PCR-detected hepatitis C virus RNA associated with human zona-intact oocytes collected from infected women for ART

A. Papaxanthos-Roche1,5, P. Trimoulet2, M. Commenges-Ducos3, C. Hocké4, H.J.A. Fleury2 and G. Mayer1

1 Laboratoire de Biologie de la Reproduction, Maternité Pellegrin, 2 Laboratoire de Virologie and 3 Service de Gynécologie–Obstétrique, Centre Hospitalier Universitaire, Hôpital Pellegrin, Place Amélie Raba Léon, 33076 Bordeaux and 4 Service de Gynécologie, Hôpital Saint-André, 1, rue Jean Burguet, 33075 Bordeaux, France

5 To whom correspondence should be addressed. e-mail: aline.papaxanthos{at}chu-bordeaux.fr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The aim of this study was to investigate the susceptibility of human oocytes from hepatitis C virus (HCV) RNA-positive women to HCV contamination during assisted reproductive technology (ART). METHODS: A reverse transcriptase–PCR assay was used to test for the presence of HCV RNA associated with 24 unfertilized oocytes 48 h after follicular fluid aspiration in 10 IVF attempts (seven conventional IVF and three ICSI). Negative and positive controls (10 unfertilized oocytes from HCV-negative women and 20 unfertilized oocytes artificially contaminated with HCV RNA-positive plasma; HCV RNA was also quantified in plasma and follicular fluid) were included. RESULTS: HCV RNA was associated with 17/24 (70.8%) oocytes (6/7 after ICSI and 11/17 after conventional IVF) and was found in 19/20 (95%) follicular fluid samples. A weak correlation was found between plasma and follicular fluid HCV RNA loads (r = 0.73, P < 0.001). CONCLUSIONS: HCV associated with unfertilized oocytes surrounded by their intact zona pellucida from anti-HCV antibody-positive and viraemic women undergoing ART raises questions concerning the safe management of medically assisted procreation for these women and good practice of oocyte/embryo cryopreservation and donation.

Key words: hepatitis C virus/IVF/oocyte


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The prevalence of hepatitis C virus (HCV) infection in the general population, estimated at 1% in France (Desenclos et al., 1996Go), raises difficult safety problems concerning assisted reproductive technology (ART) for HCV-infected patients. The handling of potentially infected body fluids, gametes or embryos could expose technicians and uninfected couples or gametes treated at the same time to the risk of HCV contamination. Indeed, two women were reported to have become infected during ancillary ART procedures (Lesourd et al., 2000Go). The risk of vertical HCV transmission or transmission to a woman from an infected man and potentially to gametes or embryos remains an open question.

Detection of HCV RNA in the semen of infected men is no longer controversial since the problem of false-negative results due to the presence of PCR inhibitors in the ejaculates has been resolved [HCV RNA was detected in 2/39 samples tested by Levy et al. (2000Go), 8/21 of those evaluated by Leruez-Ville et al. (2000Go) and 7/50 samples tested by Cassuto et al. (2002Go)]. However, >2300 intrauterine inseminations or IVF attempts with washed spermatozoa from human immunodeficiency virus (HIV)-infected men, 62% of whom were co-infected with HCV, have been acheived with no case of seroconversion in the women (Semprini et al., 2001Go).

When the women are infected, the risk of vertical HCV transmission in spontaneous pregnancies for HIV-seronegative women is evaluated at 5–10%, especially when the mothers have elevated viraemia (>106 copies/ml) (Roudot-Thoraval et al., 1993Go; Ohto et al., 1994Go; Poiraud et al., 2001Go). Maternal–fetal HCV transmission seems to occur during the perinatal period with fetal exposure to maternal blood and with vaginal secretions at the time of delivery (Poiraud et al., 2001Go). We do not yet know if an excess risk of vertical transmission of HCV exists during ART for infertile infected women. A prospective, follow-up, case–control French study on the risk of HCV vertical transmission during IVF compared with spontaneous pregnancy is ongoing (Association Nationale de Recherche sur le SIDA Study HC04, Assistance Médicale à la Procréation et Virus de l’Hépatite C: Evaluation du risque de transmission lorsque l’un des membres du couple est infecté; directed by Roudot-Thoraval) (Devaux and Roudot-Thoraval, 2002Go). Although the presence of HCV in follicular fluid has been demonstrated in several studies (Papaxanthos-Roche et al., 1999Go; Leruez-Ville et al., 2001Go; Sifer et al., 2002Go; Devaux et al., 2003Go), HCV contamination of the oocytes remains unknown. Indeed, only a few published studies have examined the risks of contaminating human oocytes or embryos. Witz et al. (1999Go) failed to detect human cytomegalovirus (CMV) DNA in 244 unfertilized oocytes and discarded embryos from 44 women with prior CMV infections. However, no one has attempted to detect CMV DNA in the oocytes or embryos of CMV DNA-positive women. Baccetti et al. (1998Go) demonstrated by transmission electron microscopy, immunocytochemistry and PCR that free HIV-1 particles are not able to bind to and penetrate artificially exposed human oocytes in vitro. However, viral contamination of oocytes or embryos has been better documented in animal models (see the review by Bielanski, 1997Go).

The aim of this study was to assess the susceptibility of oocytes collected from anti-HCV antibody-positive and viraemic women to HCV contamination during ART in order to evaluate the risk for oocytes or embryos destined for IVF/ICSI, cryopreservation and/or donation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Eight infertile couples underwent 10 IVF attempts (seven conventional IVF and three ICSI) from June 2000 to April 2002. Two couples had two complete ART cycles. Infertility was tubal for six couples, male factor for one couple and unexplained for one couple.

The women, 30–39 years old, had chronic HCV infections with detectable anti-HCV antibodies and plasma HCV RNA, and were HIV negative. They had received no antiviral treatment for 4 months. The men were anti-HCV antibody and HCV RNA negative.

Ovarian stimulation was controlled by associating the GnRH analogue triptorelin (Decapeptyl, Ipsen Biotech, Paris, France) with recombinant FSH (Gonal-F, Serono, Boulogne, France; or Puregon, Organon, Saint-Denis, France) or HMG (Menogon, Ferring, Gentilly, France). Ultrasound-guided follicular fluid aspiration was scheduled 36 h after HCG injection.

Oocytes
Unfertilized oocytes, obtained in the context of an ART programme, were tested. Prior approval by the Bioethics Committee (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale de Bordeaux) was obtained.

After follicular fluid aspiration, oocytes were isolated under a stereomicroscope. The follicular fluid aspect was classified as bloody by colour (+ for reddish orange, ++ for red, +++ for dark red) or clear (yellow). From oocyte–granulosa cell complex isolation to embryo transfer, the oocytes/embryos were handled and cultured individually and the pipette tip was changed between each manipulation. The culture medium used throughout this study was IVFTM medium (Scandinavian IVF, Göteborg, Sweden) equilibrated in 5% CO2 at 37°C before use. After two washes in 500 µl droplets of fresh medium, each isolated oocyte–granulosa cell complex in 10 µl of medium was transferred separately into 1 ml of fresh culture medium. Spermatozoa were extracted from the ejaculates by discontinuous gradient centrifugation (PureSperm; Nicadon, Göteborg, Sweden) and the final pellet was resuspended in 500 µl of Ferticult HEPES medium (Fertipro; Aalter-Lotenhulle, Belgium).

For ICSI, each oocyte–granulosa cell complex in 10 µl of medium was transferred separately into 450 µl of culture medium containing 40 IU of hyaluronidase (Synvitro-Hyadase; Medicult, Lyon, France) for 1 min, during which granulosa cells were removed by pipetting through a capillary tube, 125 µm in diameter. Then, each oocyte was washed twice in 1 ml of fresh culture medium. The absence of granulosa cells was verified under an inverted light microscope equipped with Normarski optics (Nikon Diaphot, Tokyo, Japan) at x200 magnification, which also allowed the observation of the polar body, and detection of possible cytoplasmic or zona pellucida abnormalities. The oocytes with one polar body in the perivitelline space were then placed in microdroplets (5 µl) of fresh medium for ICSI. The microinjection and the contention pipettes were changed for each oocyte. After injection, each oocyte was individually transferred into 1 ml of fresh culture medium for 48 h.

For IVF, 2–3 h after isolation, each oocyte–granulosa cell complex was inseminated separately with 50 000 motile spermatozoa in 1 ml of fresh culture medium. After 18–20 h of incubation, the oocytes were checked for the presence of pronuclei. For IVF, before examining the oocyte and looking for the pronuclei, the remaining corona cells were stripped from the oocytes by pipetting through a capillary tube, 125 µm in diameter, and the absence of granulosa cells was also verified under an inverted light microscope equipped with Normarski optics at x200. Then, each oocyte was placed individually into 1 ml of fresh IVF medium for 24 h. Regardless of the ART technique, embryos were transferred in utero 48 h after oocyte collection. The remaining unfertilized oocytes were used for viral assay. Each one was rinsed twice in 1 ml of fresh phosphate-buffered saline (PBS), transferred in 10 µl of medium into a cryotube containing 200 µl of a proteinase K solution (0.1 mg/ml) (Macherey-Nageland, Duren, Germany) and frozen at –80°C until viral assay. For each transfer, a new pipette tip was used. Because the embryo culture media of oocytes from HCV-positive women were shown previously to be negative for HCV RNA (Leruez-Ville et al., 2001Go; Sifer et al., 2002Go; Devaux et al., 2003Go), they were not tested in this study.

Twenty-four oocytes which remained unfertilized (seven after ICSI and 17 after conventional IVF) and their 20 corresponding follicular fluid samples were stored at –80°C. The unfertilized oocytes had one polar body in their perivitelline space and exhibited no cytoplasmic or zona pellucida anomalies. No immature oocyte was included. Among the 20 follicular fluid samples, 17 contained a single oocyte, two harboured two oocytes (A-1-2 and A-1-3; E-1-16 and E-1-17) and one contained three oocytes (B-1-8, B-1-9 and B-1-10). Blood samples were also collected on the day of oocyte retrieval and plasma was separated and frozen at –80°C (n = 10).

Virus assay
Oocytes from HCV RNA-positive women. HCV RNA was detected using a standardized, automated, qualitative reverse transcriptase (RT)–PCR assay (Cobas Amplicor HCV 2.0; Roche Diagnostics, Meylan, France). Briefly, all 210 µl of the proteinase K solution containing the oocyte were incubated with lysis buffer, then the RNA was precipitated with isopropanol, centrifuged, washed once with ethanol, and finally resuspended in 200 µl of specimen diluent. To prevent false-negative results due to the presence of inhibitors, an internal control was included in each amplification reaction. The HCV internal control consisted of a synthetic RNA transcript with primer-binding regions identical to those of the HCV target sequence, a randomized internal sequence, whose length and base composition were similar to those of the HCV target sequence, and a unique probe-binding region allowing one to distinguish between the internal control and the target amplicon. Each specimen or control was amplified in the thermal cycler of the COBAS analyser with primers KY78 (biotinylated) and KY80, which identify a 244 bp sequence of the highly conserved 5'-untranslated region of the HCV genome. The AmpErase enzyme (uracil N-glycosylase) was used to prevent carry-over contamination by previously amplified material (i.e. false-positive results). After amplification, the analyser automatically denatured the double-stranded, biotinylated amplicons and captured them with a suspension of magnetic particles coated with an oligonucleotide probe specific to HCV (or internal control). The unbound material was washed away, and the biotinylated amplicon was detected with an avidin–horseradish peroxidase conjugate revealed by colorometric reaction with tetramethylbenzidine–H2O2. The absorbance (660 nm) of each sample was recorded and compared with a pre-defined cut-off value to determine positive or negative results for both HCV and internal control. The sample was considered as negative when the optical density was <0.15, and positive when the optical density was >0.15. No sample was observed between 0.15 and 0.5. The expression ‘weakly positive’ was used to qualify optical density comprised between 0.5 and 2. The detection limit was 50 IU/ml.

Oocyte control groups. Unfertilized oocytes voluntarily offered by HCV-seronegative women undergoing the same ART procedures served as controls (a total of 30 oocytes from 10 patients) and were subjected to the PCR protocol. They had one polar body in their perivitelline space and did not present any cytoplasmic or zona pellucida anomalies.

Negative controls were systematically included. Two unfertilized oocytes were used in each run. A total of five runs were performed, i.e. 10 HCV-negative oocytes (six after conventional IVF and four after ICSI) were tested.

To validate the HCV RNA detection on oocytes, unfertilized oocytes from HCV-negative women, artificially contaminated with HCV RNA-positive plasma samples, were also tested. A plasma sample containing 500 000 IU/ml of HCV RNA, quantitated with the Cobas Amplicor HCV Monitor 2.0 kit, was serially diluted in HCV-seronegative normal human plasma to obtain an HCV RNA range of 50–500 000 IU/ml. Each oocyte was placed in a sterile plastic tube; 100 µl of one dilution of HCV RNA-positive plasma (undiluted, 1/10, 1/100, 1/1000, 1/10 000) were added and incubated at 37°C for 1 h. After incubation, oocytes were rinsed separately five times in 1 ml of fresh PBS.

Each oocyte in 10 µl of PBS was transferred into a cryotube containing 200 µl of a proteinase K solution (0.1 mg/ml) and frozen at –80°C until assayed for HCV. Each dilution was tested on four oocytes; then, 20 oocytes (13 conventional IVF and seven ICSI) from six HCV-seronegative women were analysed.

Plasma and follicular fluid
The HCV RNA contents in the 10 plasma samples collected on the day of oocyte retrieval and in each of the 20 follicular fluid samples from which the 24 oocytes were isolated were analysed quantitatively using the Cobas kit, according to the manufacturer’s instructions. The principle of the assay is similar to that of the first-generation Amplicor HCV Monitor 1.0 assay. The following modifications were made in the master mix: dimethylsulfoxide was added to unwind the hairpin secondary structures, and manganese was added to optimize the quantification of all HCV genotypes. This assay has a manual HCV RNA extraction step followed by automated RT amplification, amplicon detection and calculation of the number of viral RNA IU. The linearity of the assay is stated to range between 600 and 600 000 IU/ml (all samples with >600 000 IU/ml should be diluted). When the quantitative (Monitor 2.0) analysis was <600 IU/ml, a qualitative (Amplicor 2.0) analysis of HCV RNA was performed.

Statistical analysis
Spearman’s correlation coefficient was used to estimate the statistical relationship between two quantitative variables.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oocyte control groups
The 20 oocytes artificially exposed to HCV RNA-positive plasma were positive for all dilutions tested, as assessed by qualitative RT–PCR. The eight oocytes exposed to plasma with an HCV RNA load of 500 000 or 50 000 IU/ml were strongly positive, while the 12 incubated with HCV loads of 5000, 500 or 50 IU/ml were weakly positive.

HCV was not detected in the 10 unfertilized oocytes collected from HCV-seronegative women (negative controls).

Oocytes from HCV RNA-positive women
The results of HCV RNA detection and quantification in plasma, follicular fluid and oocytes are shown in Table I.


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Table I. PCR detection of HCV RNA associated with plasma, follicular fluid and individual oocytes
 
We tested 24 unfertilized oocytes from 10 ovarian stimulation cycles from which 45 oocytes were retrieved. Among the 21 embryos obtained, 17 were in utero deposited in nine transfers, yielding two pregnancies. One woman miscarried (patient ‘G’) and the other (patient ‘B’) gave birth to a child who was HCV RNA negative 3 months after birth. Among the 24 oocytes tested, 17 (70.8%) were positive for qualitative HCV RNA. HCV RNA loads ranged from 4405 to 1 456 751 IU/ml in plasma and from 2030 to 122 524 IU/ml in the 13 follicular fluid samples positive by the quantitative method. For six follicular fluid samples, HCV RNA was detected only by the qualitative method. HCV RNA was not detected in B-2-12 follicular fluid.

Oocyte positivity did not reflect the HCV RNA load in the corresponding follicular fluid; even for those follicular fluid samples negative by quantitative analysis but positive by qualitative analysis, HCV RNA detection associated with oocytes was positive. No relationship could be established between the follicular fluid aspect and HCV association with the oocyte; even the two oocytes from clear follicular fluid were HCV RNA positive. According to the type of ART, 6/7 oocytes subjected to ICSI were positive as opposed to 11/17 that underwent conventional IVF.

Among the seven HCV RNA-negative oocytes, six from three different patients had HCV RNA-positive follicular fluid. The results of qualitative HCV RNA testing of oocytes from the same attempt were consistently positive or negative, except for patient H, who had one negative and two very weakly positive oocytes. Only one follicular fluid sample (5%) was negative with the qualitative method; the corresponding oocyte was also negative, and the plasma HCV RNA load was the lowest observed in this study. A weak correlation was observed between plasma and follicular fluid HCV RNA loads (r = 0.73, P < 0.001) (Figure 1).



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Figure 1. Correlation between HCV RNA loads in plasma and follicular fluid.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We report for the first time PCR-detected HCV RNA associated with zona-intact unfertilized human oocytes obtained from HCV RNA-positive women enrolled in an ART programme. Among the 24 oocytes tested, 17 (70.8%) were positive for qualitative HCV RNA detection. Positive and negative controls included in each test allowed the verification of the PCR technique applied to individual oocytes. The positive controls were also included to identify a possible PCR inhibitor of oocyte origin. In the positive control group, HCV RNA was detected in association with all 20 oocytes, regardless of the virus-positive plasma dilution used. Six oocytes from HCV-infected women (A-1-1, A-1-2, A-1-3, A-1-4, D-1-14 and H-1-22) were virus negative despite the positivity of their corresponding follicular fluid and plasma. Because the loss of these oocytes during the last pipetting is highly improbable, this difference might be linked to the detection threshold, leading to an underestimation of the number of oocytes associated with HCV RNA.

The HCV RNA detected in association with the oocytes is probably not due to either the presence of granulosa cells on the zona pellucida (their absence was verified previously under the microscope), or to the 10 µl of medium containing the oocyte after two washes. In addition, no HCV RNA was detected in any embryo culture media in which embryos derived from HCV-positive women and selected for transfer or freezing had been incubated (Leruez-Ville et al., 2001Go; Sifer et al., 2002Go; Devaux et al., 2003Go). All the oocytes tested were unfertilized after either conventional IVF or ICSI.

Furthermore, in the three ICSI cycles, all the oocytes were mature and microinjected, then no immature oocyte was tested. The percentage of HCV RNA-positive oocytes was higher after ICSI (85.7%) than conventional IVF (64.7%). This tendency has to be confirmed with a larger number of oocytes, but raises the possibility that microinjection allows viral elements to be transported into the oocyte with the spermatozoon. Indeed this possibility has been investigated by injecting the oocyte/zygote with a purified solution of viral RNA (Gamarnik et al., 2000Go), DNA (Baskar et al., 1993Go), purified virus solution (Tebourdi et al., 2002Go) or contaminated spermatozoa (Chan et al., 2000Go). Because our patients were infected with HCV and their partners were HCV negative, the hypothesis that ICSI could be a greater source of contamination than conventional IVF requires further examination. Among the six HCV RNA-negative oocytes with positivity of their corresponding follicular fluid and plasma, five were from IVF. Devaux et al. (2003Go) have shown that on day 1, culture media were weakly positive for HCV RNA in 25% of samples, but then negative on day 2 after granulosa cells were removed. It is therefore possible that granulosa cells could partially protect the oocyte, and this could be another explanation for the differences observed between oocytes from IVF and ICSI.

PCR analysis does not allow one to determine the localization of the viral RNA; it could be on or in the zona pellucida, in the perivitelline space or inside the oocyte cytoplasm. However, based on animal experiments, which provided no evidence of viral penetration of the intact zona pellucida after artificial exposure of in vitro-derived embryos (see the review by Stringfellow and Givens, 2000Go), we suppose that the human zona pellucida can also represent a protective barrier against viral contamination. Using scanning electron and confocal laser scanning microscopy, Vanroose et al. (2000Go) examined the ultrastructure of the zona pellucida of mature bovine oocytes and in vitro-produced embryos, and demonstrated the presence of pores at the outer surface of the zona pellucida and channels of centripetally decreasing diameters in the zona pellucida. These pores are large enough for viruses, such as the bovine herpes virus-1 (BHV-1) (180–200 nm) or bovine viral diarrhoea virus (BVDV) (45–55 nm) to penetrate, but they do not pass through the inner layers of the zona pellucida to reach the embryonic cells (Vanroose et al., 2000Go). The same centripetal conical structure of zona pellucida pores has been described in humans (Nikas et al., 1994Go). HCV measures 55–65 nm in diameter; thus, it is able to enter into the external pore opening but whether it can pass completely through the zona pellucida and reach the oocyte cytoplasm remains unknown. The facilitating role of the alteration of the zona pellucida induced by the spermatozoon during fertilization in conventional IVF is not so evident.

Suzuki et al. (1994Go) described dramatic changes of the zona pellucida upon fertilization, with decreasing size and number of channels. The facilitating role of the ICSI process, like that of zona pellucida abnormalities, is still being debated (see below). In our study, the zona was intact around oocytes tested, as evaluated under the light microscope at x200 magnification. Washing oocytes or embryos could lower the quantity of virus associated with the zona pellucida but, as has been demonstrated with BVDV, washing and enzymatic treatment (trypsin) did not reliably remove all BVDV that was associated with developed IVF bovine embryos (Bielanski and Jordan, 1996Go; Trachte et al., 1998Go).

In our study, despite decoronisation with hyaluronidase, the remaining oocytes unfertilized after ICSI were also HCV RNA positive. HCV in the channels of the zona pellucida might be protected from such treatment. Additional research is needed to determine if oocyte washing effectively eliminates HCV.

In our study, women were infected with HCV during adulthood and, in light of the absence of serum or supporting cells from oocyte/embryo cultures, oocytes or only their zona pellucida were contaminated, probably by contact with blood during follicular aspiration. The level of HCV RNA in follicular fluid probably reflects both the circulating HCV RNA load and the degree of blood contamination during retrieval, as suggested by the correlation we found between plasma and follicular fluid HCV RNA loads. This is in agreement with Devaux et al. (2003Go) who also reported a significant correlation between HCV serum load and HCV follicular fluid load. The follicular fluid of oocyte B-2-12 was HCV RNA negative despite its slightly bloody aspect; however, the plasma virus load was the lowest in this study. Viral contamination of the oocyte prior to ovulation cannot be excluded although it is still impossible to demonstrate. However, the ability of HCV to cross cellular compartments was indicated by the analysis of the virus’s genome in hepatocytes (Shimizu et al., 1997Go), or by HCV RNA detection in sperm (Levy et al., 2000Go; Leruez-Ville et al., 2000Go; Cassuto et al., 2002Go).

In conclusion, we report for the first time the PCR detection of HCV associated with zona pellucida-intact oocytes from anti-HCV antibody-positive and viraemic women undergoing ART. Oocyte contamination probably occurred during ovarian puncture by blood and contamination of follicular fluid. Because of the zona pellucida structure, the HCV is probably embedded in the pores of its outer layers. The ability of HCV to pass through the zona pellucida barrier and reach the oocyte cytoplasm or embryonic cells is still unknown. For HCV-positive women undergoing ART, more studies are needed to evaluate the risk of HCV contamination to which oocytes/embryos are exposed and to establish good safety guidelines for oocyte/embryo manipulation, cryopreservation and donation.


    Acknowledgements
 
The authors thank Mrs Janet Jacobson for reviewing the English text.


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 Materials and methods
 Results
 Discussion
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Submitted on April 11, 2003; resubmitted on December 1, 2003; accepted on February 18, 2004.





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Articles by Papaxanthos-Roche, A.
Articles by Mayer, G.