1 Centre dAMP de Strasbourg, Service de Gynécologie-Obstétrique, CMCO-SIHCUS, 19 rue Louis Pasteur BP 120 67303 Schiltigheim, 2 CISIH, Clinique Médicale A, Hôpital Civil, 67091 Strasbourg Cedex, 3 Centre dAMP de Strasbourg, Service de Biologie de la Reproduction, CMCO-SIHCUS, 19 rue Louis Pasteur, 67303 Schiltigheim and 4 Institut de Virologie, Faculté de Médecine-ULP, 3 rue Koeberlé, 67000 Strasbourg, France
5 To whom correspondence should be addressed. e-mail: pierre.ohl{at}evc.net
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: assisted reproduction techniques/HIV/pregnancy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our aim here is to present our results, in terms of pregnancy and risk of infection, of 18 months of assisted reproductive techniques indicated for HIV seropositivity. Two very different profiles emerged according to whether the man or woman was HIV+.
History
After seeing women anxious to be pregnant who become infected with HIV through sexual activity, our team developed a multidisciplinary clinical research protocol in the year 2000, which was approved by the institutional Ethics Committee. Our experience began in January 2001 when we set up special areas in our laboratory to handle cases with viral risks. At that time, it was reserved for couples in which the man was infected by HIV. The decree of May 10, 2001 about assisted reproductive treatment of patients with viral risks (Journal Officiel de la République Française, May 15, 2001) made it possible to extend this treatment to serodiscordant couples in which the woman is HIV-infected and/or co-infected with hepatitis C virus (HCV) or hepatitis B virus (HBV).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All patients provided written informed consent for assisted reproductive techniques. An additional consent form, specifically acknowledging comprehension of the viral risk involved, was signed by both members of the couple.
The assisted reproductive technology laboratory used for the procedure was considered a viral risk area, geographically separated from the laboratory space used for the other couples who were negative for HIV and hepatitis B and C. All procedures were established in writing and validated by the hospital Hygiene Committee.
The assisted reproductive technologies laboratory follows recommended safety precautions (World Health Organization, 1999). Specific precautions were added against the risk of HIV, HCV and HBV contamination (Decree of May 10, 2001). General measures protected the staff (use of hand, face, and eye protection), and the potentially infected gametes and embryos were handled separately. A special biosafety cabinet workstation was used for all tasks that involved handling sperm, oocytes and embryos.
In all cases, a standard vaginal sample for bacteriological testing was taken several days before the assisted reproductive technique procedures. Semen was systematically cultured and treatment given for any infection by common bacteria, mycoplasma or chlamydiae.
The ovarian stimulation protocol was chosen according to the clinical data, the patients hormone profile, and the result of any earlier stimulations. For IVF we used the standard long protocols. A GnRH analogue (Decapeptyl®; Ipsen, France, or Suprefact®; Aventis Pharma, Germany) was given s.c. each day. Recombinant FSH (Gonal F®; Ares-Serono, UK or Puregon®; Organon, France) was given together after desensitization. The GnRH analogue was continued up to the day that hCG 5000 IU (Gonadotrophine Chorionique Endo 5000®; Organon) was administered. Only a few follicles were stimulated for the intrauterine insemination; the pure protocol used recombinant gonadotrophin (Gonal F® or Puregon®) and induction by 5000 IU hCG.
Cycles were monitored with serial transvaginal ultrasonography and serum LH and estradiol (E2) assays by the assisted reproductive technologies center nearest the couples home. For IVF, oocytes were retrieved transvaginally under ultrasound guidance. The oocyte retrieval, embryo transfer and insemination procedures all took place at our centre. Supernumerary embryos on day 3 were frozen and thawed according to the standard technique involving 1,2-propanediol and sucrose as cryoprotectants (Testart et al., 1986).
The status of the partners of seropositive men was confirmed by HIV antibody testing and viral load measurements in the 2 weeks before and 23 weeks after each assisted reproductive technique attempt. These tests were repeated 3 and 6 months after assisted reproductive techniques and at any delivery.
The principle of this technique involved verification by virological testing that the sperm population had no detectable HIV-1 provirus or genome. Only such samples, negative for HIV-1 RNA and DNA, could be used.
Sperm preparation used two successive methods: a two-step 4080% discontinuous gradient (PureSperm® 100; Nidacon International AB, Sweden) and the swim-up technique performed for 3060 min at 37°C. At the end of the incubation period, the supernatant containing the motile sperm constituted the final fraction.
A modified Amplicor HIV-1 Monitor v1.5 assay (Roche Diagnostic Systems, France) was used to detect HIV-1 RNA in seminal plasma or RNA and DNA in seminal cells. The internal quality standard (IQS) was added to 500 µl of seminal plasma or to frozen pellets of 2x106 cells before acid nucleic extraction. Extraction was performed using Nuclisens NASBA Diagnostics extraction kit (Organon Teknika SA, France). All extracted samples were then processed using the ultrasensitive Monitor assay protocol. One negative and three positives controls were included in each run. The assay detection limit was 50 RNA copies/ml in seminal plasma and 50 copies of RNA or DNA in 1x106 seminal cells.
In compliance with the May 10, 2001 decree, the results of the virologic testing of the seminal plasma and the final fraction determined the choice of assisted reproductive technologies technique (Table I).
|
Laboratory constraints made it impossible to obtain the virological results for fresh sperm during the same day. The final fraction was therefore cryopreserved for subsequent use. The sperm preparation was diluted drop by drop with the freezing medium (Spermfreeze; Irvine Scientific®, USA) was aliquoted in 0.25 ml CBS high-security strawsTM (Cryo Bio System, Group IMV Technologies, France). A programmable freezer was used to control the cooling rate. The samples were then taken to 196°C by plunging them directly into the liquid nitrogen. The straws were then stored in separate tanks dedicated to gametes with viral risks.
To thaw the sperm, the straws were taken out of the liquid nitrogen tank and warmed slowly at room temperature. After washing with culture medium, the contents were then expelled into a centrifuge tube, gently diluted with 2 volumes of culture medium and centrifuged at 600 g for 10 min. The pellet was resuspended in 0.3 ml of culture medium, and the concentration and motility of the thawed sperm were assessed in a Makler chamber.
When the woman was seropositive for HIV, HCV and/or HBV, the follicular fluid was handled in the at-risk area.
Statistical analysis was performed according to the MannWhitney U-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Seven couples were determined to be ineligible: immuno-virological stability not obtained (three men and one woman), inadequate sperm semen quality with <2x106 motile sperm recovered (two cases), not amplifiable HIV-2 (one man). Fifty-seven couples were included (47 infected men and 10 women). The 37 remaining couples were still in the process of being treated at the time of writing.
The immuno-virological characteristics of the 57 couples included are summarized in Table II.
|
|
Group of couples with HIV-1 infected female
At inclusion, seven women were receiving HAART and three no antiretroviral therapy at all. After consultation with virus disease specialists our assisted reproductive technique team decided to continue HAART for four women (seven attempts) and to stop it for the other three (five attempts). The treatment was resumed at the 21st week of pregnancy for the one who became pregnant; treatment was resumed for the second woman after four attempts failed, and was not resumed for the third.
Comparison between FSH level of seropositive women and partners of seropositive men showed a significant difference (9.0 ± 2.4 and 7.0 ± 2.9 IU/l respectively, P < 0.001). Seropositive women were significantly older than partners of seropositive men (35.9 ± 4.1 and 32.7 ± 4.5 years respectively, P < 0.001). Nine couples were treated through 15 assisted reproductive technique attempts. One pregnancy occurred which was still ongoing.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The increase in heterosexual transmission has led to the infection of young women from every socioeconomic category. At the same time, the reduction in viral load before delivery has considerably diminished the risk of maternalfetal transmission in the industrialized countries. Among patients who receive good follow-up and treatment and who deliver by Caesarean section, this frequency is <2% (Mandelbrot et al., 1998).
Patients can thus make life plans and even envisage having children. This pressing desire for children is legitimate and any other conclusion would discriminate against these patients. The fight against discrimination, social, occupational or familial, has from the beginning of the epidemic been a priority for the National Ethics Committee for the Life Sciences and Health (CCNE) and for the National AIDS Council (CNS) (opinion dated February 10, 1998). A cultural revolution is taking place, especially in the medical profession (Balet et al., 1998).
Legitimacy of assisted reproductive technologies
Antiretroviral treatments help significantly in reducing the viral load in semen but it is far from clear that undetectable levels can be reached (Zhang et al., 1998). In patients receiving treatment who have an undetectable plasma viral load, the frequency of viral RNA detection in seminal fluid is reported to be 2% (Vernazza et al., 1998
). HIV levels in blood and semen are not well correlated (Kim et al., 1999
). Decontamination of the sperm and isolation of the sperm from the seminal fluid are therefore essential and thus legitimize assisted reproductive technologies. Moreover, no intracellular penetration by the virus in the separated sperm have been reported in seropositive patients receiving treatment (Pudney et al., 1998
). Sperm do not express significant levels of HIV receptors, so they are unlikely to be major targets for HIV infection (Kim et al., 1999
). Isolation of the sperm from the seminal fluid is therefore essential and thus legitimizes assisted reproductive technologies which use only the final fraction.
The balance between the importance of the message of prevention and help for patients who want a child has tilted towards medical intervention (Englert et al., 2001). Assisted reproduction should be adopted as public policy in this situation (Drapkin Lyerly et al., 2001
). Clear legal changes in France have translated this conclusion into rights.
HIV+ women are reported to have a frequency of upper genital tract infections and their sequelae 10-fold greater than the general population (Sobel, 2000). Physicians are thus often asked to treat tubal infertility in seropositive women. Assisted reproductive technologies also allow the treatment of possible male sterility at the same time as it prevents transmission to the partner at conception.
Choice of technique
The choice of technique depended on the overall medical picture, as it does for all couples in assisted reproductive technologies. Nonetheless because of the viral specificity of these couples specificity of the indication, other considerations played a role.
For seropositive men, the decree dated May 10, 2001 requires that sperm be prepared whenever possible with two successive techniques (density gradient, then swim-up), which results in use of most if not all of the sperm in the ejaculate. Adequate testing requires 2x106 sperm in the final fraction. Below this threshold, even ICSI cannot be proposed. This was initially a problem for several patients in our centre but, except for two, was most often transient or managed by pooling two ejaculates produced the same day. The seminal plasma and final fraction were tested virologically and the quantity of virus found in seminal plasma also determined the type of assisted reproductive techniques (Table I).
Because virological findings could not be obtained the same day for fresh sperm in our laboratory, they therefore had to be frozen in straws. This combination of constraints as well as the distance from home of some couples had to be considered. ICSI rapidly became the favoured method, not for safety reasons but because freezing dramatically decreases the number of available motile sperm as already reported (Leruez-Ville et al., 2002). Despite the high cost of the ICSI process, the efficiency of this technique avoids the expensive repetitive virological semen testing and numerous cycles of ovarian stimulation.
For seropositive women, IUI was initially considered possible in the absence of severe infertility factors but was rapidly replaced by IVF to optimize the probability of success and diminish the number of attempts necessary. When interruption of the antiretroviral treatment was recommended because of the planned pregnancy, we used ICSI to avoid unexpected fertilization failure and thus to reduce the delay of conception. For one patient with Von Willebrands disease, insemination was proposed to avoid the possible risk of haemorrhage associated with the oocyte retrieval.
Virological aspects
In this study, the sperm preparation procedure effectively led to an undetectable HIV-1 genome in the semen of seropositive men. Virus was detected in the seminal fluid of 20.4% of the sperm samples tested but no final fraction was positive for HIV. The infection of the men under treatment was well controlled. Moreover, although viral levels in sperm could have been increased by genital tract infection, the systematic sperm cultures and the treatment of possible infections enabled control of this additional risk. No seminal fluid was infected to the point of having a viral load >10 000 copies/ml, the threshold that would have excluded assisted reproductive techniques (Table I). A study of 32 clinically asymptomatic men (Pasquier et al., 2000) found similar results: HIV-1 RNA was detected in 30% of the seminal plasma samples, HIV-1 genomes were found in 18% of the semen samples, but in none of the motile sperm after the density gradient and swim-up. On the other hand, Marina et al. (1998
) had to eliminate 5.6% of the final fractions tested because of detectable viral loads in a cohort of 63 seropositive patients.
None of these studies, as well as others performed using IUI, IVF or ICSI (Semprini et al., 1997; Weigel et al., 2001
; Sauer et al., 2002
) or our study reported any cases of seroconversion in either mother or child. Assisted reproductive techniques therefore appear reliable and safe.
Efficacy of assisted reproductive technologies
For the men with HIV infection, the ANRS 096 (Agence Nationale de Recherche sur le SIDA) protocol used IUI, with sperm prepared by two successive techniques (Daudin et al., 2001). A total of 174 cycles of insemination in 54 couples led to 31 pregnancies and an 18% pregnancy rate per cycle. Half the pregnancies occurred during the first two cycles.
A European experience of >2000 inseminations with washed and tested sperm has been reported (Gilling-Smith, 2000). The pregnancy rate per insemination was 14%. Our centre has minimal experience (five attempts without pregnancy) with IUI.
For the men with HIV infection, our results for IVF/ICSI were encouraging: the clinical pregnancy rate per embryo transfer was 48.8%. These results are similar to those from other protocols. The French ANRS 092 protocol concerned a pilot study where IVF/ICSI was chosen for its efficacy and to prevent contact between the female genital tract and any sperm (Guibert et al., 2001). In 97 cycles of assisted reproductive techniques, 34 pregnancies occurred giving a 35.1% pregnancy rate and 22.7% delivery rate per oocyte retrieval. Seven additional pregnancies occurred after the transfer of frozen embryos, thereby raising the pregnancy rate per transfer by 11%. Overall, 39.7% of the couples gave birth.
Another study used ICSI for 34 serodiscordant couples in which the man was HIV-positive (Sauer et al., 2002). Treatment efficacy was good, in terms of oocytes retrieved (15.8 ± 1.3), fertilization rate (64.9%), clinical pregnancies per embryo transfer (45.5%) and ongoing or delivered pregnancies per embryo transfer (30.9%). A recent study has presented preliminary data for 36 couples treated by ICSI for male HIV infection and poor semen quality (Semprini et al., 2002
). Mean number of oocytes collected per retrieval was 9.2 (SD: 5.49) and pregnancy rate per retrieval was 53.5%. Possible explanations for this excellent performance include the absence of a clinical indication for ICSI and especially the absence of infertility among the female partners.
Little work has been done with assisted reproductive techniques in seropositive women. The clinical and biological course of HIV disease during pregnancy is nonetheless well known. In asymptomatic women whose infection is well controlled, pregnancy does not aggravate HIV disease (Weisser et al., 1998; Saada et al., 2000
). In the industrialized countries, prospective studies confirm the efficacy of HAART in preventing motherchild transmission (Mandelbrot et al., 2001
; Cooper et al., 2002
). Knowing that this transmission occurs late in pregnancy due to fetalmaternal exchanges at the end of pregnancy and especially during labour (Rouzioux et al., 1995
), here assisted reproductive techniques should cause no harm at all.
Couples in which the woman is seropositive have often attempted self-insemination at home, before turning to assisted reproductive techniques (Delfraissy et al., 2002). The sterility factor is predominant here, unlike in the couples with HIV+ men. The assisted reproductive technique results are accordingly more disappointing.
Moreover, the seropositive women seeking assisted reproductive techniques were significantly older than the partners of the seropositive men. Some patients had waited a long time for this treatment made possible by medical progress and by the recent legislative changes in France. Their hormone levels, with elevated FSH, confirm the trend towards ovarian depletion. The respective impacts on ovarian function of both infection and HAART are still not well-known. Some studies suggested a negative effect of the infection on menstrual cycle length (Harlow et al., 2000) and on ovulation (Clark et al., 2001
).
In conclusion, treatment progress and clear changes in social attitudes now make it possible for assisted reproductive techniques to be offered to HIV serodiscordant couples. Assisted reproductive techniques are performed with the double benefit of controlling the risk of viral transmission and of treating possible sterility. Our results suggest that screening and washing sperm are a safe risk-reduction option for couples who want to conceive. Assisted reproductive techniques are efficacious for HIV+ men, when their primary indication is the presence of the virus. The pregnancy rates were not as good among the seropositive women, who were often older and had other infertility problems.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Balet, R., Lower, A.M., Wilson, C., Anderson, J. and Grudzinskas, J.G. (1998) Attitudes towards routine human immunodeficiency virus (HIV) screening and fertility treatment in HIV positive patientsa UK survey. Hum. Reprod., 13, 10851087.[Abstract]
Clark, R.A., Mulligan, K., Stamenovic, E., Chang, B., Watts, H., Andersen, J., Squires, K. and Benson, C. (2001) Frequency of anovulation and early menopause among women enrolled in selected adult AIDS clinical trials group studies. J. Infect. Dis., 184, 13251327.[CrossRef][ISI][Medline]
Cooper, E., Charurat, M., Mofenson, L., Hanson, C., Pitt, J., Diaz, C., Hayani, K., Handelsman, E., Smeriglio, V. and Hoff, R. et al. (2002) Combination antiretroviral strategies for the treatment of pregnant HIV-1-infected women and prevention of perinatal HIV-1 transmission. J. AIDS, 29, 484494.[ISI][Medline]
Daudin, M., Pasquier, C., Izopet, J., Kallman, M., Lachendowier, C., Lanusse, P., Morucci, M., Mercadier, B., Labeyrie, E. and Seguela, G. et al. (2001) Le protocole ANRS 096: prise en charge en assistance médicale à la procréation des couples sérodifférents dont lhomme est infecté par le VIH. Résultats préliminaires de Toulouse. Reprod. Hum. Horm., 14, 365369.
Delfraissy, J.F., Rouzioux, C, Bujan, L., Faucher, J.M., Heard, I., Jouannet, P., Le Mercier, Y., Leruez, M., Meier, A. and Prestel, T. (2002) Désir denfant et assistance médicale à la procréation. In Prise en charge thérapeutique des personnes infectées par le VIH. Paris Rapport 2000. Ministère de lemploi et de la solidarité, Secrétariat dEtat à la santé et à laction sociale. Médecine-Sciences, Flammarion, p. 282.
Drapkin Lyerly, A. and Anderson, J. (2001) Human immunodeficiency virus and assisted reproduction: reconsidering evidence, reframing ethics. Fertil. Steril., 75, 843858.[CrossRef][ISI][Medline]
Englert, Y., Van Vooren, J.P., Place, I., Liesnard, C., Laruelle, C. and Delbaere, A. (2001) ART in HIV-infected couples: has the time come for a change of attitude? Hum. Reprod., 16, 13091315.
Gilling-Smith, C. (2000) HIV prevention. Assisted reproduction in HIV-discordant couples. AIDS Read, 10, 581587.[Medline]
Guibert, J., Merlet, F., Le Dû, A., Mandelbrot, L., Leruez-Ville, M., Costagliola, D., Kunstmann, J.M., De Almeida, M., Heard, I. and Dulioust, E. et al. (2001) Prise en charge des couples séro-différents pour le VIH. Résultats du protocole NECO (ANRS 092) à Paris. Reprod. Hum. Horm., 14, 363364.
Harlow, S., Schuman, P., Cohen, M., Ohmit, S., Cu-Uvin, S., Lin, X., Anastos, K., Burns, D., Greenblatt, R. and Minkoff, H. et al. (2000) Effect of HIV infection on menstrual cycle length. J. AIDS, 24, 6875.[ISI][Medline]
Kim, L.U., Johnson, M.R., Barton, S., Nelson, M.R., Sontag, G., Smith, J.R., Gotch, F.M. and Gilmour, J.W. (1999) Evaluation of spermatozoa washing as a potential method of reducing HIV transmission in HIV-discordant couples. AIDS, 13, 645651.[CrossRef][ISI][Medline]
Leruez-Ville, M., de Almeida, M., Tachet, A., Dulioust, E., Guibert, J., Mandelbrot, L., Salmon, D., Jouannet, P. and Rouzioux, C. (2002) Assisted reproduction in HIV-1-serodifferent couples: the need for viral validation of processed semen. AIDS, 16, 22672273.[CrossRef][ISI][Medline]
Mandelbrot, L., Le Chenadec, J., Berrebi, A., Bongain, A., Benifla, J.L., Delfraissy, J.F., Blanche, S. and Mayaux, M.J. (1998) Perinatal HIV-1 transmission: interaction between zidovudine prophylaxis and mode of delivery in the French Perinatal Cohort. J. Am. Med. Assoc., 280, 5560.
Mandelbrot, L., Landreau-Mascaro, A., Rekacewicz, C., Berrebi, A., Benifla, J.L., Burgard, M., Lachassine, E., Barret, B., Chaix, M.L. and Bongain, A. (2001) Lamivudinezidovudine combination for prevention of maternalinfant transmission of HIV-1. J. Am. Med. Assoc., 285, 20832093.
Marina, S., Marina, F., Alcolea, R., Exposito, R., Huguet, J., Nadal, J. and Verges, A. (1998) Human immunodeficiency virus type 1-serodiscordant couples can bear healthy children after undergoing intrauterine insemination. Fertil. Steril., 70, 3539.[CrossRef][ISI][Medline]
Pasquier, C., Daudin, M., Righi, L., Berges, L., Thauvin, L., Berrebi, A., Massip, P., Puel, J., Bujan, L. and Izopet, J. (2000) Spermatozoa washing and virus nucleic acid detection to reduce HIV and hepatitis C virus transmission in serodiscordant couples wishing to have children. AIDS, 14, 20932099.[CrossRef][ISI][Medline]
Pudney, J., Nguyen, H., Xu, C. and Anderson, D.J. (1998) Microscopic evidence against HIV-1 infection of germ cells or attachment to spermatozoa. J. Reprod. Immunol., 41, 301306.[CrossRef][ISI][Medline]
Rouzioux, C., Costagliola, D., Burgard, M., Blanche, S., Mayaux, M.J., Griscelli, C. and Valleron, A.J. (1995) Estimated timing of mother-to-child human immunodeficiency virus type I transmission by use of a Markov model. Am. J. Epidemiol., 142, 13301337.[Abstract]
Saada, M., Le Chenadec, J., Berrebi, A., Bongain, A., Delfraissy, J.F., Mayaux, M.J. and Meyer, L. (2000) Pregnancy and progression to AIDS: results of the French prospective cohorts. AIDS, 14, 23552360.[CrossRef][ISI][Medline]
Sauer, M.V. and Chang, P.L. (2002) Establishing a clinical program for human immunodeficiency virus 1-seropositive men to father seronegative children by means of in vitro fertilization with intracytoplasmic spermatozoa injection. Am. J. Obstet. Gynecol., 186, 627633.[CrossRef][ISI][Medline]
Semprini, A.E., Fiore, S. and Pardi, G. (1997) Reproductive counselling for HIV-discordant couples. Lancet, 349, 850851.[CrossRef][ISI][Medline]
Semprini, A.E., Vucetich, A., Oneta, M., Rezek, D., Rubino, P., Scarselli, F. and Hollander, L.H. (2002) Spermatozoa washing and ICSI in HIV discordant couples: >50% pregnancy rate. Hum. Reprod., 17 (Abstract Book 1), P-340.
Sobel, J.D. (2000) Gynecologic infections in human immunodeficiency virus-infected women. CID, 31, 122533.
Testart, J., Lassalle, B., Belaisch-Allart, J., Hazout, A., Forman, R., Rainhorn, J.D. and Frydman, R. (1986) High pregnancy rate after early human embryo freezing. Fertil. Steril., 46, 268272.[ISI][Medline]
Vernazza, P.L., Troiani, L., Flepp, M., Cone, R., Shock, J. and Roth, F. (1998) Potent antiretroviral treatment (ART) results in marked suppression of seminal HIV-RNA and DNA sheddings. AIDS European Meeting. Glasgow (abstract). Drug Ther. HIV Infect., 15, 36.
Weigel, M.M., Gentili, M., Beichert, M., Friese, K. and Sonnenberg-Schwan, U. (2001) Reproductive assistance to HIV-discordant couplesthe German approach. Eur. J. Med. Res., 6, 259262.[ISI][Medline]
Weisser, M., Rudin, C., Battegay, M., Pfluger, D. and Kully, C. (1998) Does pregnancy influence the course of HIV infection? Evidence from two large Swiss cohort studies. J. Acquir. Immun. Defic. Syndr. Hum. Retrov., 17, 404410.[ISI][Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SemenCervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Zhang, H., Dornadula, G., Beumont, M., Livornese, L., Van Uitert, B., Henning, K. and Pomerantz, R.J. (1998) Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy. N. Engl. J. Med., 339, 18031809.
Submitted on November 25, 2002; accepted on March 3, 2003.