropositive. Today, HIV-seropositive individuals live active and productive lives despite their illness, largely a result of prescribed highly active antiretroviral therapy (HAART). Most individuals are of reproductive age, and many desire to have children. Various assisted reproductive techniques have been used to reduce or eliminate infectious elements known to be present in semen so that isolated spermatozoa can be safely inseminated or used for intracytoplasmic sperm injection into oocytes. Presently, several European centres and a few US groups offer assistance to HIV seropositive men and their seronegative partners by performing either intrauterine insemination (IUI) or IVF. Since 1987, more than 3600 published attempts have been reported in which processed spermatozoa from HIV-seropositive men were used to establish pregnancy in HIV-seronegative women. Although the data remain observational, sperm washing techniques appear to be relatively safe and effective, offering HIV-serodiscordant couples an opportunity to have children. Keywords: HIV, IUI, IVF ICSI, serodiscordant, sperm washing Introduction It is estimated that nearly 40 million people are presently infected with HIV. Most are under the age of 25 years (UNAIDS, 2004). HIV infection occurs primarily in young, reproductively healthy individuals. The epidemic has affected hundreds of thousands of American men (CDC, 2001). Although many of these men are gay, others are engaged in heterosexual relationships and may desire children. A report of 2864 HIV-infected adults in the United States interviewed as part of the HIV Cost and Services Utilization Study confirmed this presumption, with approximately one-third of participants stating a strong desire to have children (Chen et al., 2001). However, safe sexual practices require the constant use of condoms, which reduces the risk of transmitting virus to the uninfected partner, yet also precludes any hope of pregnancy. Reproductive choices for HIV-serodiscordant couples are limited. Providing assisted reproductive care to couples in whom the male partner is known to be HIV-seropositive remains a subject of intense controversy (Anderson, 1999; Englert et al., 2001; Sauer, 2003). Donor sperm insemination and adoption represent the only recommended safe options for couples wishing to have a family. Yet, reproductive drive is very strong, and patients are known to take unreasonable risks in order to have a baby. It is therefore not surprising that HIV seroconversions of uninfected partners have occurred as a result of timed intercourse without a condom. Sperm washing and insemination techniques Sperm preparation techniques, commonly referred to as sperm washing, followed by intrauterine insemination (IUI), 135

2 has been suggested as a means of reducing the likelihood of horizontal transmission of HIV. Its clinical use in HIV infected men was first reported over a dozen years ago (Semprini et al., 1992). Although subsequent published results from Milan (Semprini et al., 1997, 1999) were encouraging, few practitioners in the United States were willing to offer IUI therapy for fear of infecting the seronegative partner and child. Early on, the Centres for Disease Control (CDC) recommended against treating HIV-serodiscordant couples following an alleged seroconversion in a woman inseminated with washed spermatozoa from her HIV-seropositive husband (CDC, 1990). That position was recently reiterated (Duerr, 2003). Renewed calls for prohibition relate more to concerns regarding the safety of sperm washing and its quality control, as well as the relative absence of long-term follow-up data on outcomes. In many locales within the United States, intentionally inseminating a woman with spermatozoa from an HIV-seropositive man constitutes a criminal act. Unfortunately, many prohibitions were based upon information regarding the natural history of HIV infection prior to 1990, a time in which the disease was typically considered to be a terminal illness. Thus, the professional, civil, and possible criminal liabilities associated with treating HIV-serodiscordant couples continue to dissuade many clinicians from providing care. Processed spermatozoa for IUI have been used in Europe, and numerous series (Table 1) document pregnancy success without HIV seroconversions (Brechard et al., 1997; Semprini et al., 1997; Vernazza et al., 1997; Marina et al., 1998; Tur et al., 1999; Bujan et al., 2001; Daudin et al., 2001; Marina, 2001; Weigel et al., 2001; Delvigne et al., 2003; Gilling-Smith et al., 2003) The large number of patients successfully treated is impressive, and these results may have influenced the American College of Obstetricians and Gynecologists (ACOG) and the American Society for Reproductive Medicine (ASRM) to revise earlier statements against caring for HIVserodiscordant couples. Both groups now recommend adopting more tolerant policies of non-discrimination (ACOG Committee Opinion, 2002; Ethics Committee of the ASRM, 2002). Separating virus from spermatozoa The basis for IUI treatment rests on the premise that isolated motile spermatozoa used in washed inseminations do not carry HIV. Spermatozoa lack the CD4 receptor and the CCR5 and CXCR4 co-receptors needed for the virus to gain entry into the host cell. Non-motile cells, particularly CD4 positive lymphocytes and macrophages, are known to exist in seminal plasma, and are principle targets for infection with HIV (Van Voorhis et al., 1991; Quayle et al., 1997). Density gradient centrifugation combined with sperm swim-up has been used in centres offering assisted reproduction for years in order to permit the intrauterine placement of spermatozoa. Washed swim-up techniques effectively separate the motile fraction of spermatozoa from the seminal plasma and non-motile cells, and have been reported to reduce HIV RNA and proviral DNA to undetectable concentrations (Kim et al., 1999; Hanabusa et al., 2000). Most practitioners of fertility care are familiar with the technique, as IUI therapy is commonly performed for the treatment of unexplained infertility. However, concerns do exist regarding the ability of a HIV surface glycoprotein (gp120) to bind to galactosyl-alkylacylglyerol (GalAAG), a glycolipid structurally related to galactosylceramide present on the surface membrane of spermatozoa (Brogi et al., 1998). This molecular complex has been associated with HIV infection in cells that lack CD4, CCR5, and CXCR4 proteins (i.e. neurons and colonic epithelium), and could potentially implicate spermatozoa as vectors of infection. This might also explain the mechanism for how HIV-like particles become associated with spermatozoa when the virus is co-cultured with gametes, as Table1. Summary of published results on HIV-1 serodiscordant couples undergoing washed sperm intrauterine inseminations. All values are numbers. Study Cycles Patients Pregnancies Births Ongoing Infection treated Semprini et al. (1997) Marina et al. (1998; 2001) Tur et al. (1999) Gilling-Smith et al. (2003) Vernazza et al. (1997) Weigel et al. (2001) Bujan et al. (2001) Daudin et al. (2001) Brechard et al. (1997) Delvigne et al. (2003) Total Cycles = attempts at treatment. Pregnancies = clinical pregnancies established, including miscarriages and ectopic pregnancies. Births = deliveries post-treatment. Ongoing = ongoing pregnancies beyond 20 weeks gestation. Infection = infections with HIV-1 resulting in seroconversions in mothers or offspring.

3 viewed by electron microscopy (Baccetti et al., 1998). However, other investigators have failed to demonstrate these relationships (Politch and Anderson, 2002; Pudney et al., 1998). HIV-1 proviral DNA or RNA has not been identified in spermatozoa taken from the highly motile fraction of processed spermatozoa (Politch et al., 2004), and it is presumed that the single case report of HIV infection resulting from an IUI (CDC, 1990) occurred as a result of contamination from non-motile cellular elements that exist in the specimen post-wash. Prior to swim-up, up to 8% of processed specimens have been noted to contain detectable HIV-RNA (Hanabusa et al., 2000). IUI or IVF There are advantages and disadvantages to both IUI and IVF approaches. Although more cases of IUI have been reported, it remains undetermined whether one approach is superior to the other in terms of safety. Intrauterine insemination is technically easier, much less expensive, and with repetitive applications approaches the efficacy of IVF in carefully selected patients. However, IUI therapy requires millions of sperm cells to be placed above the natural immunological barrier of the cervix. Catheters navigating the endocervix and endometrial cavity may create bleeding, which potentially could further increase risk. As previously mentioned, it is difficult to ensure that all CD4 positive cells are eliminated from the washed preparation. Paired semen samples obtained from HIV-seropositive men commonly express virus, even when plasma viral counts are very low (Coombs et al., 1998). Men with chronic HIV infection often have abnormal semen profiles (Pena et al., 2003a). Hypogonadism and endocrine disorders are relatively common in men with HIV (Dobs et al., 1988; Sellmeyer and Grunfeld, 1996). In other cases, men may be prescribed androgens to improve well-being and lessen muscle wasting (Bhasin et al., 2000). In such instances, the choice of IUI therapy may be questioned, since the number of procedures necessary to achieve pregnancy is greater and the overall success of IUI is generally reduced in men with persistently abnormal semen analyses (Ohl et al., 2003). Finally, as mentioned previously, several jurisdictions in the United States have regulations that prohibit insemination of HIV-infected material. Thus, although IUI therapy may be more simplistic and less expensive than IVF, and could be used in fertile couples or those with less severe fertility problems (i.e. anovulation), physicians may not be willing to inseminate women for fear of civil or criminal liability. Intracytoplasmic sperm injection (ICSI) has commonly been used to address male factor infertility and is available at most centres providing assisted reproduction. Similar to preparing spermatozoa for IUI, discontinuous density gradient centrifugation techniques are used prior to ICSI and only the resultant motile spermatozoa found in the supernatant following swim-up are selected. Typically, fewer than 20 spermatozoa are injected per case of IVF ICSI, compared with tens of millions inseminated during an IUI. Furthermore, since IVF ICSI represents a mechanical means to fertilize oocytes, it may be argued that insemination is not performed. This may be helpful in locations where laws prohibit insemination procedures. Thus, IVF ICSI represents another alternative to reduce the risk of horizontal transmission of HIV. However, IVF ICSI is associated with high cost, and has its own unique risks and complications. Therefore, patients choosing this technique must be aware of the increased incidence of multiple births and ovarian hyperstimulation syndrome reported in serodiscordant couples undertaking IVF ICSI (Pena et al., 2003b). The published results from clinical studies utilizing IVF or IVF ICSI are reviewed and summarized in Table 2. (Semprini et al., 1999; Semprini, 2000; Journet et al., 2001; Loutradis et al., 2001; Weigel et al., 2001; Delvigne et al., 2003; Gilling- Smith et al., 2003; Marina et al., 2003; Ohl et al., 2003; Pena et al., 2003c; Morshedi et al., 2003). Table 2. Summary of published results on IVF or IVF ICSI in HIV-1 serodiscordant couples. All values are numbers. Study Cycles Patients Pregnancies Births Ongoing Infection treated Semprini et al. (1999, 2000) Gilling-Smith et al. (2003) Weigel et al. (2001) Delvigne et al. (2003) Total IVF ICSI therapy Marina et al. (2003) Weigel et al. (2001) Jonannet et al. (2001) Loutradis et al. (2001) Ohl et al. (2003) Pena et al. (2003) Morshedi et al. (2003) Gilling-Smith et al. (2003) Total See Table 1 for definitions of parameters. 137

4 Establishing a programme for treatment In 1997, the Ethics Committee of Columbia Presbyterian Medical Centre first considered and granted a request to allow HIV-serodiscordant couples access to assisted reproductive care (Sauer, 2003). A protocol was next approved by the Institutional Review Board of the medical centre, allowing a study of safety and treatment efficacy (Sauer and Chang, 2002). Enrolled subjects were under active medical surveillance for their HIV illness by infectious disease specialists in internal medicine and where appropriate, they were prescribed antiviral medications. Plasma HIV RNA viral counts and CD4 status were ascertained. Men were infected through a variety of ways, including transfusion therapy, sex and intravenous drug use. Female partners were HIV tested and required to be seronegative. Couples stated that they were using condoms and practicing safe sex. Women underwent thorough pelvic examinations including PAP smears and cervical cultures, and serum oestradiol and follicle stimulating hormone concentrations were drawn to evaluate the appropriateness of IVF therapy. Table 3 profiles the first 50 consecutive couples entering the programme and tallies their attitudes regarding various ways of having a child together (Klein et al., 2003). IVF ICSI and follow-up Standard pre-cycle procedures routinely used in assisted reproduction were provided. Needle aspiration of the oocytes was timed h following human chorionic gonadotrophin (HCG) injection. A fresh semen sample was used for ICSI and processed by centrifuging it through a discontinuous density gradient. After centrifugation of the gradient tubes, the pellet was resuspended in human tubal fluid (HTF), washed and again suspended in HTF. The specimen was then subjected to a final swim-up procedure in order to recover the most motile fraction for use in ICSI. Semen preparation was performed using a class II biological hood located in a separate laboratory outside of embryology. Following swim-up, the purified specimen was brought into the IVF ICSI suite for use. Ultrasound guided transcervical embryo transfers at 72 h occurred in all cases. Patients were tested for pregnancy 12 days following transfer. Serial blood testing (HIV-proviral DNA or ultrasensitive HIV-RNA polymerase chain reaction; PCR) was repeated throughout each trimester of pregnancy. At delivery and 3 months postpartum, mothers were again tested using HIV-DNA or HIV- RNA PCR, sensitive enough to detect virus down to the level of <50 copies/ml blood. Newborns were also tested at birth and 3 months age. Patients failing to become pregnant or women who experienced spontaneous abortion were asked to repeat their HIV tests 3 and 6 months later. Results during the initial 5 years In preliminary published experience (Sauer, 2002) with IVF ICSI there were no seroconversions in women (n = 34) or offspring (n = 25) following 55 embryo transfers. Treatment efficacy was similar to normal controls undergoing IVF ICSI for the treatment of male factor infertility. Most pregnancies occurred within the first three attempts at IVF ICSI. Half (17/34) of all couples making it to retrieval successfully achieved a viable pregnancy. There were no seroconversions in any of the 34 patients receiving one or more of the 55 embryo transfers. All 25 delivered infants were HIV negative at birth and at 3 months of age. A follow-up series of over 100 attempts noted similar results (Pena et al., 2003c). Table 4 describes the clinical outcomes. Again, pregnancies typically occurred within 3 cycles of treatment, with cumulative pregnancy rates per couple greater than 50%. Not all serodiscordant couples have performed well, however, as patients who are poor responders to ovarian stimulation and women over 40 years of age have not produced as many pregnancies as younger patients. Therefore, couples with fertility problems that are associated with poor IVF success rates should be made aware of the financial costs of care and the low success rate of treatment given the unknown risk of HIV transmission Table 3. Demographic characterization of HIV-1 serodiscordant couples interested in assisted reproductive care. Profile of 50 consecutive couples (Klein et al., 2003). Figures in parentheses are percentages. Age of men (years; mean ± SD) 38.0 ± 6 (range 26 51) Age of women (years; mean ± SD) 34.5 ± 5 (range 24 45) Married 4/50 (88) Duration of relationship (years; mean ± SD) 9.0 ± 5 (range 1 20) Couples with child together prior to presentation 9/50 (18) Children conceived after knowing HIV status 3/9 (33) Couples attempting timed intercourse to conceive 4/50 (8) Couples preferring donor sperm to timed intercourse 24/50 (48) Favour posthumous attempts to conceive if partner died 22/50 (44) Couples discussed single parenting risk 45/50 (90) Couples discussed adoption of child if both parents died 29/50 (58) Couples desiring to have multiple children through assisted reproduction 33/50 (66) Couples willing to discuss experience with other discordant couples 36/50 (72) 138

5 Table 4. Clinical results of the first 113 consecutive treatment attempts using IVF ICSI in HIV-1 serodiscordant couples (Pena et al., 2003). No. of couples treated 61 No. of initiated cycles 113 No. attempts per couple a 1.9 ± 1.1 (1 6) Cancellation rate due to poor ovarian response (%) 11.5 No. oocytes aspirated per retrieval a 17.1 ± 9.5 (2 47) No. mature oocytes suitable for ICSI a 13.9 ± 8.1 (1 42) No. fertilized oocytes per ICSI case a 9.3 ± 5.5 (0 24) No. embryos transferred per attempt a 3.5 ± 1.1 (1 6) Couples with cryopreserved embryos (%) 32.2 No. embryos cryopreserved per couple a 5.1 ± 3.5 (1 19) Overall clinical pregnancy rate per embryo transfer (%) 44.8 Ongoing and delivered pregnancy rate per embryo transfer (%) 36.5 Cumulative pregnancy rate for couples over repeated attempts (%) 54.1 No. of seroconversions in treated women 0 No. of seroconversions in delivered offspring 0 a Values are mean ± SD (range). that accompanies IVF ICSI. Although controversial, oocyte donation may be yet another alternative to allow couples at high risk for IVF failure the opportunity for pregnancy with their husbands spermatozoa (Pena et al., 2003d). Conclusions The purpose of all clinical trials involving various sperm separation techniques is to provide HIV-serodiscordant couples an opportunity to have a child without risk of viral transmission. Both IUI and IVF have been suggested as preventive measures to avoid infection in HIVserodiscordant couples intent on reproducing. It remains undetermined as to whether one procedure is superior to the other with respect to both safety and efficacy. The commonly accepted principles of health care ethics include consideration of respect for autonomy, nonmaleficence, beneficence, fidelity and justice (Beauchamp and Childress, 1994; Sauer, 2003). Each of these tenets should be individually considered in making a decision to treat. Informed and rationale decision-making must occur in every case of intervention. A lengthy discussion of the natural history of HIV infection, and the biology of transmission should precede attempts at treatment. Reproductive alternatives, including artificial insemination with donor spermatozoa, adoption, and childless living should also be offered, as not all patients will choose sperm preparation techniques once cognizant of the potential danger for infection. It is important to engage patients in a frank dialog with respect to the investigational nature of this form of treatment while providing them enough information to exercise informed consent. It is essential to safeguard the patient s right to act intentionally and without controlling influences. Most importantly, participants need to understand that no procedure is risk free, as all carry a possibility for transmitting infection. References Anderson DJ 1999 Assisted reproduction for couples infected with the human immunodeficiency virus type 1. Fertility and Sterility 72, Bacetti B, Benedetto A, Collodel G et al The debate on the presence of HIV-1 in human gametes. Journal of Reproductive Immunology 41, Beauchamp T, Childress J 1994 Principles of Biomedical Ethics, 4th edition. Oxford University Press, New York. Bhasin S, Storer TW, Javanbakht M et al Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testosterone levels. Journal of the American Medical Association 283, Brechard N, Galea P, Silvy F, Amram M et al Etude de la localization du VIH dans le sperme. Contraception Fertility and Sexuality 25, Brogi A, Presentini R, Moretti E et al New insights into the interaction between the gp120 and the HIV receptor in human sperm. Journal of Reproductive Immunology 41, Bujan L 2001 Reproduction, laboratory and HIV-1. Fourth International Symposium on AIDS and Reproduction, Genoa. Centers for Disease Control 1990 Epidemiologic notes and reports. HIV-1 infection and artificial insemination with processed semen. MMWR Morbidity Mortality Weekly Report 249, Centers for Disease Control and Prevention 2001 HIV/AIDS Surveillance Report. CDC and Prevention, Atlanta, 13, Chen JL, Phillips KA, Kanouse DE et al Fertility desires and intentions of HIV-positive men and women. Family Planning Perspectives 33, Committee on Ethics of the American College of Obstetricians and Gynecologists (ACOG) 2002 Ethics in Obstetrics and Gynecology, ACOG, Washington, D.C., pp Coombs RW, Speck CE, Hughes JE et al Association between culturable human immunodeficiency virus type 1 in semen and HIV-1 RNA levels in semen and blood: evidence for compartmentalization of HIV-1 between semen and blood. Journal of Infectious Disease 177, Daudin M, Pasquier C, Izopet J et al Le Protocole ANRS 096: prise en charge en assistance medicale a la procreation des couples serodifferents don t l homme est infecte par le VIH. Reproduction Human Hormones 14, Delvigne A, Barlow P, Manigart Y et al Fertility treatment in 139

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