Volume 110 Number 8 August 2011 Formosan Medical Association Taipei, Taiwan ISSN 0929 6646 Resveratrol for prophylaxis of ischemic stroke Microglia and chronic pain ART in HIV-1-discordant couples in Taiwan Prognostic evaluation of patients with multicentric papillary thyroid microcarcinoma J Formos Med Assoc 2011;110(8):495 500 Contents lists available at ScienceDirect Journal of the Formosan Medical Association Journal homepage: http://www.jfma-online.com Journal of the Formosan Medical Association Original Article Outcomes of Assisted Reproductive Techniques for HIV-1-discordant Couples Using Thawed Washed Sperm in Taiwan: Comparison With Control and Testicular Sperm Extraction/Microscopic Epididymal Sperm Aspiration Groups Ming-Yih Wu, Li-Jong Chang, Mei-Jou Chen, Kuang-Han Chao, Yu-Shih Yang, Hong-Nerng Ho* Background/Purpose: An increasing number of human immunodeficiency virus-1 (HIV-1)-discordant couples in Taiwan have been seeking fertility help. We conducted the first clinical trial in Taiwan of assisted reproductive technology (ART) using sperm washing and viral load measurement. Methods: From 2005 to 2009, we performed 22 ART cycles on 14 HIV-1-discordant couples. The sperm washing involved density gradient centrifugation followed by swim-up method. HIV-1 RNA was checked by real-time reverse transcription-polymerase chain reaction with a sensitivity of 40 copies/ml. In addition, we enrolled two other groups of ART recipients using frozen sperm to compare the clinical outcomes. Results: There were five pregnancies in the fresh cycles (23.8%) of HIV-1-discordant couples and the cumulative pregnancy per couple was 42.9% (6/14). The data were comparable with normal controls and testicular sperm extraction/microscopic epididymal sperm aspiration groups. The nine babies and the 14 women in this study showed no seroconversion. Conclusion: The preliminary data showed good ART results in HIV-1-discordant couples. Fertility services should not be withheld from individuals with HIV-1, although larger series are needed to reach conclusions about safety. Key Words: assisted reproductive technology, human-immunodeficiency-virus-1-discordant, sperm washing In Taiwan, the first case of acquired immunodeficiency syndrome (a foreigner in transit) was identified in December 1984, and recently Taiwan has shown an increasing incidence of human immunodeficiency virus-1 (HIV-1) infection. 1 Meanwhile, there has been continual improvement in survival among HIV-infected individuals through the use of newer forms of highly active antiretroviral therapy. 2 With the achievement of improved survival rates, married couples now 2011 Elsevier & Formosan Medical Association................................................... Department of Obstetrics and Gynecology, College of Medicine, National Taiwan University, Taipei, Taiwan. Received: October 8, 2009 Revised: April 6, 2010 Accepted: July 21, 2010 *Correspondence to: Dr Hong-Nerng Ho, Department of Obstetrics and Gynecology, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 100, Taiwan. E-mail: hnho@ntu.edu.tw J Formos Med Assoc 2011 Vol 110 No 8 495
M.Y. Wu, et al often wish to have children safely. The clinical use and risk of sperm washing and its risks were first reported in 1992 by Semprini et al, 3 and since then it has been confirmed worldwide. 4,5 The guidelines of the American Society for Reproductive Medicine in 2006 6 also recommended Semprini s widely used sperm-washing procedure: the use of gradient centrifugation followed by a sperm swim-up procedure. In the first trial of this procedure in Taiwan, we here present the clinical data of 14 HIV-1-discordant couples and compare the results with two other groups [normal controls and testicular sperm extraction/microscopic epididymal sperm aspiration (TESE/MESA)] who were also using frozen sperm. Materials and Methods Subject couples The subject couples were seeking reproductive services and were referred to the Department of Obstetrics and Gynecology of National Taiwan University Hospital. HIV-1 infection status of the couples including CD4 + cells and plasma viral load was assessed first. Gynecological examinations, including pelvic ultrasound, and hormone profiles, and semen analysis were subsequently performed to decide which couples would receive intrauterine insemination (IUI, not discussed here) or in vitro fertilization (IVF) cycles. From 2005 to 2009, a total of 14 couples (22 stimulation cycles) were enrolled in this study. The institutional review board approved this study (22MD02) in 2003, and signed informed consent was obtained from each couple before they entered the treatment cycle. The indications for IVF in these HIV-1-discordant couples were the same as for other patients. Semen pretreatment Semen samples were obtained by masturbation and then examined for sperm concentration, motility and morphology. The samples were processed using a density gradient (three layers at 50%, 70% and 90%, from top to bottom; SpermGrad; Vitrolife, Kungsbacka, Sweden) to separate motile spermatozoa from non-sperm cells, immotile spermatozoa, and seminal plasma. The ejaculate was layered over the gradient and centrifuged at 300g for 20 minutes. After centrifugation, the 50% and 70% layers were eliminated, and the sperm pellet recovered and resuspended in 2 ml of fresh human tubal fluid medium (home-made, quality control by mouse embryo culture), divided into four parts, placed in four test tubes and centrifuged at 200g for 10 minutes. The supernatant was discarded, 0.5 ml of human tubal fluid medium was subsequently gently layered on the pellet, and the tube was incubated at 37 C for 1 hour. After swimup, a supernatant volume of 350 400 μl was recovered per test tube (i.e. 1.4 1.6 ml). One of the aliquots was tested for detectable HIV-1 RNA. Detection of HIV-1 RNA in washed semen A sample of extract (50 μl) was mixed manually with 50 μl COBAS TaqMan HIV-1 premix reagent and amplified using the real-time polymerase chain reaction (PCR) COBAS TaqMan 48 instrument (COBAS Ampliprep analyzer; Roche Diagnostics, Meylan, France). Target and internal quality standards amplified DNA in each sample were detected and assayed using specific TaqMan probes. Results were visualized on a computer using the Amplilink 3.01 system connecting the COBAS TaqMan 48 and COBAS Ampliprep instruments. The sensitivity ranged from 40 copies/ml to 10,000,000 copies/ml. Assisted reproductive technology (ART) Controlled ovarian hyperstimulation was performed mostly by a long gonadotropin-releasing hormone agonist protocol or by short protocols for some poor responders, as previously reported. 7 Recombinant follicle-stimulating hormone (Puregon; Organon, the Netherlands) at 200 IU/day was given starting from Day 3 of the long protocol or Day 5 of the short protocol. Ultrasound-guided transvaginal oocyte retrieval (TVOR) was performed at 34 36 hours after human chorionic gonadotropin (hcg) administration. Subsequently, intracytoplasmic sperm 496 J Formos Med Assoc 2011 Vol 110 No 8
ART in HIV-1-discordant couples in Taiwan injection (ICSI) was performed in all 14 cases 6 hours later. 8 Embryo transfer (ET) was conducted on the Day 3 after TVOR. According to the March 2006 guidelines of the Taiwan Society of Reproductive Medicine, no more than four embryos could be transferred. Luteal phase was supported with 1500 IU hcg on Day 2, Day 5 and Day 8 after transfer, and 25 mg/day progesterone in oil for 14 days. Clinical pregnancy was defined by sonographic visualization of a gestational sac, combined with elevation of serum β-hcg levels. When focusing on the embryo quality, the average embryo score 9 was calculated by cumulative embryo score divided by embryo numbers. Seroconversion tests Before ART, each patient was screened for HIV-1 negativity. Therefore, all female partners were HIV negative at the beginning. After ART, all patients regardless of whether they were pregnant were tested for HIV-1 antibodies 6 months later. Furthermore, newborn babies were also tested at birth or later. Control couples To verify the pregnancy rates of the HIV-1- discordant couples, we enrolled another two groups using frozen sperm for ICSI during the same period (2005 2009). The first group was the normal control group who were using frozen sperm for personal reasons because the husband was often abroad for business. The few cases where we applied IVF were excluded, so we only enrolled ICSI cases. The second group was the TESE/MESA group. The sperm specimens were always frozen because the operation by the urologists might not match our TVOR schedule. These tiny samples were stored in aliquots for possible repeated therapies. All cases in this group received ICSI at our hospital. Statistical analysis For comparison among the three groups, the Kruskal Wallis test was applied to most data followed by the Mann Whitney U test. A p value < 0.05 was considered to be significantly different. When looking at the success of the pregnancy, Pearson s χ 2 test was performed to check the significance. Results Twenty-two fresh cycles of ovarian stimulation were performed in the 14 HIV-1-discordant couples (Table 1). Of these, there was one case in which an oocyte was fertilized but did not show cleavage, so only 21 cycles of ET were done. The clinical pregnancy rate per ET was 23.8% (5/21), which was lower than our routine IVF [in 2008, our fresh pregnancy rate per ET was 41.9% (218/ 520), unpublished data]. All the 14 male partners had received highly active antiretroviral therapy for 1 10 years, so all except one had undetectable plasma viral load (< 40 copies/ml). To date, no babies or women showed seroconversion during the follow-up from 14 months to 52 months. Table 1. Clinical data of in vitro fertilization/ intracytoplasmic sperm injection in 14 human-immunodeficiency-virus-discordant couples Patient Spouse (n = 14) (n = 14) Age at first visit (yr) 33.3 ± 4.9 36.1 ± 3.6 Stimulation cycles 22 Fresh clinical pregnancy 23.8% (5/21) a rate/et Thaw clinical pregnancy 33.3% (3/9) rate/et Cumulative pregnancy 42.9% (6/14) rate/couple Multiple pregnancy rate 25.0% (2/8) Ectopic pregnancy rate 12.5% (1/8) CD4 + > 250/mm 3 6/6 b Serum HIV-1 6/7 c < 40 copies/ml Maternal seroconversion 0 Delivered offspring 0 seroconversion a One case had only one oocyte and did not transfer due to no cleavage of the 2PN embryo; b only six cases with available CD4 data near the time of in vitro fertilization; c only seven cases had HIV-1 plasma viral load data near the time of in vitro fertilization. ET = embryo transfer; HIV-1 = human immunodeficiency virus-1. J Formos Med Assoc 2011 Vol 110 No 8 497
M.Y. Wu, et al Table 2. Comparison of the first in vitro fertilization/intracytoplasmic sperm injection cycle among three groups using frozen sperm a Frozen semen HIV-discordant TESE/MESA p No. of couples 68 14 36 Age (yr) 35.7 ± 5.7* 33.3 ± 4.9 31.2 ± 4.2* < 0.001* Day 2 FSH (miu/ml) 7.5 ± 4.8 5.1 ± 3.1 6.7 ± 3.0 NS Total r-fsh (IU) 2,088 ± 1,045 2,002 ± 486 1,725 ± 804 NS E2 on day of hcg 2,251 ± 1,483 2,715 ± 2,027 3,294 ± 2,402 NS Retrieved oocytes 11.2 ± 7.4 14.1 ± 8.7 14.3 ± 8.7 NS Mature oocytes 9.4 ± 6.0 12.9 ± 8.4 12.0 ± 7.1 NS No. of 2-PN 6.9 ± 4.7 8.7 ± 6.5 8.3 ± 5.1 NS Fertilization rate (%) 73.1 ± 20.5 66.8 ± 14.9 72.8 ± 19.2 NS AES (Day 2) 9.4 ± 3.5 9.5 ± 3.5 9.1 ± 2.6 NS Transferred embryos 3.4 ± 1.3 3.1 ± 0.9 3.7 ± 1.7 NS CES 52.1 ± 29.1 53.8 ± 25.7 54.7 ± 27.8 NS Clinical pregnancy (%) 30/68 (44.1) 4/14 (28.6) 20/36 (55.6) NS Implantation rate (%) 17.6 ± 26.3 9.5 ± 16.7 18.2 ± 24.5 NS No. of embryos frozen 2.3 ± 3.6 4.7 ± 5.9 3.4 ± 4.4 NS a Data are presented as n, n (%) or mean ± standard deviation. Kruskal Wallis test was applied on most data followed by Mann Whitney U test. Clinical pregnancy per transvaginal oocyte retrieval cycle was compared by Pearson s χ 2 test. *p > 0.05. HIV = human immunodeficiency virus; TESE/MESA = testicular sperm extraction/microscopic epididymal sperm aspiration; FSH = follicle-stimulating hormone; NS = not significant; r-fsh = recombinant FSH; hcg = human chorionic gonadotropin; AES = average embryo score; CES = cumulative embryo score. The PCR results for HIV-1 in washed semen were available after 12 24 hours. Therefore, we froze those samples for ICSI. We compared those samples to those in another two groups using frozen sperm (Table 2). To eliminate individual variation, only the first trial of IVF cycles was included in these three groups. In the TESE/MESA group, the patients were younger than in the control groups (p < 0.05) and the pregnancy rates seemed to be better than in the control and HIV groups (but p > 0.05 by Pearson s χ 2 test). Discussion The risk of male-to-female transmission of HIV-1 is approximately 0.1% per coital act with HIVprevalent index partners. 10 However, pregnancy sometimes cannot be achieved in only one heterosexual contact. With unprotected sexual contact near the ovulation period in women in HIV-1-discordant couples, there is a 4% chance of viral transmission to achieve one pregnancy. 11 If performing IUI using semen from HIV-1- infected donors, the risk is 3.52% in these couples. 12 HIV-1 is found primarily in white blood cells and as cell-free virions in semen; 13 therefore, sperm-washing techniques can reduce HIV-1 levels markedly before insemination. The clinical value of sperm washing without seroconversion was reported by Semprini et al in 1992, 3 and since then, several thousand published attempts at IUI and IVF have been reported, to achieve pregnancy and result in uninfected partners and offspring. 14 16 Sperm-washing techniques that separate motile sperm from the round cell and seminal fluid fractions include gradient centrifugation and swimup methods. 3 Some modifications such as the double-tube gradient method, 17 the revised swimup method, 18 or trypsin exposure 19 can also remove HIV-1 completely. However, the efficiency of sperm washing in removing HIV-1 varied according to the seminal viral load in a recent study. 20 The authors estimated the upper limit of HIV-1 to be 5 10 4 copies/ml. Therefore, several solutions 498 J Formos Med Assoc 2011 Vol 110 No 8
ART in HIV-1-discordant couples in Taiwan have been suggested in cases of high plasma viral load: (1) to treat genital infections; (2) to modify highly active antiretroviral therapy; and (3) to evaluate the viral load in washed sperm. In our subjects, we measured the viral load in the washed sperm, but not in the raw semen. We only referred to the plasma viral load as an indicator, because usually there are subtle differences in blood and seminal plasma HIV-1 RNA viral loads. 21 In this study, we used a real-time reverse transcription-pcr to detect HIV-1 RNA to < 40 copies/ml. Some believe that HIV-1 might be present in seminal cells in the form of DNA. 22 Yet, the presence of HIV-1 in spermatozoa itself has been a matter of debate. Using immunocytochemistry, in situ hybridization at the electron microscope level, and PCR, it has been demonstrated that human sperm can incorporate HIV-1 using special receptors different from the commonly used CD4. 23 Moreover, a recent study 24 using nested PCR detected virus in 69.2% of purified sperm from HIV-1-positive men; in contrast, these purified sperm were all negative for HIV-1 RNA. However, at the same time, a Japanese study 5 using the same nested PCR failed to detect any proviral DNA from all 36 samples of washed sperm. On an epidemiological level, in > 5,000 ART cycles for HIV-1-discordant couples, none has had seroconversion. However, on the molecular level, the evidence seems to suggest some seroconversion risk still exists when these couples use ART. One explanation for this discrepancy is that the absence of HIV-1 in the women was probably due to their limited exposure to infected sperm, rather than to the absence of HIV-1 in the washed sperm themselves. The pregnancy rates for our Taiwanese HIV-1- discordant couples are compatible with other reports around the world. Nine babies were born and none was seroconverted. We also compared the clinical outcomes between HIV-1-discordant couples and HIV-1-negative controls. Men in the TESE/MESA group had a significant fertility deficit that forced them to seek help earlier. They were relatively younger in age and had the highest pregnancy rates (although still not significantly different from the other two groups; perhaps due to the small case number in the HIV-1 group) (Table 2). This is believed to be the first study in Taiwan to provide ART to HIV-1-discordant couples. If we can afford safe and efficient fertility services for these people, they might consult us earlier and achieve better results. Acknowledgment The authors are indebted to Dr Chien-Ching Hung of the Department of Internal Medicine, National Taiwan University Hospital, for his full support with HIV-1 viral detection. References 1. Yang CH, Huang YF, Hsiao CF, et al. Trends of mortality and causes of death among HIV-infected patients in Taiwan, 1984 2005. HIV Med 2008;9:535 43. 2. Lima VD, Hogg RS, Harrigan PR, et al. Continued improvement in survival among HIV-infected individuals with newer forms of highly active antiretroviral therapy. AIDS 2007;21:685 92. 3. Semprini AE, Levi-Setti P, Bozzo M, et al. Insemination of HIV-negative women with processed semen of HIVpositive partners. Lancet 1992;340:1317 9. 4. Bujan L, Hollander L, Coudert M, et al. Safety and efficacy of sperm washing in HIV-1-serodiscordant couples where the male is infected: results from the European CREAThE network. AIDS 2007;21:1909 14. 5. Kashima K, Takakuwa K, Suzuki M, et al. Studies of assisted reproduction techniques (ART) for HIV-1-discordant couples using washed sperm and the nested PCR method: a comparison of the pregnancy rates in HIV-1-discordant couples and control couples. Jpn J Infect Dis 2009;62:173 6. 6. Guidelines for reducing the risk of viral transmission during fertility treatment. Fertil Steril 2006;86:S11 7. 7. Ho CH, Chen SU, Peng FS, et al. Prospective comparison of short and long GnRH agonist protocols using recombinant gonadotrophins for IVF/ICSI treatments. Reprod Biomed Online 2008;16:632 9. 8. Chen SU, Shieh JY, Wang YH, et al. Successful pregnancy achieved by intracytoplasmic sperm injection using cryopreserved electroejaculate sperm in a couple both with spinal cord injury: a case report. Arch Phys Med Rehabil 2005;86:1884 6. J Formos Med Assoc 2011 Vol 110 No 8 499
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