1 IVI Valencia, Valencia, 2 FIVI, (Fundación IVI) Valencia, 3 Departments of Paediatrics, Obstetrics and Gynaecology, Valencia University School of Medicine, Valencia and 4 Department of Obstetrics and Gynecology Hospital Dr Peset, Valencia, Spain
5 To whom correspondence should be addressed at: Instituto Valenciano de Infertilidad, Andrology Laboratory and Semen Bank; Plaza de la Policía Local 3, Valencia 46015, Spain. Email: nicolas.garrido{at}ivi.es
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Abstract |
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Key words: HCV/HIV/ICSI/nested PCR/semen wash/serodiscordant
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Introduction |
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Given the low transmission risk, established as one to three infections per 1000 acts of sexual intercourse (or semen exposures) for HIV (De Vincenzi 1994) and even less for HCV (Garrido et al., 2004), all the accumulated data regarding viral transmission through semen with assisted reproduction techniques (mainly intrauterine insemination) do not reflect the real situation. To avoid any accidental infection of the partner, offspring and laboratory staff through assisted reproduction treatments, we must employ the safest techniques available.
HIV belongs to the retroviruses family, having the capacity to synthesize reverse transcriptase, and convert the RNA into DNA and to insert its genome in this manner into the hosts, while HCV is a RNA virus unable to act in this way. This issue is relevant to the detection methods employed in this work (Meseguer et al., 2002).
Commonly, the routine detection methods found in the literature are based on the amplification of defined sequences of the viral nucleic acids, and artificial insemination is the first treatment to be performed, if the gynaecological findings are normal (Semprini et al., 1992; Marina et al., 1998
). We have previously demonstrated that the available technologies can be improved in order to detect even a single RNA or DNA viral copy with nested PCR (or reverse transcription and nested PCR for RNA, Meseguer et al., 2002
). Nevertheless, an assisted reproduction procedure without using any detection method to determine the virus presence in the washed sperm has been reported despite the possible risk of transmission (Garrido et al., 2002a
).
Also, adequate equipment and trained personnel to work with these samples are mandatory. Strict virology protocols must be implemented in the laboratory (Garrido et al., 2004).
The aim of our study was to determine: (i) the efficiency of the sperm wash, in terms of presence or absence of viral load for HIV and HCV viruses after the wash of the samples, (ii) to determine the results of the assisted reproduction techniques with ICSI in serodiscordant couples for HIV and/or HCV employing washed sperm, (iii) to analyse seroconversion rates by employing these procedures.
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Materials and methods |
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As a necessary condition to be included in this programme, the men were requested for a full report of their disease by the physician providing care for their infection, although many HCV patients were not followed at all.
For HIV positive males, CD4+ T-cell counts in blood, blood viral load, treatment received, co-infections, and a general description of the male patient status were detailed.
The results are presented in a global manner, without considering the infection. In the following analysis, different subgroups were constituted depending on the presence of each virus. The HIV-infected patients displayed all the stages of the infection (A, B or C) according to the Centers for Disease Control classification (CDC) (Table I), the A2 group being the most represented with 18 patients. Six of the HIV or HIV/HCV (10.3%) were not receiving anti-retroviral treatment. The mean viral load was 48 623 copies/ml (range 78525 000 IU/ml), with 46 patients without detectable blood viral load. The mean CD4 cell count was 502.7 CD4/ mm3 (range 261664). Finally the time of evolution of the disease was 11 years (range 320).
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All the samples were processed in a laboratory separate from the laboratories where non-infectious samples were prepared, and all the safety guidelines followed have been published elsewhere (Bellver et al., 2002). Briefly, the viral risk area was only dedicated to these biological samples, including biosafety cabinet workstation, exclusive centrifuge, and nitrogen tank.
Semen parameters were analysed according to World Health Organization (WHO, 1999) criteria. Total count and motility before and after the wash were recorded. Sperm morphology was not analysed for safety reasons: it is not recommended to work with glass or blades when treating HCV/HIV positive samples. Sperm wash was only performed in those samples with >2x106 of total progressive motile sperm in the whole semen sample.
The HIV infection was acquired by parenteral drug addiction in 25 of patients (48.8%), plasma donation in one (0.2%), sexual transmission in 11 (21.6%), blood transfusion in six (11.7%), and eight infection acquisitions (15.5%) were unknown. The infection source for HCV was never known.
Regarding the female population, different gynaecological findings were observed: 59 were considered as normal (64.8%), 15 were aged >36 years (16.4%), five were low responders (5.5%), eight had endometriosis (8.6%). Depending on the patient's characteristics we treated these women with two assisted reproduction procedures: ICSI with their own oocyte or with oocytes obtained from young healthy donors. In HIV serodiscordant couples, 11 procedures were performed with donated oocytes, while seven procedures were performed with donated oocytes in HCV serodiscordant couples.
Only females with demonstrated absence of HIV and HCV antibodies were accepted in the study. They were also requested to practice sex with condoms.
HCV seropositive males presented a median viral load of 125 000 copies (range 31 7502 500 000); of these, 35 had detectable viral load for HCV (47.9%), and only two had liver biopsy revealing liver damage. In all, only five males have been treated with different interferon protocols.
Sperm wash
Ejaculates obtained after a sexual abstinence of 35 days were allowed to liquefy, and then were diluted 1:1 (vol:vol) with Sperm Medium (MediCult, Denmark). They were then pelleted at 400 g for 10 min, and the supernatants were discarded. A volume of Sperm Medium equal to the initial volume of semen was added, and then layered onto a triple concentration gradient (90, 70 and 45%, PureSperm; Nidacon, Sweden) of 11.2 ml of each layer, and centrifuged 20 min at 300 g.
Each pellet was obtained and washed with 5 ml of Sperm Medium, and re-pelleted again. Supernatants were discarded and a swim-up of 0.50.7 ml was done. After 45 min, the upper 0.35 ml of each tube supernatant was aspirated, pooled and split into two samples. One half was immediately submerged in liquid nitrogen for PCR determinations, and the other half was frozen with Sperm Freezing Medium (MediCult), according to the manufacturer's instructions, and stored until their use after a negative result for viral presence (Meseguer et al., 2002).
PCR techniques for HIV and HCV applied to sperm
Nucleic acids were extracted from the washed sperm using the Nuclisense® method (Organon Teknika, Spain) following the manufacturer's instructions. Two extractions were run in parallel, one from the sperm sample and the other with the sperm samples after the addition of HIV RNA obtained from HIV-infected plasma, to detect the presence of transcription or amplification inhibitors after the nucleic acid extraction procedure. Both extracted samples were used for two HIV RNA transcriptions to detect genes from the gag and pol region, followed by a nested DNA amplification. The same samples were used to amplify HIV proviral DNA by a nested amplification to detect both genes (gag and pol). The other extraction, run in parallel with added HIV RNA before nucleic acid extraction, was used as a positive control to detect the presence of inhibitors of the transcription or amplification. Negative controls to detect the presence of amplicon contamination were also performed.
For HIV RNA transcription we used the antisense external primers to anneal with nucleotides 1696 to 1676 and 3286 to 3265 for gag and pol genes respectively. Standardized conditions for transcription were followed using 100 mmol/l dithiothreitol, 1 mmol/l each dNTP, 0.2 mmol/l antisense primer, 20 IU RNAasin (Promega, Spain) and 5 IU Avian Myeloblastosis Viruses reverse transcriptase (AMV) transcriptase (Promega) in a final volume of 20 ml. Nested DNA amplification used the external primers to anneal with nucleotides (from ARV2/SF2 sequence) 12241243 and 16961676 and internal primers to anneal with nucleotides 13161335 and 15241504 for the gag region. External primers annealing with nucleotides 26232642 and 32863265 and internal primers annealing with nucleotides 27162741 and 32503227 for the pol region were used. PCR standardized conditions were followed including 6 µl of RT or previous PCR product, 2.5 and 2 mmol/l MgCl2 for gag and pol region respectively, 0.2 mmol/l each dNTP (Amersham Pharmacia, Spain), 0.5 mmol/l each primer and 2 IU Taq polymerase (Promega) in a 50 ml final volume. -Actin gene amplification was performed to confirm the presence of DNA in the extraction from sperm suspension. Results were read from a 2% agarose gel electrophoresis after ethidium bromide staining. In all the samples a consistent result (either positive or negative) was obtained. The assay failure rate was zero.
HCV nested PCR protocols were described in a previous work, in order to amplify viral Non-coding Region, (NRC) region in a protocol comparable to reverse transcription and nested PCR for HIV but with the adequate specific primers (Meseguer et al., 2003).
Ovarian stimulation and ovum donation
Protocols for ovarian stimulation
For ovarian stimulation, both GnRH agonist and antagonist protocols were used. For GnRH agonist, long protocol was employed as previously described (Díaz et al., 2000). Briefly, patients started administration of 0.1 mg of leuprolide acetate (Procrin; Abbott S.A., Spain) or triptorelin (Decapeptyl; Ipsen Pharma; Spain) in the mid-luteal phase of the previous cycle, until negative vaginal ultrasound defined ovarian quiescence. The dose of GnRH analogue was then decreased to 0.05 mg until the day of hCG administration. GnRH antagonists were used following the low dose daily protocol recently described (Bosch et al., 2003
): starting on stimulation day 6, 0.25 mg of the GnRH antagonist Cetrotide (Cetrorelix; Serono S.A., Spain) was administered on a daily basis until the day of hCG administration.
Recombinant FSH (Gonal-F; Serono S.A., Spain; or Puregon; Organon Española, Spain) and hMG (Lepori; Farma Laboratories, Spain; or Menopur; Ferring, Spain) were used for ovarian stimulation. Initial doses were determined according to patients' age and basal serum FSH and estradiol (E2) levels. On stimulation day 3, serum E2 level was assessed and gonadotrophin doses adjusted according to a step-up or step-down protocol. hCG (Profasi 10 000 UI; Serono S.A., Spain) was administered when three or more follicles reached 18 mm in diameter and oocyte retrieval was scheduled 36 h later.
Oocyte donation
Oocyte donor recruitment and management has been previously described, they were screened for sexually transmitted diseases (HIV and HCV included) (Garrido et al., 2002b). The protocol for steroid replacement has been also described. It included pituitary desensitization with one dose of 3.75 mg i.m. triptorelin (Decapeptyl; Ipsen Pharma, Spain) beginning in the secretory phase of the previous cycle. Patients started administration of E2 valerate (Progynova; Schering, Spain) on cycle day 1; 2 mg were administered from day 1 to day 8, 4 mg from day 9 to day 11, and 6 mg from day 12 onwards. On day 14, vaginal ultrasound and serum E2 determination were performed to evaluate endometrial receptivity, and E2 valerate doses were continued until donation became available. On the day of the donation, 800 mg/day of micronized intravaginal progesterone (Progeffik; Laboratories Effik, Spain) were added.
IVF with ICSI
The microinjection was performed as previously described (Meseguer et al., 2003). Briefly, morphologically normal sperm were sought in the sperm droplet, and then immobilized and aspirated, tail first, into the tip of a microinjection pipette. A metaphase II oocyte was held on the holding pipette, and the injection pipette was pushed through the zona pellucida injecting a single spermatozoon.
Injected oocytes were incubated in 20 ml drops and fertilization was assessed after 18 h, and embryo cleavage 24 h thereafter. Embryos were transferred into the uterine cavity 4872 h after ICSI. The remaining embryos were frozen for eventual future transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heartbeat at 7 weeks of pregnancy. Embryo freezingthawing protocols can be found elsewhere (Cobo et al., 2002).
Statistical analysis
t-Tests were employed for comparisons between groups when the data followed a normal distribution. Non-parametric tests were used to compare study parameters in fertile and infertile males when the data did not follow a normal distribution. Subsequently, in these cases, MannWhitney U-tests were carried out. Significance was defined as P<0.05. The statistical analysis was performed using MedCalc Software (Belgium).
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Results |
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Semen parameters
The results of the WHO analysis of the semen parameters are shown in Table III. Similar results were obtained in all groups.
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Sperm motility seems to be slightly impaired in comparison to what WHO states as normal, given that the mean does not reach in any case 50% of A+B types, although the lowest values were displayed by those males with only HIV infection.
After the sperm wash procedure, results were again similar among groups, and means of >70% of A+B forms is always found, suggesting that these semen samples display an adequate preparation, and behave in a similar way to those from non-infected males.
Comparing between groups, only the recovery rate, expressed as the percentage of recovered sperm after preparation in comparison to the initial amount, was different between patients infected with HIV and HCV versus patients with only HCV.
If this finding represents that co-infection is diminishing sperm ability to capacitate is still unknown and needs further investigation.
Cycle results
The results of treatment according to the viral infection are shown in Table IV. The total number (all patients grouped) of follicular aspirations was 124, with a total number of 1541 oocytes obtained, of which 1256 were metaphase II. Cancellation rate was 10.1% (14 cancellations). To decide cancellation, less than three follicles >18 mm or <450 pg/ml E2 on the day of hCG administration had to be found.
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Regarding frozen embryos, four pregnancies were achieved out of 16 attempts (25.0%).
The viral infection results demonstrate similar success rates with respect to the different parameters analysed, with fertilization and pregnancy rates comparable among groups, except for the results of frozenthawed embryos, where the low numbers available made the sample unrepresentative.
Comparing the results in each group, the parameters analysed were similar except for fertilization rates. The success of fertilization per metaphase II oocyte was significantly higher in patients with only HIV than in couples where the male partner was infected with HCV.
Seroconversion tests
Seroconversion tests for the partner were programmed 3 and 6 months after finishing every treatment (each embryo transfer). To date, none of them has been seroconverted, neither for HIV nor for HCV.
Women were tested in our clinic (n=41) for the same infection as their respective male partner, whereas the others were controlled in their respective hospitals every 6 months, as a routine screening for women at risk of infection. None reported a positive result, the transmission rate thus being zero.
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Discussion |
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These viruses must also be considered from a different point of view, regarding the methods employed to detect them. While HIV is a retrovirus, that can be present as an RNA molecule or as a part of the sequence of the host cell, after the reverse transcription to DNA. HCV is only present as RNA.
Subsequently, for RNA tests of viral presence, different positive and negative controls to both RNA extraction and reverse transcription must be included (Meseguer et al., 2002) in order to avoid false positive/negative results.
The analysis of HIV DNA, free or integrated inside the human cells, is also necessary for HIV. With our analysis, we only are able to determine the viral presence in a representative population of sperm cells. The results obtained will be extended to the half sample that will be employed for assisted reproduction if virus sequences are not found.
When a positive result is yielded, we are unable to determine whether this is a consequence of the presence of seminal plasma residues, immune cells that escaped from the purification protocols, or even whether single sperm cells are carrying the viruses inside them.
Nevertheless, using single sperm to inseminate oocytes, instead of several thousands in classic IVF, or even millions with intrauterine insemination (IUI) techniques, is theoretically safer, since the woman's exposure to potentially dangerous sperm is minimized. This is against other authors' theories which claim that we could be introducing viral particles into the oocyte. We believe that sperm cells carrying viruses (if any) and fertilizing an oocyte by IVF/IUI also act in this way, introducing the viral genome inside.
Both situations (HCV/HIV) converge to nearly the same protocols in the andrology laboratory: the sperm wash. With this treatment of the samples, we can eliminate round cells, seminal plasma and the majority of immotile sperm efficiently. Moreover, we will overcome PCR technical difficulties linked to seminal plasma polymerase inhibitors.
Once this semen wash is performed, several procedures to detect HIV and HCV presence have been historically employed with different sensitivity. Nested PCR has been demonstrated to be able to detect one single viral copy (Meseguer et al., 2002). Moreover, this methodology is probably responsible for the positive rates after wash of
10%. This is higher than any value we can find in the literature (Semprini et al., 1992
; Marina et al., 1998
); obviously these studies used tests with lower sensitivity.
The next step is to decide which kind of assisted reproduction treatment will be applied. Several considerations must be taken into account.
Given the low risks, artificial insemination has been the method of choice for some time (Semprini et al., 1992; Marina et al., 1998
). This technique has the advantages of low economic cost and less inconvenience for the woman, but nevertheless presents some inconveniences, such as the need for a sperm wash result on the same day (and, if a positive result is found, the cycle will be cancelled), the low numbers of sperm inseminated, and the high risk of viral transmission (due to the exposure to thousands of potentially infecting sperm). Also the whole sperm preparation must be employed within the same cycle.
We support a different approach with these couples, including ICSI. Although more expensive, safety reasons, as well as cost-effectiveness issues, encouraged us to follow this protocol.
First, with regard to safety, we are exposing the woman to an infinitesimal risk: we employ just one single sperm for each oocyte, from a previously negative sample with nested PCR. Semen samples are frozen, and nPCR can be confirmed as many times as needed, given that the sample is not necessarily employed the same day. Also, sperm wash can be performed before the cycle, thus not cancelling any cycle because of a positive result.
Second, regarding the costs, pregnancy rates are two to three times higher with ICSI than IUI, and a sperm wash that resulted negative can be employed in as many cycles as required, thus avoiding the need for new washes. Many IUI patients unable to achieve pregnancy will subsequently proceed to IVF cycles. Also, many males will provide semen samples that will not be adequate for IUI after the extensive procedure of sperm wash, since the recovery of sperm is very low in order to avoid possible positives (see Table III).
Pregnancy rates are acceptable in all the groups, and are as expected for patients attending our centre for assisted reproduction undergoing ICSI cycles. Also we must consider that, although it was presumed that the women were not an infertile population, gynaecological study showed that some clearly infertile women were included in these protocols which may have had a negative influence on the results. More than 60% had a gynaecological finding to classify them as subfertile.
Another consideration is the source of patients. HIV serodiscordant couples form a cohort of patients with infertility features equal to the general population. Conversely, HCV-infected males are not counselled to avoid procreation due to the transmission risk: these are already infertile couples infected with HCV. This may account for the low pregnancy rates displayed.
Finally, different studies have excluded patients regarding several disease features such as variable viral blood load in the past 6 months, elevated viral loads, not receiving antiretroviral treatment, low CD4 levels, etc. We strongly support the idea that every patient requesting this kind of help must be offered a sperm wash and the subsequent treatment, given that there are no current scientific or ethical reasons to reject them on the basis of their status. Only two conditions must be considered: one, the female partner should be seronegative, and second, the impossibility of employing a washed sample that resulted positive. The issue of accepting seronegative women is under debate, since treatment can also be recommended for seropositive women in order to avoid complicating her disease by transmitting a different or mutated strain from her partner (Williams et al., 2003).
The high incidence of positive samples in comparison with other works is clearly due to the extreme sensitivity of the technique. This was previously demonstrated by our group (Meseguer et al., 2002) in a study comparing the sensitivity of the PCR techniques applied to sperm. It was demonstrated that samples testing positive with nested PCR were negative when the results were confirmed with a single round PCR procedure, thus indicating that there are samples below the detection limit of a single round of PCR that still have viral presence.
In summary, we provided information supporting sperm wash, nested PCR and ICSI as the safest and most reasonable method to treat HIV and HCV serodiscordant couples seeking progeny.
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Acknowledgements |
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References |
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Submitted on December 10, 2003; resubmitted on April 15, 2004; accepted on July 20, 2004.