In several countries, couples in

Transcription

1 Intracytoplasmic sperm injection with microsurgically retrieved spermatozoa in azoospermic men infected with human immunodeficiency virus 1 or hepatitis C virus: the EP43 AZONECO ANRS study Marianne Leruez-Ville, Ph.D., a Nicolas Thiounn, M.D., b Catherine Poirot, M.D., c Odile Launay, Ph.D., d Philippe Sogni, M.D., e Sophie Grabar, Ph.D., f and Emmanuel Dulioust, Ph.D. g a Laboratoire de Virologie, H^opital Necker-Enfants Malades, and b Service d'urologie, H^opital Europeen George Pompidou, Assistance Publique de Paris, Universite Paris Descartes, Sorbonne Paris Cite, Paris; c Laboratoire de Biologie de la Reproduction, H^opital Pitie Salp^etriere, Assistance Publique de Paris, Universite Pierre et Marie Curie, Paris; d CIC de Vaccinologie Cochin-Pasteur, e Service d'hepatologie, f Service de Biostatistiques et Epidemiologie, and g Laboratoire de Biologie de la Reproduction, H^opital Broca-Cochin-H^otel Dieu, Assistance Publique de Paris, Universite Paris Descartes, Sorbonne Paris Cite, Paris, France Objective: To evaluate the viral contamination of sperm obtained after sperm extraction (TESE) and microsurgical sperm aspiration (MESA) in men with azoospermia and human immunodeficiency virus (HIV) or hepatitis C virus infection. Design: Prospective study. Setting: Fertility clinic, and reproductive technology and virology laboratories. Patient(s): Six men with azoospermia: two HIV-1 infected with undetectable blood viral load and four HCV infected with detectable blood viral load. Intervention(s): Processing by gradients density centrifugation and washing of surgically recovered sperm (TESE and MESA); virological analysis; in vitro fertilization with intracytoplasmic sperm injection (ICSI). Main Outcome Measure(s): Detection of HIV-1 RNA or gradient supernatants, s and spermatozoa, and of HIV-1 DNA in s. Result(s): Gradient supernatants and s tested HCV RNA positive in all cases while processed spermatozoa always tested negative. Gradient supernatants, s, and processed spermatozoa tested HIV-1 RNA negative. HIV-1 DNA was detectable in one. All female partners tested HCV or HIV negative after ICSI. Conclusion(s): Density gradient and washing suppressed virus detection in final suspensions of and spermatozoa. ICSI after MESA or TESE appears to be feasible and could be offered in azoospermic men infected by HCV or HIV. (Fertil Steril Ò 2013;99: Ó2013byAmericanSocietyfor Reproductive Medicine.) Key Words: Azoospermia, HCV, HIV, ICSI, infertility Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/leruez-villem-icsi-azoospermia-hiv-hcv/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Received August 9, 2012; revised and accepted November 7, 2012; published online November 30, M.L.-V. has received payment for presentations on congenital CMV at the Diasorin Meeting June 2011, Turino, and the Diasorin Workshop, China, May 2012 (outside the submitted work); and travel expenses for the CMV Workshop, May 2011, Germany, funded by Argene (outside the submitted work). N.T. has nothing to disclose. C.P. has received payment for lectures from Merck Serono (outside the submitted work). O.L. has nothing to disclose. P.S. is on the board of and has received travel expenses from Gilead, Roche, BMS, Janssen, and MSD (outside the submitted work). S.G. has nothing to disclose. E.D. has nothing to disclose. Supported by the ANRS (French National Agency for Research on AIDS and viral hepatitis) (EP43 AZONECO). Reprint requests: Marianne Leruez-Ville, Ph.D., Hospital Necker-Enfants-Malades, 149 rue de Sevres, Paris, France ( marianne.leruez@nck.aphp.fr). Fertility and Sterility Vol. 99, No. 3, March 1, /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. In several countries, couples in whom the man is infected with human immunodeficiency virus (HIV) or hepatitis C virus (HCV) can now benefit from assisted reproductive technologies (ART). In HIVserodiscordant couples, ART with sperm washing is recommended to prevent viral transmission and/or to treat infertility (1 3). In couples with an HCV-infected man, ART is only VOL. 99 NO. 3 / MARCH 1,

2 ORIGINAL ARTICLE: ANDROLOGY recommended to treat infertility (4 6). Azoospermia is found in about 10% of infertile couples; its prevalence in men infected with HIV or HCV is unknown but is likely to be similar to the rest of the population. Since the mid 1990s, the retrieval of spermatozoa from epididymis or testis through microsurgical sperm aspiration (MESA) and sperm extraction (TESE) has enabled in vitro fertilization (IVF) via intracytoplasmic sperm injection (ICSI) in men with azoospermia (7). However, azoospermic men who are infected by HIV or HCV are usually denied this option. The main concern has been that the biological samples obtained through MESA or TESE will be contaminated with blood and thus at high risk of containing viral particles. In the Agence Nationale de Recherche sur le SIDA et les hepatites virales (ANRS) AZONECO study, HIV- or HCVinfected men with azoospermia were offered TESE (and/or MESA, when possible). A protocol for washing and sperms was designed. The presence of HIV RNA and/or HCV RNA was assessed in the sperm preparations before and after washing as well as in the remaining tissues from the testis biopsy samples. We performed ICSI after the final sperm preparation had tested negative for detection of HIV RNA or HCV RNA. MATERIALS AND METHODS Population The population consisted of four HCV-infected and two HIVinfected men seeking ART. The HCV patients included one tetraplegic man with anejaculation (case 1), two men with obstructive azoospermia (cases 2 and 3), and one man with cryptozoospermia (case 4). All had detectable blood viral loads (Table 1), and their female partners were HCV negative. The two HIV-infected men had nonobstructive azoospermia, and both had undetectable blood viral loads under highly active antiretroviral therapy (HAART) with CD4 counts at 929/ mm 3 (case 5) and 169/mm 3 (case 6). The female partner was HIV positive in case 5 and HIV negative in case 6. The study was approved by the institutional review board (reference no A ), and all men gave written consent for participation in the study. Surgical Methods Microsurgical spiration (MESA) performed under general anesthesia. Open biopsies were performed after opening the scrotal skin and tunica vaginalis; small incisions were made in the tunica albuginea in different regions of each testicle, and small pieces of extruding tissue were excised. Epididymal Spermatozoa Preparation At retrieval, the sample was diluted in 1 ml of Ferticult medium (JCD). The sperm count and motility were evaluated from a 50 ml aliquot of this diluted sample. The sperm suspension was then layered on a two-phase (45% to 90%) density gradient (Puresperm; JCD) and centrifuged at 300 g for 20 minutes. After centrifugation, the sperm pellet TABLE 1 Assisted reproduction results obtained in the four men infected with hepatitis C virus. No. ICSI cycles Pregnancy left testis tissue (IU/mL) right testis tissue (IU/mL) gradient supernatant of sperm (IU/mL) sperm (IU/mL) a No. sperm/field a gradient supernatant of sperm (IU/mL) sperm (IU/mL) a No. HCV RNA in blood (IU/mL) Age (y) Patient , /ml 10% < /10 < ,000 ND ,000 10/field 4/10 < /7 < POS b POS b ,000 ND ND ND ND 4 1/4 <240 1, ,600,000 ND ND ND ND 1 0/20 <240 1, Note: HCV ¼ hepatitis C virus; ICSI ¼ intracytoplasmic sperm injection; ND ¼ not done; POS ¼ positive. a After gradient selection and washing. b Detectable but under threshold value. Leruez-Ville. ICSI in azoospermic HIV or HCV infected men. Fertil Steril VOL. 99 NO. 3 / MARCH 1, 2013

3 Fertility and Sterility was carefully collected and resuspended in 5 ml of Ferticult medium. It was then centrifuged for 10 minutes at 600 g. This washing step was reiterated once. After the two washings, the sperm pellet was resuspended in 0.5 to 1.5 ml of Ferticult, and the sperm number and motility were evaluated again. Then, the appropriate proportion of freezing medium (Sperm Freeze; JCD) was added to the sperm suspension to obtain a final concentration of 15% glycerol, and a variable number of straws were filled for cryopreservation. The upper layer of the gradient as well as an aliquot of the sperm final suspension equivalent to the volume used for one straw were collected for virologic analysis. Testicular Spermatozoa Preparation The pieces of tissue were washed in Ferticult-HEPES medium to eliminate blood and were transferred to Petri dishes containing 2 ml of the same medium. Sterile needles were used for seminiferous tubules dilacerations. A 20 ml aliquot of the medium was examined to evaluate the number of spermatozoa, other cells, and the percentage of sperm showing static motility. The suspension was then centrifuged at 300 g for 20 minutes on a 1 ml layer of 45% Puresperm. The sperm pellet was processed as for samples. The sperm number and motility were evaluated on a 20 ml aliquot of the final suspension. The remaining pieces of tissue, the upper layer or the gradient, and a one-strawequivalent aliquot of the final suspension were kept for virologic analyses. HIV RNA Quantification We extracted the HIV RNA from semen samples with COBAS Ampliprep Total Nucleid Acid Isolation Kit and amplified it with COBAS TaqMan HIV-1 Test (HPS) (Roche Diagnostics), as previously described elsewhere (2, 4). For the tissue samples, a pretreatment was performed before the automated extraction by adding volume to the volume lysis buffer (containing guanidine thiocyanate) of the RNeasy Micro kit (Qiagen). The threshold value of the technique as determined by the manufacturer is 20 IU/mL when 1 ml of sample is tested. Here, the threshold value was 100 IU/mL as we tested 200 ml of the different semen samples. Blood plasma samples were assessed with Cobas Taqman HIV-1 version 2.0 with a threshold value of 12 copies/ml. HIV DNA Quantification The HIV DNA was extracted from the using the DNA Blood Mini kit (Qiagen), following the manufacturer recommendations. We amplified the HIV DNA with an HIV-1 DNA real-time polymerase chain reaction (Biocentric). HCV RNA Quantification We assessed the HCV RNA with the Real Time HCV (Abbott). For samples, a pretreatment was performed before the automated extraction, as described previously. The threshold value of the technique determined by the manufacturer was 12 copies/ml when 1 ml of sample was tested. Here, the threshold value ranged from 12 copies/ml to 240 copies/ ml, depending on the amount of semen sample available for testing (1 ml to 50 ml). RESULTS The results for the four HCV-infected men are presented in Table 1. The first two men (cases 1 and 2) had MESA; HCV RNA was not detected in the sperm recovered after washing but was detected in the supernatant of the two-layer gradient. The four men had a bilateral testis biopsy; HCV RNA was not detected in the fter washing but was detected in the supernatant of the one-layer gradient and in the s. The results for the two men with HIV are presented in Table 2. For case 5, no spermatozoa could be recovered from the testis biopsy; however, the pellet obtained after gradient, the gradient supernatant, and the remaining pieces of tissue were sent for virologic analysis, and no HIV RNA was detected in any of those samples. We detected HIV DNA in the tissue from the left testis, but it was undetectable in the tissue from the right testis. For case 6, the TESE was positive, and no HIV RNA was detected in the spermatozoa or the gradient supernatant. The remaining pieces of tissue were not tested for the latter case. We performed ICSI for five couples (cases 1, 2, 3, 4, and 6), with a number of ICSI cycles ranging from one to four. A pregnancy was achieved by one couple, and a healthy child was born. The four women (partners of cases 1 to 4) tested HCV negative between 2 and 12 months after the last ICSI cycle, and the woman from case 6 remained HIV negative after the ICSI cycle. DISCUSSION In this study, we analyzed the efficiency for infectious risk reduction of a sperm-washing protocol after MESA or TESE with open surgery in men infected with HIV-1 or by HCV. We found testis biopsy tissues and in the media containing and sperm before washing. The presence of those samples was likely to be a result of blood contamination during the surgical procedure. The washing protocol was effective for risk reduction: HCV RNA was undetectable in all sperm preparations. We performed ICSI cycles with washed sperm in two cases and with washed sperm in the other two cases. No HCV contamination occurred among the four female partners. One case of ICSI after spiration in a man with a high HCV RNA blood load has been reported (8). In that case, the sample was only washed twice by centrifugation, no density gradient was used, HCV RNA was found to be undetectable in the zygotes and embryos, and the woman tested HCV negative after the ICSI procedure. Safe ICSI cycles after TESE have also been reported for two couples in whom the men had positive HCV serology but undetectable blood (9). Two cases of safe ICSI after MESA in HIV-infected men with obstructive azoospermia have been reported (10, 11) with no contamination of the women or the newborns. VOL. 99 NO. 3 / MARCH 1,

4 ORIGINAL ARTICLE: ANDROLOGY TABLE 2 Assisted reproduction results obtained in two men infected with human immunodeficiency virus. No. ICSI cycles Pregnancy HIV DNA in left (copies/million of cells) HIV DNA in right (copies/million of cells) HIV RNA in left (copies/ml) HIV RNA in right (copies/ml) HIV RNA in gradient supernatant of sperm (copies/ml) HIV RNA in sperm (copies/ml) a No. sperm/field a HIV RNA in blood (copies/ml) Patient Age (y) 5 43 < <100 <100 <100 <100 <70 POS b <12 1 1/25 <400 <100 ND ND ND ND 1 0 Note: HIV ¼ human immunodeficiency virus; ICSI ¼ intracytoplasmic sperm injection; ND ¼ not done; POS ¼ positive. a After selection gradient and washing. b Detectable but under threshold value. Leruez-Ville. ICSI in azoospermic HIV or HCV infected men. Fertil Steril Here, we used TESE to allow ICSI in two HIV-infected men with nonobstructive azoospermia. For one man, no spermatozoa could be retrieved from the ; for the other man, TESE was successful. In both cases, HIV RNA was undetectable in the and in the sperm samples before and after washing. It is interesting that HIV DNA was detectable at a weak level in the tissue of one man. The presence of HIV DNA in his may have been linked to the contamination of the biopsy material by blood-infected leukocytes or the presence of infected macrophages in the testis interstitial tissue. Macrophages of interstitial tissue are the only cells of the testis expressing sufficient amounts of HIV cell receptors (12, 13) to allow HIV-1, as has been reported via the organotypic culture of human testis explants from HIV-1 infected men (13). Sperm processing before IVF usually involves centrifugation over a two-layer gradient, which is efficient to retain macrophages or lymphocytes and to recover sperm pellets without integrated virus (2). To facilitate the recovery of sperm from, we used one-layer gradients. This technique may be less efficient in separating mononuclear cells from spermatozoa, which could be a matter of concern in HIV-infected patients, but this possibility was limited by careful rinsing and elimination of blood vessels from the biopsy pieces before the tissue dilacerations. Indeed, when aliquots of the final sperm suspension were examined, we observed red blood cells but no mononuclear cells. Moreover, in the context of ICSI, the presence of a few macrophages in the final preparation of spermatozoa would be of no consequence, because sperm is picked up individually under microscope control to be injected in the oocyte. An additional and important security measure was that only patients in whom viral replication was efficiently repressed could participate in this protocol. Azoospermia is rarely observed in patients with HIV-1 or HCV infection, and data are lacking on the outcome of ICSI in such population. The AZONECO study showed that a careful processing of and samples is efficient for obtaining negative virologic testing of the final sperm suspensions and that ICSI cycles using such sperm suspensions are safe. However, we did not compare ejaculated sperm with / sperm, so this study does not indicate that retrieval is preferable over the use of ejaculated sperm, and it should be limited to azoospermic men. Although the study results need to be confirmed by other groups, we should no longer be denying ICSI with microsurgically retrieved spermatozoa to azoospermic men infected by HIV and/or by HCV. REFERENCES 1. Kim LU, Johnson MR, Barton S, Nelson MR, Sontag G, Smith JR, et al. Evaluation of sperm washing as a potential method of reducing HIV transmission in HIV-discordant couples wishing to have children. Aids 1999;13: Leruez-Ville M, de Almeida M, Tachet A, Dulioust E, Guibert J, Mandelbrot L, et al. Assisted reproduction in HIV-1-serodifferent couples: the need for viral validation of processed semen. Aids 2002;16: Bujan L, Hollander L, Coudert M, Gilling-Smith C, Vucetich A, Guibert J, et al. Safety and efficacy of sperm washing in HIV-1-serodiscordant couples 716 VOL. 99 NO. 3 / MARCH 1, 2013

5 Fertility and Sterility where the male is infected: results from the European CREAThE network. Aids 2007;21: Leruez-Ville M, Kunstmann JM, De Almeida M, Rouzioux C, Chaix ML. Detection of hepatitis C virus in the semen of infected men. Lancet 2000;356: Bourlet T, Levy R, Maertens A, Tardy JC, Grattard F, Cordonier H, et al. Detection and characterization of hepatitis C virus RNA in seminal plasma and spermatozoon fractions of semen from patients attempting medically assisted conception. J Clin Microbiol 2002;40: Bourlet T, Lornage J, Maertens A, Garret AS, Saoudin H, Tardy JC, et al. Prospective evaluation of the threat related to the use of seminal fractions from hepatitis C virus-infected men in assisted reproductive techniques. Hum Reprod 2009;24: Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Manno M, Marchesan E, Crovatto M, Martelli P, Tomei F, Adamo V. Preliminary evidence on the safety of ICSI with spermatozoa in HCV-infected male: a case report. Hum Reprod 2003;18: Garrido N, Gil-Salom M, Martinez-Jabaloyas JM, Meseguer M. First report of the absence of viral load in sperm samples obtained from men with hepatitis C and HIV after washing and their subsequent use. Fertil Steril 2009;92: Nicopoullos JD, Frodsham LC, Ramsay JW, Almeida PA, Rozis G, Gilling- Smith C. Synchronous sperm retrieval and sperm washing in an intracytoplasmic sperm injection cycle in an azoospermic man who was positive for human immunodeficiency virus. Fertil Steril 2004;81: Bujan L, Daudin M, Moinard N, Plante P, Parinaud J, Pasquier C. Azoospermic HIV-1 infected patients wishing to have children: proposed strategy to reduce HIV-1 transmission risk during sperm retrieval and intracytoplasmic sperm injection: Case Report. Hum Reprod 2007;22: Habasque C, Aubry F, Jegou B, Samson M. Study of the HIV-1 receptors CD4, CXCR4, CCR5 and CCR3 in the human and rat testis. Mol Hum Reprod 2002;8: Roulet V, Satie AP, Ruffault A, Le Tortorec A, Denis H, Guist'hau O, et al. Susceptibility of human testis to human immunodeficiency virus-1 infection in situ and in vitro. Am J Pathol 2006;169: VOL. 99 NO. 3 / MARCH 1,