1 Service dHistologie-Embryologie-Cytogénétique, 2 Service de Virologie, 3 Service de Gynécologie-Obstétrique Hôpital Bichat-Claude Bernard, AP-HP, Paris and 4 Département de Biostatistique INSERM U 444, Hôpital Saint-Louis, AP-HP, Paris, France
5 To whom correspondence should be addressed at: Service dHistologie-Embryologie-Cytogénétique, Hôpital Bichat-Claude Bernard, 46 rue Henri Huchard, 75877 Paris, Cedex 18, France. e-mail: aviva.devaux{at}bch.ap-hop-paris.fr
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Abstract |
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Key words: culture media/follicular fluid/HCV RNA/hepatitis C virus/IVF
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Introduction |
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In this context, the study aim was to: (i) search for HCV RNA in FF and in culture media at each step of IVF undergone by HCV+ women; (ii) investigate the impact of blood contamination of the FF induced by vascular injury associated with the transvaginal ovarian puncture; (iii) evaluate the risk for the embryo and the impact on the contamination rate of the newborn; and (iv) determine the viral risk presented by these fluids, in order to define guidelines in the laboratory.
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Materials and methods |
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The origin of HCV contamination was professional in two cases (11.8%), following blood transfusion in two cases (11.8%), due to intravenous drug addiction in four cases (23.5%) and unknown in nine cases (52.9%).
The median age of the women was 37.5 (range 2943) years, and 10 of them were aged over 38 years (59%). Causes of infertility were Fallopian tube disease (n = 11), dysovulation (n = 1), mixed (n = 3) and undetermined (n = 2).
Sample collection
Samples were collected during 22 conventional IVF attempts. On day 0, at 3637 h after hCG administration, FF containing oocytecumulus complexes were recovered by transvaginal ultrasound-guided needle aspiration using a prewarmed syringe. Syringes were classified and pooled depending on the macroscopic blood contamination; they were labelled A0 when FF was clear, A1 when FF was lightly coloured, and A2 for bloody FF. Oocyte collection and identification were carried out using a stereomicroscope equipped with a warm working area (37°C). Once identified, oocytecumulus complexes were rinsed several times and incubated in new multi-well dishes containing prewarmed B2 Upgraded Medium (INRA, CCD, France) labelled B0, B1, B2 depending on the macroscopic blood contamination of FF. Samples A0, A1 and A2 of FF were stored at 80°C until the viral tests. The selected oocytes were inseminated with prepared spermatozoa for the fertilization step and stored at 37°C under 5% CO2.
At 1719 h after insemination (day 1), all oocytes were transferred from B0, B1 and B2 into new multi-well dishes containing prewarmed B2 Upgraded Medium. Granulosa cells surrounding oocytes were removed. Samples of B0, B1 and B2 fluids were stored at 80°C until the viral tests. Oocytes were checked for maturity and evidence of normal fertilization (two pronuclear). Fertilized oocytes were rinsed again and transferred into new multi-well dishes of fresh prewarmed medium labelled C0, C1 and C2, depending on the origin of the oocytes.
At 48 or 72 h after insemination (day 2 or 3), embryo development (cell number and degree of fragmentation) was recorded. Two or a maximum of three embryos were transferred, while excess embryos were cryopreserved in liquid nitrogen. Samples of C0, C1 and C2 fluids were stored at 80°C until the viral analysis.
Three types of FF were collected (A0, A1, A2) in six of the 21 IVF procedures, but only two of the three FF types were collected in 11 of 21 IVF procedures. Only bloody FF (A2) was collected in four IVF procedures. A total of 153 samples was collected, including blood plasma (n = 24), A0 (n = 17), A1 (n = 15), A2 (n = 15), B0 (n = 17), B1 (n = 15), B2 (n = 15), C0 (n = 15), C1 (n = 12) and C2 (n = 8).
Viral tests
HCV RNA was detected using a reverse transcription PCR assay in plasma, FF and in the different media collected at each IVF step. Assays were carried out using the same commercial kit as for serum or plasma and according to the manufacturers instructions (Cobas Amplicor HCV 2.0; Roche, Meylan, France). Plasma and FF were tested undiluted; the sensitivity of the method was 0.05x103 IU/ml. When the available volume of culture media at day 1, 2 or 3 was insufficient, the samples were diluted 1:2 before being tested; the sensitivity in these cases was 0.1x103 IU/ml. Two methods were used to control the results. Uracil DNA glycosylase was included for prevention of false-positive results, while false-negative results due to Taq inhibitors were evaluated by use of an internal control introduced into each sample, co-extracted and co-amplified with target RNA (Amplicor Internal Control Detection Kit; Roche). When HCV RNA was detected in a sample, quantification was performed using a Cobas Monitor HCV RNA kit (Roche). In the first batch of tested samples, Taq inhibitors were present once in FF during qualitative detection and four times in FF and culture media during quantitative tests. These samples were submitted to high-speed centrifugation (23 500xg for 1 h at 4°C) before extraction, and then tested using the same protocol as described above. No inhibitor was found after this procedure. Subsequently, all samples except plasma were centrifuged before extraction and no inhibitor was detected.
Statistical analysis
As HCV load data in the samples could clearly not be considered as Gaussian, all analyses were based on non-parametric rank statistics and tests. The association between HCV load in FF and in the serum was assessed through Spearmans correlation coefficient, separately for A0, A1 and A2. FF HCV loads were compared according to the bloody status using Friedmans two-way analysis of variance, the subject being the blocking factor, in order to account for non-independence between measures on the same subject. This test compared the three groups. However, to identify where the difference was post-hoc, a 2x2 comparison was performed using Wilcoxons signed rank sum test for paired data, with Bonferronis correction for multiple testing to guarantee a global type 1 error risk at 0.05. The frequency of patients with detectable HCV load in FF, and after oocyte washing in B0, B1 and B2 collected at day 1, was compared using McNemars test. All tests were two-sided, with a P-value of 0.05 being considered statistically significant. Analyses were performed using SAS v8.2 (SAS Institute, Cary, NC, USA) and S-Plus 2000 (Mathsoft Inc., Seattle, WA, USA) software. For the analyses (see Table III), when the qualitative detection was negative (below the 100 IU/ml threshold), the value was noted zero (0); when the qualitative detection was positive with a quantitative detection below the 0.6x103 IU/ml threshold, a value of 0.5x103 IU/ml was arbitrarily attributed.
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Results |
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HCV RNA detection was positive in the other 16 sera samples on the day of ovarian puncture. A total of 140 samples was collected from these 16 HCV+ women and analysed during 21 IVF procedures (Table I). A total of 44 FF samples was tested, with HCV RNA detection being positive in 13 of 14 A0 (93%), 12 of 15 A1 (80%) and 14 of 15 A2 (93%). At day 1, HCV RNA was positive in 1 of 14 B0 (7%), 2 of 15 B1 (7%) and 5 of 15 B2 (47%); negative in 11 of 14 B0 (79%), 12 of 15 B1 (80%) and 8 of 15 B2 (53%), and doubtful in two B0, one B1 and two B2. On the day of embryo transfer, all the tested samples C0, C1 and C2 were negative. HCV RNA detection was positive in 89% of all FF samples (39/44), irrespective of the degree of macroscopic blood contamination, but was negative in one A0, two A1 and one A2, even though it was positive in the corresponding serum sample. Rinsing of the oocytecumulus complexes before transfer into the culture media on the day of ovarian puncture, and a second rinsing after checking of pronuclear status at day 1, reduced the detection of HCV RNA (B0, B1 and B2) which became undetectable at the day of embryo transfer in all media (C0, C1 and C2).
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HCV RNA was quantified in five of eight positive culture media (one B0, one B1, three B2). For the other three HCV RNA-positive samples (one B1, two B2) and for the five qualitatively doubtful samples (two B0, one B1, two B2) the available volume was insufficient. The virus load was below threshold (600 IU/ml) in four of five cases, and just above it in the last sample (608 IU/ml).
The virus load increased with the degree of macroscopic blood contamination (see Table III). In 13 of 39 FF samples (33%), the HCV RNA quantification was <0.6x103 IU/ml, while in the other samples the virus load ranged from 2.84 to 325.0x103 IU/ml. There was a significant correlation between HCV serum load and HCV FF load (r = 0.64, P = 0.033 for A0; r = 0.92, P = 0.0014 for A1; and r = 0.65, P = 0.032 for A2). Friedmans two-way analysis of variance yielded a significant FF bloody status effect (P = 0.016), thus indicating that the FF HCV load differed according to the FF blood contamination status.
Post-hoc comparisons gave the following P-values: P = 0.016 for A0 versus A2; P = 0.023 for A1 versus A2; and P = 0.12 for A0 versus A1. When applying a full Bonferronis correction, the significance level had to be taken at 0.017 to account for multiplicity of tests, yielding a significant difference only between A0 and A2. However, this comparison between A0 and A1, A0 and A2, A1 and A2 was limited respectively to 11, seven and seven pairs of samples.
The frequency of samples with detectable HCV load was significantly higher before (A0, A1, A2) than after oocyte washing (B0, B1 and B2), as shown by McNemars test (P < 0.0001). HCV RNA detection was always negative in C0, C1 and C2. Rinsing appeared effectively to reduce the HCV load in the culture media.
IVF outcome
Twenty-two conventional IVF were performed, and 21 embryo transfers were made. In total, six pregnancies were obtained (five singles, one twin), four deliveries and five newborn. In two of the single pregnancies and in the twin pregnancy with a normal delivery, four babies remained HCV RNA PCR-negative at 6 months after birth. In one case the mother developed an acute hepatitis with an increasing HCV RNA load at 7 months gestation. A non-scheduled Caesarean for a single premature baby was performed at 8 months; HCV RNA detection by PCR assay was positive in the infant at 6 months after birth. Control investigations will be made at 12 months after the birth, and two pregnancies are still on-going.
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Discussion |
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HCV RNA detection
Using the present technique, HCV RNA can be clearly identified in FF (A0, A1, A2) and in culture media (B0, B1, B2), and this confirms the present authors initial results (Devaux et al., 2001). No PCR inhibitor was detected in any of the samples. HCV RNA has been detected in a variety of body secretions (Liou et al., 1992
; Tang et al., 1996
; Ackerman et al., 1998
), but not in saliva (Fried et al., 1992
), though the latter study was conducted in only nine patients. More recently, HCV RNA was detected in 33% of saliva samples (Kage et al., 1997
) and in 36% of normal cervical smears of viraemic women (Manavi et al., 2002
). Contradictory results have also been obtained in colostrum and breast milk. In colostrum, a 10% positive detection was reported (Aizaki et al., 1996
; Morales et al., 1996
), despite the viral load being very low in one study (Hunt et al., 1997
) and even negative in others (Kage et al., 1997
; Polywka et al., 1997
). In a prospective study of 16 HCV+ women, HCV RNA was detected in only one sample of clear amniotic fluid (Delamarre et al., 1999
). Although in earlier studies HCV could not be detected in sperm samples from HCV+ men (Semprini et al., 1998
; Debono et al., 2000
), more recent investigations have shown HCV to be present in 510% of sperm samples analysed (Leruez-Ville et al., 2000
; Levy et al., 2000
; Pasquier et al., 2000
; Cassuto et al., 2002
). HCV RNA detection methods and the low number of samples tested might provide an explanation for these contradictory results (Bourlet et al., 2003
). In the present study, the use of uracil DNA glycosylase and an internal control prevented false responses. In order to be considered as infectious, FF must present infectious characteristics. Although the present viral tests were based on particles of genome detection after RNA amplification, this technique reveals the presence of virus genomes, but not their infectivity (Dore, 2000
). It is unclear whether the viral particles in FF have an infectious nature, and as HCV culture is difficult to perform in vitro there are at present no data available to answer this question. Hence, at present it is impossible to evaluate precisely the viral risk presented by FF.
Nevertheless, because HCV RNA was detected in most of the follicular aspirates, FF aspirated in HCV+ women should be considered as potentially infected, irrespective of the degree of macroscopic blood contamination. This observation raises some questions about the original HCV localization inside the follicle in vivo, or to what extent this was induced by the ovarian puncture.
Origin of HCV in follicular fluid
In order to evaluate the viral risk induced by ART and to inform the couples, knowledge is required of the localization of the virus in vivo. Follicular fluid cannot be collected without follicle rupture, and this might involve a risk of contamination as a result of vascular injury occurring during the ovarian puncture.
HCV RNA was not detected in four FF samples, but might have been undetectable as three of these HCV+ women had viral replication (Table II: patients 3, 6 and 11) and one showed no HCV replication (patient 17). If HCV penetrates inside the follicle in vivo, its load is likely to be very low and below the viral test threshold. FF is secreted by granulosa cells. In the ovary, a thick basement membrane surrounds the follicle and separates granulosa cells and the surrounding stromal cells which form a layer containing the vessels with ill-defined outer limits called the theca folliculi. Until ovulation, this membrane separates the follicle components and the vessels. The basement membrane is considered to be a barrier which protects the follicle and principally the oocyte (Bloom and Fawcett, 1986). No data are available concerning the possible compartmentalization of HCV in the follicle in vivo. However, a compartmentalization might be evoked in both the male genital tract (Leruez-Ville et al., 2000
; Pasquier et al., 2000
; Cassuto et al., 2002
) and amniotic fluid (Delamarre et al., 1999
).
Cytological FF analysis has shown the constant presence of peripheral cells (lymphocytes, granulocytes and erythrocytes) (unpublished data), and this provides proof of the constant association of blood contamination with the puncture. Viral loads increase significantly with the presence of macroscopic blood contamination. A similar study on 33 clear amniotic fluid samples from 22 HCV+ women showed that HCV RNA could also be detected in one of 16 viraemic women (Delamarre et al., 1999). These authors invoked the impact of amniotic puncture on the contamination of amniotic fluids. In IVF, the high HCV RNA detection rate in FF (89%) can most likely be explained by the vessel injuries associated with ovarian puncture.
In conclusion, the presence of HCV was highly unlikely in the ovarian follicles in vivo, but when it did occur it was below the threshold of the present technique. HCV RNA detection appears to be induced by vascular injuries that are always associated with ovarian puncture, and the possible consequences of this blood contamination must be considered.
Viral risk for oocytes and embryos
As HCV RNA was detected in 89% of FF samples, all FF samples should be considered as potentially infected, and consequently the viral risk for retrieved follicles must also be considered. Following ovarian puncture, FF is retrieved which contains oocytes surrounded by granulosa cells. Two HCV receptors for HCV have been identified, namely the CD 81 molecule and the low-density lipoprotein receptor (rLDL) (Pileri et al., 1998; Flint et al., 1999
; Germi et al., 2001
), the rLDL having been reported present on granulosa cells (La Voie et al., 1999
). The possible adsorption of HCV onto granulosa cells during ovarian puncture cannot be eliminated. Consequently, HCV present as a result of blood contamination might also be adsorbed onto the granulosa cells, and this might explain the presence of HCV RNA in spite of the first follicle washing. HCV RNA was never detected in the embryo culture media after washing, removal of granulosa cells and refreshing the culture mediaa result which seems to indicate that HCV RNA might be adsorbed onto granulosa cells in FF during the IVF procedure, though further studies are needed to verify this hypothesis. Furthermore, until implantation the oocytes and embryos are surrounded by the zona pellucida (ZP), which is considered to be a physiological protective barrier. Evaluation of the viral risk for oocyte and embryo during IVF has not yet been completely defined, though it cannot be ignored and deserves future study. However, whilst awaiting further information this risk can be evaluated indirectly by monitoring the contamination rate of the offspring after IVF compared with spontaneous pregnancies in HCV+ women.
Impact on contamination of the offspring
Although vertical mother-to-infant HCV transmission in spontaneous pregnancy has been well documented (Tibbs, 1995; Dienstag, 1997
; Hunt et al., 1997
; Michielsen and Van Damme, 1999
; Yeung et al., 2001
), the mode and moment of transmission are not well known. The transmission rate was only 6% in patients with a viraemia below 106copies/ml (Poiraud et al., 2001
), and contamination occurred most frequently at the time of delivery (Abergel et al., 1995
; Moriya et al., 1995
; Sabatino et al., 1996
). Hepatitis C appears to have no impact on pregnancy and fetal development (Marcellin et al., 1993
). However, acute hepatitis C contracted during the third trimester of pregnancy might induce contamination of the child (Hunt et al., 1997
), and 50% of children infected present with a clinical infection, though this is usually benign. Pregnancy has never been contraindicated in HCV+ women.
In the present study, the HCV RNA titre was >106 IU/ml in four of 16 serum samples, but never reached this level in FF samples. After washing and refreshing the culture media, HCV RNA decreased and was apparently discarded in the embryo media. The viral risk which could be induced by IVF procedures appeared to be low. A total of 21 embryo transfers was performed in 16 women, from which six pregnancies were obtained with five newborns (four HCV-negative and one HCV-positive). In the latter case the mother had developed acute hepatitis during the third trimester of pregnancy, the serum viral load had increased from 1.9x106 to 15.3x106copies/ml, and the delivery was a non-scheduled Caesarean. A viral load beyond 106copies/ml leads to contamination of the child in 50% of cases (Ohto et al., 1994; Lin et al., 1994
). Moreover, HCV RNA detection was negative in the amniotic fluid sampled for prenatal diagnosis, which would suggest that contamination on delivery and the impact of amniotic puncture must be further studied.
At present, the number of newborn babies is too small to evaluate whether IVF either decreases, increases or does not modify offspring contamination rate. In France, a national follow-up casecontrol study on the offspring of HCV+ mothers after IVF compared with spontaneous pregnancy is on-going (Protocol ANRS HC 04, Roudot-Thoraval, 2000). The follow-up involves 400 children for each group at 3, 6 and 12 months after birth. After 140 IVF procedures performed in France, 25 pregnancies were obtained with 12 HCV-negative babies and one PCR-positive baby (Devaux and Roudot-Thoraval, 2002). Half of the pregnancies in these preliminary results were obtained at the present authors centre, and the other pregnancies are still on-going.
HCV undergoes very rapid mutation. Hence, in order to determine the time at which the child is contaminated, HCV sequencing should be conducted on the maternal blood (which is stored on the day of puncture) as well as on the infected infants blood.
HCV and guidelines for IVF centres
The risk of nosocomial infection in ART has been discussed (Botta-Fridlund, 1994; McKee et al., 1996
; Steayert et al., 2000
). Two cases of HCV nosocomial infection have been described during ancillary procedures for ART (Lesourd et al., 2000
). Although such contamination has occurred in the operating theatre, until now it has never been described in an IVF laboratory. Two types of risk in the laboratory must be considered, namely contamination of the HCV couples embryos, and contamination of laboratory workers during ART. In this respect, the high prevalence of hepatitis C (as mentioned above) must be taken into account, as well as the high detection rate of HCV RNA in FF, and hence all FF samples should be considered as potentially contaminated. Strict adhesion to universal guidelines (CDC Atlanta NMWR no. 2, 1987) can prevent gamete and embryo contamination of HCV couples. Moreover, in France specific statements have been prepared for IVF laboratories that perform ART in a viral context (Arrêté du 10 Mai, 2001). A separation of the IVF procedures for carrier and non-carrier virus couples (for HIV, HCV and HBV) in time or in space must be respected. In addition, the cryopreservation of embryos must be carried out in high-security straws which are stored in a specific liquid nitrogen tank. However, this procedure goes against the universal laboratory guidelines, where all samples must be considered as potentially infected and no specific circuit is organized. These specific laboratory procedures, which have been in operation in France since 1999 (Arrêté du 12 Janvier, 1999), should be discussed when the impact of ART on embryo viral contamination risk is to be assessed. Until such discussion has been concluded, all FF must be considered as potentially infected and treated according to the laws in France.
Discarding oocytes from bloody FF, as has been proposed by some practitioners in order to prevent a viral risk, appears unnecessary. This procedure might affect IVF outcome by decreasing the number of oocytes for the fertilization step. During the IVF procedure, the decreasing HCV RNA titre might be due to a dilution of the virus during washing of the oocytes, removal of the granulosa cells, and refreshing of the media. It is possible to compare the oocyte washing procedure with a selection procedure using a gradient of PureSperm®, followed by the washing of spermatozoa, from HCV+ and HIV+ men (Bourlet et al., 2003). The HCV RNA titre was decreased drastically after the selection and washing of spermatozoa from infected men, and preliminary results have confirmed an absence of contamination in both the negative female partner and the newborn (Cassuto et al., 2002
). It could therefore be expected that, if the embryo culture media is negative, then the viral risk for viraemic womens embryos is very low and, at most, almost absent. The present results have shown that thorough rinsing of the oocytes and embryos and refreshing of the media appear to be effective and that, as a consequence, all oocytes could be used. None the less, oocytes which were included in a clot of blood and could not be thoroughly washed, must be discarded. Attention must also be paid to the impact of HCV+ genital secretions on the contamination rate of the embryo when the transfer is associated with a light cervical bleeding. The risk incurred after the embryo transfer cannot be ignored, and the impact on the offspring contamination rate must be studied in order to evaluate exactly the consequences.
Using serum from an HCV+ mother sera for embryo cryopreservation must also be avoided. Consequently, the use of a tested negative serum albumin, or the male partners serum is recommended.
The viral risk for the laboratory personal is the same as that described in all laboratories. ART does not increase the occupational risk; rather, the strict respect of universal guidelines prevents occupational contamination.
In conclusion, HCV RNA was detected in 89% of FF and 25% of culture media on the day after the ovarian puncture in attempted IVF procedures in HCV+ women. Follicular fluid must be considered as potentially infected. A blood contamination during puncture increases HCV loads in FF. After washing of the oocytes and embryos and refreshing the media, the HCV viral load becomes undetectable, irrespective of the original FF bloody status, and consequently all oocytes can be inseminated. A compartmentalization between the blood and FF in vivo could be invoked, but further studies are required in order to assess the exact impact of ovarian puncture on FF viral status and a possible coupling of the virus with the oocyte and embryo. At present, it is too early to assess the impact of IVF on the mother-to-child transmission rate of HCV, and the follow-up of offspring must be continued. Likewise, there is a need for a prospective study to be conducted on the impact of HCV+ genital secretions on transmission rate. Consequently, after counselling HCV+ women on the potential risk for the offspring, an IVF attempt is justified. ART procedures do not specifically increase the risk to IVF professionals, and strict adherence to universal guidelines can prevent the nosocomial infection of other negative couples and laboratory workers alike.
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Acknowledgements |
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Submitted on June 1, 2003; resubmitted on June 16, 2003; accepted on July 10, 2003.