Effects of hepatitis C virus on the apoptosis percentage of granulosa cells in vivo in women undergoing IVF: preliminary results

C. Sifer1,2,6, J.L. Benifla2,5, M. Branger3, A. Devaux1, F. Brun-Vezinet3, P. Madelenat4 and G. Feldmann1,2

1 Service d'Histologie, 2 Unité Inserm U327, 3 Service de Virologie, 4 Service de Gynécologie–Obstétrique, CHU Bichat-Claude-Bernard, and 5 Service de Gynécologie–Obstétrique, Hôpital Rothschild, Paris, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The aim of this study was to investigate the relationship between the apoptosis percentage of human luteinized granulosa cells (GC) and the presence of hepatitis C virus (HCV) in follicular fluid (FF). METHODS: GC were isolated from FF of 12 women undergoing 12 IVF cycles: six were HCV+ with active viral replication and six HCV– serving as controls. No male partner was HCV+. HCV detection and quantification were assessed by reverse transcriptase–polymerase chain reaction in serum, FF and embryo-incubation medium. GC were analysed by flow cytometry after propidium iodide staining to measure the percentages of apoptotic GC. Routine IVF parameters were tabulated. RESULTS: Mean ± standard deviation (SD) serum and FF HCV viral loads were 3.58 ± 4.25x106 and 0.14 ± 0.10x106 IU/ml respectively. Mean percentages of apoptotic GC from HCV+ and HCV– women were 3.08 ± 1.14 and 3.14 ± 1.40% respectively. No statistically significant difference was found between these two groups concerning GC apoptosis and when we compared all IVF parameters. No HCV RNA was detected in embryo incubation media after 2 days of culture. CONCLUSIONS: Comparing GC apoptosis percentages and usual IVF parameters in the HCV+ group versus the HCV– group, our preliminary study shows that active chronic HCV infection does not affect follicle development and IVF outcome in HCV+ women undergoing IVF. Furthermore, the risk of newborns becoming HCV-infected might not be increased by assisted reproductive technologies when performed in couples in which women are HCV+ and men HCV–.

Key words: apoptosis/hepatitis C virus/human granulosa cell/IVF


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Hepatitis C virus (HCV) is known to increase both the in-vivo serum level of interferon gamma (IFN{gamma}) in HCV+ patients with active viral replication (Piazzolla et al., 2000Go) and the in-vitro IFN{gamma} production by peripheral blood mononuclear cells (PBMC) (Izuma et al., 2000Go). Furthermore, it has been recently demonstrated that HCV is a positive regulator of Fas-mediated apoptosis occurring in PBMC (Hahn et al., 2000Go; Taya et al., 2000Go). This could be mediated by an increase of circulating soluble Fas-Ligand induced by HCV. Fas-Ligand is known to induce apoptosis in sensitized cells when bound to the Fas receptor (Nagata, 1997Go). Enhanced caspase-3 activity in HCV–infected cells has been also reported without alteration of Fas-receptor expression (Hahn et al., 2000Go).

HCV tropism is controversial and the mechanisms of virus entry into cells remain unknown. It was recently advanced that the family of low-density lipoprotein (LDL) receptors might serve as HCV receptors (Agnello et al., 1999Go). LDL receptors are present on the plasma membrane of granulosa cells (GC) and they are up-regulated by GnRH agonists, human FSH or HCG (Bramley et al., 1987Go; Foster et al., 1993Go). In addition, each maturing ovarian follicle is perfused by a follicle artery, thus suggesting intimacy among PBMC, HCV, the oocyte and its cumulus. Primary follicular fluid (FF) is produced by the antral follicle until shortly before ovulation and essentially represents a plasma transudate plus GC secretions (Gosden et al., 1988Go). We recently confirmed that isolated human GC were sensitive to apoptosis induced by IFN{gamma} and an agonistic anti-Fas antibody (Benifla et al., 2002Go), as previously reported by Quirk et al. (Quirk et al., 1995Go). Thus, we hypothesize that HCV could induce GC apoptosis, by acting directly on GC or by modifying local concentrations of pro-apoptosis substances synthesized by PBMC.

The aim of this study was to test the hypothesis that HCV+ women, recently included in our centre for assisted reproductive technologies, could present an increased spontaneous GC apoptosis percentage compared with HCV– women, because of the presence of HCV in vascularized ovarian follicles.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
Twelve patients undergoing IVF were stimulated with recombinant FSH (Puregon®; Organon, Saint-Denis, France, or Gonal F®; Serono, Boulogne, France) after pituitary down-regulation with a GnRH agonist (triptoreline; Ipsen-Biotech, Paris, France) until at least four follicles had attained a mean diameter of 16 mm. Transvaginal oocyte retrieval was scheduled 36 h after administration of 10 000 IU of HCG. Six women (mean age = 35.1 ± 3.6 years) were HCV+ with active viral replication assessed by reverse transcriptase–polymerase chain reaction (RT–PCR) in serum (see below). Four of them underwent liver biopsies, which showed lesions consistent with active chronic viral hepatitis. Indeed, the Knodell score, a histological activity index of liver lesions such as periportal necrosis, intralobular degeneration, focal necrosis, portal inflammation and fibrosis (Knodell et al., 1981Go), was 3, 5, 5 and 6 respectively. Five of these women had liver alanine–aminotransferases twice the normal value. Modes of HCV infection were blood transfusion for four women, i.v. drug use for one and undetermined for one patient. Active chronic hepatitis was diagnosed in two patients in 1992 and 1993 and in the four others in 1998. These women had not received antiviral treatment for at least 6 months before the IVF attempt. Six HCV– women served as controls (mean age = 34.6 ± 3.2 years). Indications for infertility treatment were either bilateral tubal obstruction or a male factor. All women had HCV– male partners and gave their informed consent for the use of their FF in this study.

GC isolation and flow cytometry
After oocyte identification and isolation from FF under a dissecting microscope, each woman's fluid was pooled (n = 12). Aliquots of FF and serum (1 ml each) from each patient were stored at –80°C for subsequent virological analysis. After FF centrifugation at 500 g for 30 min, red blood cells were removed from follicular aspirates by density gradient centrifugation through 10 ml 50% Percoll solution (Pharmacia Biotech, Uppsala, Sweden) for 30 min at 100 g GC were aspirated from the interface and washed in phosphate-buffered saline solution. PBMC were depleted by treatment with anti-CD45-coated magnetic beads (Dynabeads M-450 CD45®; Dynal, Oslo, Norway) according to the manufacturer's instructions. Luteinized GC were dissociated by trypsinization (GIBCO BRL, Glasgow, UK), which was stopped after 5 min by the addition of minimum essential medium (MEM) with Earle's salts and glutamax (GIBCO BRL), supplemented with 10% fetal calf serum (Valbiotech, Paris, France). Purified GC were assessed for apoptosis by a cell cycle analysis of DNA content, as previously described by Makrigiannakis et al. (Makrigiannakis et al., 2000Go). Briefly, GC suspensions were fixed in ice-cold 70% ethanol for at least 16 h and centrifuged for 5 min at 500 g. GC pellets were treated for 15 min at room temperature with RNase A (180 µg/ml; Roche, Meylan, France), stained for 30 min with propidium iodide (50 µg/ml; Sigma, Saint Quentin Fallavier, France) and analysed by flow cytometry on a EPICS XL (Beckman Coulter, Fullerton, CA, USA). Hypodiploid GC, containing <2n DNA in the cell cycle analysis profile determined by the EXPO 32 software (Beckman Coulter), were considered to be apoptotic. As a positive control, flow cytometry was applied to GC from HCV– women. After purification as described above, GC were incubated for 12 h with the anthracycline anti-tumour drug, doxorubicin (100 nmol/l/ml), known to be pro-apoptotic for ovarian cells (Bellarosa et al., 2001Go). The cell cycle analysis detected 85% of hypodiploid GC, permitting us to validate our protocol (data not shown).

HCV RNA extraction
HCV RNA was extracted from serum, FF and embryo incubation medium using the Amplicor HCV Specimen Preparation Kit® (Roche) according to the manufacturer's instructions. Internal controls were added to validate the extraction and amplification steps and to assure the absence of false-negative results.

HCV RNA tests
HCV RNA was detected with Cobas Amplicor HCV 2.0 (Roche), which has a sensitivity of 50 IU/ml. Serum and FF virus loads were quantified using Cobas Amplicor HCV Monitor (Roche), which has a sensitivity of 600 IU/ml. To prevent false-positive results due to cDNA contamination, dUTP-uracil-DNA-glycosylase was added to the samples.

Sperm preparation, culture and embryo transfer
Discontinuous PureSperm® (Nidacon, Göteborg, Sweden) centrifugation (90 and 45% layers) was used to separate semen samples and the 90% layer was washed in B2 UpGraded INRA medium (CCD, Paris, France) by centrifugation at 500 g for 20 min. Retrieved cumulus oocyte complexes from FF were gently washed twice in B2 UpGraded INRA medium (CCD), as routinely made in our laboratory, especially to discard blood and clots. Oocytes were inseminated with 100 000 motile sperm/ml and fertilization was verified 18–20 h later, by the presence of pronuclei and polar bodies, observed under an inverted microscope after complete removal of cumulus cells surrounding the oocytes. ICSI was performed when there was severe oligoasthenozoospermia (3/6 in both groups). Embryos from ICSI or from conventional IVF were cultured for 2 more days, with development monitored daily. We recorded the mature oocyte rate, defined as the percentage of oocytes reaching metaphase II, the fertilization rate, the cleavage rate, the embryo quality (good defined as embryos with <20% cytoplasmic fragmentation and even-sized blastomeres) and the pregnancy rate. The day 2/3 culture medium from each embryo to be transferred or frozen was stored at – 80°C until tested for HCV.

Statistical analysis
Values are expressed as mean ± standard deviation (SD). Wilcoxon rank sum test was used with the level of significance set at 5%. The sample size of our study was evaluated on the basis of 3.0 ± 1.5% apoptotic cells in the control group (unpublished data) and at least twice as many apoptotic cells in the HCV+ group, i.e. 6.0%, with the same SD. For a Wilcoxon rank sum test, this led to an inclusion of six subjects in each group.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
HCV RNA was detected in the sera and FF of all six HCV+ women, but not in the sera and FF of HCV– women. Internal controls were always amplified, demonstrating that no PCR inhibitors were present in FF. In HCV+ women, mean ± SD HCV RNA load was 3.58 ± 4.25x106 IU/ml in serum and 0.14 ± 0.10x106 IU/ml in FF. No HCV RNA was detected in all the embryo culture media in which embryos selected for transfer or freezing had been incubated.

The percentages of apoptotic GC for HCV+ and HCV– groups were 3.08 ± 1.14 and 3.14 ± 1.40% respectively. The two groups did not differ significantly for this or any of the other routine IVF parameters, especially concerning the pregnancy rates, since two pregnancies occurred in each group (Table IGo).


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Table I. Comparison of IVF parameters and percentages of apoptotic GC in groups of women according to HCV infection status
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In France, HCV+ patients with active virus replication have only been eligible for assisted reproduction for 1 year. A multicentric clinical trial is in process to determine whether assisted reproduction increases the risk of newborns becoming infected by HCV. Furthermore, the risk of contamination potentially exists not only for the embryos, but also for the embryos resulting from IVF performed in a HCV– couple but incubated at the same time in the same incubator as those of a HCV-infected couple, and also for biologists and technicians performing the different steps of IVF procedures.

However, it remains unknown if HCV status modifies ovarian follicle development during the IVF stimulation procedure and, consequently, IVF outcome. Because the results of recent studies implicated HCV in apoptosis (Hahn et al., 2000Go; Izuma et al., 2000Go; Piazzolla et al., 2000Go; Taya et al., 2000Go), and it was previously demonstrated that women who became pregnant after IVF treatment had significantly lower percentages of apoptotic luteinized GC, as assessed by flow cytometry, than those who did not become pregnant despite normal basal serum FSH levels (Oosterhuis et al., 1998Go), we thought it important to determine whether HCV could have an impact on the percentage of GC apoptosis. Furthermore, Nakahara et al. have reported that a better oocyte outcome from individual follicles and a higher pregnancy rate in women undergoing IVF were associated with a low GC apoptosis percentage, as estimated by fluorescence microscopy (Nakahara et al., 1997aGo,bGo). In this preliminary study, on a very limited number of women, we demonstrated that, despite the possible exposure of the follicular micro-environment to HCV and PBMC which might trigger apoptosis via the Fas-mediated pathway (Hahn et al., 2000Go; Taya et al., 2000Go), GC were not affected, as no statistically significant difference between the GC apoptosis percentages was seen as a function of HCV status. Furthermore, none of the other IVF or ovarian parameters examined differed significantly between groups. The low mean virus load in FF compared with that measured in the sera of our patients seems to indicate that HCV does not replicate in GC from FF, perhaps because the virus does not penetrate into follicular GC. Thus, the presence of HCV in follicular aspirates is probably the consequence of blood contamination during ovarian puncture. However, because GC express LDL receptors on their plasma membrane and it has been postulated that these receptors might enable virus entry into cells (Agnello et al., 1999Go), the ability of GC to replicate HCV cannot be excluded and merits investigation.

In conclusion, our preliminary results suggested, for the first time to the best of our knowledge: (i) that in-vivo active chronic HCV infection does not affect follicle development and IVF outcome in women undergoing IVF; and (ii) routine IVF procedures including washing of oocytes and changing the culture media of embryos until transfer seems, based on our RT–PCR results, to eliminate HCV. Thus, assisted reproduction might not contribute to increasing the risk of newborns being infected by maternal HCV, a preliminary finding that needs to be confirmed on a larger number of HCV+ women. However, the presence of HCV in FF, as demonstrated in our study, implicates a possible risk of nosocomial contamination, as recently reported by Lesourd et al. in a case of contamination by HCV during assisted reproduction (Lesourd et al., 2000Go). Thus, in order to prevent this risk, strict security rules (Steyaert et al., 2000Go) must be employed in IVF laboratories which cater for infertile couples with HCV+ women.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Drs S.Alvarez, M.Naouri, A.Neuraz and C.Poncelet for their help in data collection, R.Porcher for providing helpful statistical technical expertise and Mrs J.Jacobson for reviewing the English translation.


    Notes
 
6 To whom correspondence should be addressed at: Service d'Histologie, Embryologie et Cytogénétique, CHU Bichat-Claude-Bernard, Université Paris 7 Denis-Diderot, 46, rue Henri-Huchard, 75877 Paris Cedex 18, France. E-mail: histo.survgen{at}bch.ap-hop-paris.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Agnello, V., Abel, G., Elfahal, M., Knight, G.B. and Zhang, Q.X. (1999) Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc. Natl Acad. Sci. USA, 96, 12766–12771.[Abstract/Free Full Text]

Benifla, J.L., Sifer, C., Bringuier, A.F., Blanc-Leyrac, G., Camus, E., Madelenat, P. and Feldmann, G. (2002) Induced apoptosis and expression of related proteins in granulosa cells from women undergoing IVF: a preliminary study. Hum. Reprod., 17, 916–920.[Abstract/Free Full Text]

Bramley, T.A., Stirling, D., Swanston, I.A., Menzies, G.S., McNeilly, A.S. and Baird, D.T. (1987) Specific binding sites for LH/chorionic gonadotrophin, low-density lipoprotein, prolactin and FSH in homogenates of human corpus luteum. II: Concentrations throughout the luteal phase of the menstrual cycle and early pregnancy. J. Endocrinol., 113, 317–327.[Abstract]

Bellarosa, D., Ciucci, A., Bullo, A., Nardelli, F., Manzini, S., Maggi, C.A. and Goso, C. (2001) Apoptotic events in a human ovarian cancer cell line exposed to anthracyclines. J. Pharmacol. Exp. Ther., 296, 276–283.[Abstract/Free Full Text]

Foster, J.D., Strauss, J.F. III and Paavola, L.G. (1993) Cellular events involved in hormonal control of receptor-mediated endocytosis: regulation occurs at multiple sites in the low density lipoprotein pathway, including steps beyond the receptor. Endocrinology, 132, 337–350.[Abstract]

Gosden, R.G., Hunter, R.H.F., Telfer, E., Torrance, C. and Brown, N. (1988) Physiological factors underlying the formation of ovarian follicular fluid. J. Reprod. Fertil., 82, 813–825.[Medline]

Hahn, C.S., Cho, Y.G., Kang, B.S., Lester, I.M. and Hahn Y.S. (2000) The HCV core protein acts as a positive regulator of fas-mediated apoptosis in a human lymphoblastoid T cell line. Virology, 276, 127–137.[ISI][Medline]

Izuma, M., Kobayashi, K., Shiina, M., Ueno, Y., Ishii, M., Shimosegawa, T., Toyota, T., Kakimi, K. and Miyazawa, M. (2000) In vitro cytokine production of peripheral blood mononuclear cells in response to HCV core antigen stimulation during interferon-beta treatment and its relevance to sCD8 and sCD30. Hepatol. Res., 18, 218–229.[ISI][Medline]

Knodell, R.G., Ishak, K.G., Black, W.C., Chen, T.S., Craig, R., Kaplowitz, N., Kiernan, T.W. and Wollman, J. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology, 1, 431–435.[ISI][Medline]

Lesourd, F., Izopet, J., Mervan, C., Payen, J.L., Sandres, K., Monrozies, X. and Parinaud, J. (2000) Transmissions of hepatitis C virus during the ancillary procedures for assisted conception. Hum. Reprod., 15, 1083–1085.[Abstract/Free Full Text]

Nagata, S. (1997) Apoptosis by death factor. Cell, 88, 355–365.[ISI][Medline]

Makrigiannakis, A., Coukos, G., Christofidou-Solomidou, M., Montas, S. and Coutifaris, C. (2000) Progesterone is an autocrine/paracrine regulator of human granulosa cell survival in vitro. Ann. N.Y. Acad. Sci., 900, 16–25.[Abstract/Free Full Text]

Nakahara, K., Saito, H., Saito, T. Ito, M., Ohta, N., Takahashi, T. and Hiroi, M. (1997a) The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil. Steril., 68, 312–317.[ISI][Medline]

Nakahara, K., Saito, H., Saito, T., Ito, M., Ohta, N., Sakai, N., Tezuka, N., Hiroi, M. and Watanabe, H. (1997b) Incidence of apoptotic bodies in membrana granulosa of the patients participating in an in vitro fertilization program. Fertil. Steril., 67, 302–308.[ISI][Medline]

Oosterhuis, G.J., Michgelsen, H.W., Lambalk, C.B., Schoemaker, J. and Vermes, I. (1998) Apoptotic cell death in human granulosa-lutein cells: a possible indicator of in vitro fertilization outcome. Fertil. Steril., 70, 747–749.[ISI][Medline]

Piazzolla, G., Tortorella, C., Schiraldi, O. and Antonaci, S. (2000) Relationship between interferon-gamma, interleukin-10, and interleukin-12 production in chronic hepatitis C and in vitro effects of interferon-alpha. J. Clin. Immunol., 20, 54–61.[ISI][Medline]

Quirk, S.M., Cowan, R.G., Joshi, S.G. and Henrikson, K.P. (1995) Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol. Reprod., 52, 279–287.[Abstract]

Steyaert, S.R., Leroux-Roels, G.G. and Dhont, M. (2000) Infections in IVF: review and guidelines. Hum. Reprod. Update, 6, 432–441.[Abstract/Free Full Text]

Taya, N., Torimoto, Y., Shindo, M., Hirai, K., Hasebe, C. and Kohgo, Y. (2000) Fas-mediated apoptosis of peripheral blood mononuclear cells in patients with hepatitis C. Br. J. Haematol., 110, 89–97.[ISI][Medline]

Submitted on November 10, 2001; resubmitted on December 20, 2001; accepted on March 7, 2002.