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Sperm washing techniques address the fertility needs of HIV-seropositive men: a clinical review
RBMOnline - Vol 10. No 1. 2005 135-140 Reproductive BioMedicine Online; www.rbmonline.com/Article/1541 on web 26 November 2004
Review Sperm washing techniques address the fertility needs of HIV-seropositive men: a clinical review 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 1995. 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. Dr Mark Sauer 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: +1 212 3059175; Fax: +1 646 7568280; e-mail: [email protected]
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),
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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.
serodiscordant couples. Both groups now recommend adopting more tolerant policies of non-discrimination (ACOG Committee Opinion, 2002; Ethics Committee of the ASRM, 2002).
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.
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 HIV-
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
Separating virus from spermatozoa
Table1. Summary of published results on HIV-1 serodiscordant couples undergoing washed sperm intrauterine inseminations. All values are numbers.
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Study
Cycles
Patients treated
Pregnancies
Births
Ongoing Infection
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
1954 458 155 92 46 143 62 93 11 5 3019
623 233 67 36 16 64 28 39 – 5 1111
272 116 32 12 5 19 14 18 5 4 497
242 86 – 10 3 14 2 – – 4 361
– 20 – 0 1 – 11 – – – 32
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.
0 0 0 0 0 0 0 0 0 0 0
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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; GillingSmith 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
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
70 22 11 7 110
62 22 6 7 97
20 7 6 3 36
219 32 97 2 49 113 12 19 543
156 20 68 2 39 61 6 19 353
92 11 33 2 20 35 6 2 201
See Table 1 for definitions of parameters.
Patients treated
Pregnancies
Births
Ongoing
Infection
–
– – – – –
0 0 0 0 0
7 4 3 14 58 4 22 2 14 26 3 2 131
9 1 – – – 12 1 0 23
0 0 0 0 0 0 0 0 0
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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 34–36 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 HIVRNA 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) Age of women (years; mean ± SD) Married Duration of relationship (years; mean ± SD) Couples with child together prior to presentation Children conceived after knowing HIV status Couples attempting timed intercourse to conceive Couples preferring donor sperm to timed intercourse Favour posthumous attempts to conceive if partner died Couples discussed single parenting risk Couples discussed adoption of child if both parents died Couples desiring to have multiple children through assisted reproduction Couples willing to discuss experience with other discordant couples
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38.0 ± 6 (range 26–51) 34.5 ± 5 (range 24–45) 4/50 (88) 9.0 ± 5 (range 1–20) 9/50 (18) 3/9 (33) 4/50 (8) 24/50 (48) 22/50 (44) 45/50 (90) 29/50 (58) 33/50 (66) 36/50 (72)
Review - Assisted reproduction and HIV-serodiscordant men - MV Sauer
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 No. of initiated cycles No. attempts per couplea Cancellation rate due to poor ovarian response (%) No. oocytes aspirated per retrievala No. mature oocytes suitable for ICSIa No. fertilized oocytes per ICSI casea No. embryos transferred per attempta Couples with cryopreserved embryos (%) No. embryos cryopreserved per couplea Overall clinical pregnancy rate per embryo transfer (%) Ongoing and delivered pregnancy rate per embryo transfer (%) Cumulative pregnancy rate for couples over repeated attempts (%) No. of seroconversions in treated women No. of seroconversions in delivered offspring
61 113 1.9 ± 1.1 (1–6) 11.5 17.1 ± 9.5 (2–47) 13.9 ± 8.1 (1–42) 9.3 ± 5.5 (0–24) 3.5 ± 1.1 (1–6) 32.2 5.1 ± 3.5 (1–19) 44.8 36.5 54.1 0 0
aValues 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.
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Received 7 September 2004; refereed 12 October 2004; accepted 18 November 2004.
Review Sperm washing techniques address the fertility needs of HIV-seropositive men: a clinical review 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 1995. 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. Dr Mark Sauer 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: +1 212 3059175; Fax: +1 646 7568280; e-mail: [email protected]
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),
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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.
serodiscordant couples. Both groups now recommend adopting more tolerant policies of non-discrimination (ACOG Committee Opinion, 2002; Ethics Committee of the ASRM, 2002).
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.
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 HIV-
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
Separating virus from spermatozoa
Table1. Summary of published results on HIV-1 serodiscordant couples undergoing washed sperm intrauterine inseminations. All values are numbers.
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Study
Cycles
Patients treated
Pregnancies
Births
Ongoing Infection
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
1954 458 155 92 46 143 62 93 11 5 3019
623 233 67 36 16 64 28 39 – 5 1111
272 116 32 12 5 19 14 18 5 4 497
242 86 – 10 3 14 2 – – 4 361
– 20 – 0 1 – 11 – – – 32
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.
0 0 0 0 0 0 0 0 0 0 0
Review - Assisted reproduction and HIV-serodiscordant men - MV Sauer
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; GillingSmith 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
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
70 22 11 7 110
62 22 6 7 97
20 7 6 3 36
219 32 97 2 49 113 12 19 543
156 20 68 2 39 61 6 19 353
92 11 33 2 20 35 6 2 201
See Table 1 for definitions of parameters.
Patients treated
Pregnancies
Births
Ongoing
Infection
–
– – – – –
0 0 0 0 0
7 4 3 14 58 4 22 2 14 26 3 2 131
9 1 – – – 12 1 0 23
0 0 0 0 0 0 0 0 0
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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 34–36 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 HIVRNA 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) Age of women (years; mean ± SD) Married Duration of relationship (years; mean ± SD) Couples with child together prior to presentation Children conceived after knowing HIV status Couples attempting timed intercourse to conceive Couples preferring donor sperm to timed intercourse Favour posthumous attempts to conceive if partner died Couples discussed single parenting risk Couples discussed adoption of child if both parents died Couples desiring to have multiple children through assisted reproduction Couples willing to discuss experience with other discordant couples
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38.0 ± 6 (range 26–51) 34.5 ± 5 (range 24–45) 4/50 (88) 9.0 ± 5 (range 1–20) 9/50 (18) 3/9 (33) 4/50 (8) 24/50 (48) 22/50 (44) 45/50 (90) 29/50 (58) 33/50 (66) 36/50 (72)
Review - Assisted reproduction and HIV-serodiscordant men - MV Sauer
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 No. of initiated cycles No. attempts per couplea Cancellation rate due to poor ovarian response (%) No. oocytes aspirated per retrievala No. mature oocytes suitable for ICSIa No. fertilized oocytes per ICSI casea No. embryos transferred per attempta Couples with cryopreserved embryos (%) No. embryos cryopreserved per couplea Overall clinical pregnancy rate per embryo transfer (%) Ongoing and delivered pregnancy rate per embryo transfer (%) Cumulative pregnancy rate for couples over repeated attempts (%) No. of seroconversions in treated women No. of seroconversions in delivered offspring
61 113 1.9 ± 1.1 (1–6) 11.5 17.1 ± 9.5 (2–47) 13.9 ± 8.1 (1–42) 9.3 ± 5.5 (0–24) 3.5 ± 1.1 (1–6) 32.2 5.1 ± 3.5 (1–19) 44.8 36.5 54.1 0 0
aValues 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.
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Received 7 September 2004; refereed 12 October 2004; accepted 18 November 2004.