Human Reproduction, Vol.26, No.1 pp. 143–147, 2011
Advanced Access publication on November 26, 2010 doi:10.1093/humrep/deq317
ORIGINAL ARTICLE Infertility
ICSI outcome in women who have
positive PCR result for hepatitis C virus
N.F. Hanafi 1,2,*, A.H. Abo Ali 3, and H.F. Abo el kheir 2,4
1
3
Department of Medical Microbiology, Alexandria University, Alexandria, Egypt 2Faculty of Medicine, Alexandria University, Alexandria, Egypt
Madin Fertility Center, Alexandria University, Alexandria, Egypt 4Department of Tropical Medicine, Alexandria University, Alexandria, Egypt
*Correspondence address. E-mail: [email protected]
Submitted on June 28, 2010; resubmitted on October 9, 2010; accepted on October 15, 2010
background: Hepatitis C virus (HCV) carriers are often accepted into the assisted reproduction technique programme of fertility
centres. Studies showed that HCV RNA was detected in the follicular fluid of HCV PCR positive females. The objective of this study
was to assess the impact of HCV active on the outcome of ICSI.
methods: This study was conducted on 40 women who proved to be positive for HCV, using RT – PCR. Two control groups (both
n ¼ 40), who were negative for HCV by PCR were also included. The first control group was HCV sero-positive and the second was HCV
sero-negative. We compared the three groups regarding the ovarian response to stimulation, embryo quality and pregnancy rates.
results: The number of failed cycles (lack of ovarian response to stimulation) was higher in HCV RT –PCR positive and sero-positive
females than sero-negative controls (P ¼ 0.0001). There were no differences in embryo cleavage or morphology between the study and
control groups. The pregnancy rate was significantly reduced in the HCV–PCR-positive group compared with the PCR negative/HCV
sero-positive and HCV sero-negative control groups (5, 3 and 48%, respectively; P ¼ 0.001). There was a negative correlation between
number of oocytes and viral load (0.419; P ¼ 0.007).
conclusions: Our results suggest that HCV infection in females undergoing ICSI has a negative impact on the outcome, and the impact
is higher in PCR positive cases: this might be attributed to hormonal disturbance associated with viral liver cirrhosis coinciding with active viral
replication.
Key words: viral load / pregnancy rate / retrospective study / ICSI / hepatitis C virus
Introduction
Hepatitis C is a blood-borne liver disease, caused by hepatitis C virus
(HCV) which was first identified in 1989. Currently, the worldwide
prevalence of HCV infection reaches a figure of 3% with 70% of
cases developing chronicity (Pearlman, 2004). Detection of HCV
antibodies in serum is considered to be the test of choice for screening. Detection of HCV RNA using the PCR technique is recommended to confirm positivity and diagnose active replication.
Quantitative PCR is known to be beneficial in identifying the level
of viraemia and deciding upon interferon therapy. In the majority of
cases, the disease is discovered accidentally while the patient is
free of symptoms. In the inactive stage of the disease, patients are
found to be HCV antibody positive and PCR negative with normal
liver function tests. On the other hand, patients with high-level viraemia and/or high liver function values are considered to be in the
active stage, where interferon therapy may be considered. Viraemia
in hepatitis C passes through alternating stages of remission and
relapse throughout the chronic course of the disease, which may
remain for decades without marked symptoms (Santantonio et al.,
2008). Studies show that active chronic HCV infection does not
affect ovarian follicle development despite the detection of HCV
RNA in the follicular fluid of 89% of HCV PCR positive females, irrespective of the degree of viraemia (Sifer et al., 2002; Devaux et al.,
2003). Studies have demonstrated that patients suffering from liver
cirrhosis might have higher estrogen levels, regardless of the severity
of their disease. Furthermore, these patients might have added
abnormalities in progesterone metabolism (Brown et al., 1964).
The abnormalities in estrogen level and progesterone metabolism
related to HCV replication may affect fertility and conception (Levy
et al., 2002; Englert et al., 2004; Englert et al., 2007). Infertile HCV
carrier females have usually been accepted into the assisted reproduction technique (ART) programme of many fertility centres
(Jabeen et al., 2000; Gilling-Smith et al., 2003). ICSI could be performed in such cases when precautions are taken to avoid HCV
transmission inside the laboratory. No studies in the fertility field
conducted on HCV patients have distinguished between HCV –
PCR-positive patients (viraemic) and antibody positive/PCR negative
& The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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144
Hanafi et al.
(aviraemic) patients (Floreani et al., 1996; Kumar et al., 2007). The
objective of this case-controlled retrospective study was to assess
the impact of HCV active replication, as shown by a positive HCV
RT – PCR result, on the outcome of ICSI.
U-test was used, and for two-group parametric data the t-test was
applied. The Pearson coefficient of rank correlation was used to determine the correlation between the different variables. The level of significance was set at P ≤ 0.05.
Materials and Methods
Results
The authors were advised by the Local Regional Ethics Committee that
ethical review was not required for this retrospective study. Data were
rendered anonymous according to the Guidelines of the Data Protection
Act and we did not interfere with the patients’ care.
One hundred and twenty female patients were included in the study and
were classified into three groups, of 40 patients each as follows:
A total of 120 cases were included in our study. Patients were divided
into three groups of 40 cases each. The mean + SD age was 31.3 +
4, 30.7 + 4.2 and 29.7 + 4.7 years, and the mean FSH level on Day 3
of the menstrual cycle was 5.3 + 2.4, 5.1 + 1.8 and 5.9 + 1.45 IU/
ml for Groups A, B and C, respectively. There were no differences
among the three groups for age or Day 3 FSH level. The body mass
index (kg/m2) was also comparable in all the three groups (range,
mean + SD): Group A: 18 –45, 27.3 + 4.78; Group B: it 194,
27.7 + 5.5; Group C: 19 46, 26.4 + 5.4 (F ¼ 0.311, P ¼ 0.734-NS).
The infertility data and characteristics of patients are shown in
Table I.
Viral load in HCV PCR positive females (Group A) ranged between
100 and 42 000 copy/ml. Most of the cases (75%) had a serum viral
(i) Group A (the study group): women underwent ICSI cycles in
the period January 2004 to January 2008. All subjects were HCV RT–
PCR-positive at least 6 months after conclusion of interferon therapy.
(ii) Group B (first control group): HCV sero-positive/PCR negative
females.
(iii) Group C (second control group): HCV sero-negative/PCR negative
females.
Subjects included in control Groups B and C were chosen from couples
who were undergoing ICSI on a date close to that of Group A (study
group). Cases have been selected in such a way that age, tubal condition
and status of the male factor were matched among the different groups
(no endometriosis cases were included in this study). Before starting the
stimulation cycle, as part of the preparation protocol for (ART), the presence of HCV antibodies in sera of all cases was determined using a third
generation test (enzyme-linked immunosorbant assay).
Quantitative estimation of viral load using real-time quantitative
RT– PCR was performed in all HCV antibody positive cases using a
Stratagene real-time thermal cycler (Livak et al., 1995; Alfaresi et al., 2007)
All 120 cases were scheduled for fresh ICSI cycles (neither frozen
oocytes nor frozen embryo transfers were included in this study).
All the ICSI cycles were performed with standard long protocol procedures using GnRH and injections of human menopausal gonadotrophin,
until at least two to four follicles reached a diameter of 18 mm. The GnRH
dose was adjusted according to sonographic findings and estradiol (E2)
blood levels during the ovarian stimulation period. Hormone analysis for
the studied cases was conducted using a completely automated electrochemiluminescence system (ELECYS, Roche). None of the enrolled
cycles was cancelled prior to oocyte retrieval. All cases were compared
regarding the ovarian response to stimulation (assessed by the peak E2
level), the number and quality of oocytes retrieved, and the number and
quality of embryos viable for transfer 48 h after ICSI. Clinical pregnancy
was assessed by positive fetal pulsation at 6 weeks of gestational age.
ICSI micromanipulation and embryo transfer were performed using a
G5 series culture medium. On Day 2, embryos were observed under an
inverted microscope equipped with Hoffman modulation contrast optics
(Nikon-TE2000-S) for cleavage, equality of blastomeres and fragmentation
(Fauque et al., 2007). A minimum number of one and a maximum of three
best embryos were selected for transfer. Embryo transfer was performed
under ultrasound guidance using a Labotect embryo transfer catheter.
Statistical analysis
Significant differences were determined using the Statistical Package
for Social Sciences (SPSS/version 15) software. The statistical tests
employed were arithmetic mean and SD for categorized parameters,
and x2 for numerical data. For two-group non-parametric data, the
Table I Summary of infertility data in the study of ICSI
outcome in women with HCV.
Group
A
n 5 40
B
n 5 40
C
n 5 40
P-value
........................................................................................
3.1 + 1.34
3.45 + 1.5
3.2 + 1.63
0.23
Number of cases
with male factor*
12 (30%)
19 (47.5%)
17 (42.5%)
0.103
Number of cases
with female
factor**
18 (45%)
12 (30%)
10 (25%)
0.215
Number of cases
with unknown
cause
10 (25%)
9 (22.5%)
13 (32.5%)
0.11
Duration of
infertility
mean + SD (years)
Cause of infertility
No of cases with previous ICSI trials***
Failed trial
7
4
9
0.023#
Successful trial
0
5
7
0.018#
Group A: women who were HCV RT –PCR positive at least 6 months after the
conclusion of interferon therapy.
Group B: first control group, HCV sero-positive/PCR negative.
Group C: second control group, HCV sero-negative/PCR negative.
*Sperm count ,20 million sperm/ml, motile sperm ,40% or sperm with abnormal
morphology .60%.
**Included tubal obstruction, ovarian dysfunction and endometriosis.
***Data of HCV status during the previous trials (collected via phone calls):
Group A: in five previous trials, cases were HCV PCR positive; two cases failed to be
contacted.
Group B: in two failed trials patients were HCV PCR positive and in one successful
trial the female was HCV PCR negative/antibody positive. Remaining cases could
not be contacted.
Group C: as all cases in this group were HCV PCR/antibody negative at the time of
the study (i.e. never experienced HCV infection), it is assumed that during the
previous trials also they were HCV antibody and PCR negative (relying on the fact
that once HCV infection occurs, antibodies remain for life).
#
P-value significant t-test was used. P-value refers to Groups A and B in comparison
with Group C.
145
ICSI outcome in women with hepatitis C virus
load below 5000 copy/ml (low-level viraemia) with a mean of 5704 +
SD 10 078.5 copy/ml. Figure 1 shows the viral load in HCV PCR positive females.
The number of failed cycles resulting from failure of ovarian response
(absence of mature oocytes, 34 –36 h after HCG administration) was
significantly higher in Groups A (52.5%) and B (30%) compared with
Group C (5%) (P ¼ 0.0001 for both comparisons, Table II).
The peak E2 levels reached and the number of oocytes retrieved
were significantly lower in the HCV–PCR positive study group
(Group A) compared with the two control groups (B and C).
Table II shows the characteristics of ovarian response to stimulation.
The mean fertilization rate was significantly lower in HCV PCR positive and HCV sero-positive females, compared with the HCV seronegative control group (28, 32 and 67%, respectively). There was
no significant difference between Groups A, B and C with regard to
embryo cleavage. Total number of cleaved embryos viable for transfer
was 718: Group A 78, Group B 257 and Group C 383. There was no
significant difference regarding morphology in the three groups (the
percentage of equal non-fragmented embryos out of total number
Figure 1 Scatter chart showing different levels of viral load in
serum (viral copy/ml of serum) for HCV PCR-positive females in
the study.
of embryos yielded was 60, 61.5 and 61% for Groups A, B and C,
respectively). Pregnancy rates, defined by positive fetal pulsation,
were significantly adversely affected in the study group (Group A)
compared with the two control groups (B and C) (P ¼ 0.001).
Table III shows the details of ICSI cycle outcomes.
In Group A, there was no significant correlation between the incidence of pregnancy and the viral load; however, there was a significant
correlation between viral load and ovarian failure. There was a significant negative correlation between the number of oocytes and viral
load (the Pearson correlation 20.419; two-tailed P ¼ 0.007).
Discussion
In a review of the literature, we could not find a study that discriminates between HCV PCR positive and HCV antibody positive/PCR
negative groups of patients with regard to the impact of hepatitis C
infection on reproductive function and the outcome of ICSI. In
Egypt, HCV affects around 4% of the population (Arafa et al., 2005).
Infertile couples with one HCV positive partner are not uncommon
at any of the Egyptian fertility centres. HCV positive females suffering
from infertility are treated by ICSI with precautions taken to ensure
effective sterilization of the clinical tools and to avoid iatrogenic transmission to other cases by using separate supplies, such as injectors,
holders and flexipets (Steyaert et al., 2000). It has been noted that
the pathophysiology of HCV infection encompasses, besides effects
on the liver, a multitude of extra-hepatic changes potentially affecting
other body systems (Hadziyannis, 1997; Schulze zur Wiesch et al.,
2003). Researchers investigating HCV infection and its effects on
ovarian follicle development during stimulation for ART reported
higher incidence of apoptosis, which may have a negative impact on
pregnancy rates (Oosterhuis et al., 1998; Hahn et al., 2000; Izuma
et al., 2000; Piazzolla et al., 2000; Taya et al., 2000). HCV receptors
were identified on granulosa cells surrounding the oocytes, but not
on the oocyte itself (Flint et al., 1999; LaVoie et al., 1999; Germi
et al., 2001). A few studies showed that active chronic HCV infection
does not affect follicle development (Lesourd et al., 2000), although
the virus was identified in the follicular fluid (Devaux et al., 2001).
Some authors assigned this contradiction to the use of different
HCV RNA detection methods and to the low number of samples
tested (Dore, 2000; Bourlet et al., 2003). Some authors related the
Table II Ovarian response to stimulation in the study of ICSI outcome in women with HCV.
Mean + SD
Group A
Group B
Group C
P-value
.............................................................................................................................................................................................
Patients’ age (years)
31.3 + 4
30.7 + 4.2
29.7 + 4.7
0.106
Number of gonadotrophin ampoules
31.6 + 8.3
28.4 + 4.6
26.1 + 6.54
0.015*
Days of ovarian stimulation
13.16 + 1.6
12.5 + 1.1
12 + 1.5
0.311
Peak estradiol level (pg/ml)
1280 + 1316.6
2890 + 1586.2
4670 + 1693
0.012*
Number of follicles
11.6 + 6.3
13.5 + 4.6
13.9 + 4.8
0.046*
0.001*
Number of eggs retrieved
6 + 3.4
11.4 + 2.8
13 + 4.2
Number of females with no mature oocytes
21 (52.5%)
12 (30%)
2 (5%)
0.0001*
Number of mature eggs retrieved
4 + 4.2
9.4 + 4.2
12 + 5.2
0.001*
Tests used were: x2 for numerical data, the U-test for non-parametric data and the t-test for parametric data. P-value refers to comparison of Groups A and B versus Group C.
*Significant difference.
146
Hanafi et al.
Table III ICSI cycle outcome in women with HCV.
Group A
Group B
Group C
P-value
........................................................................................
Mean number of
fertilized oocytes/
retreival
1.12 + 1.17 3 + 1.2
8 + 3.5
Total fertilized
oocytes
28%
32%
67%
Total no. of embryo 18
transfer/group
27
38
Implantation rate (%) 33.3%
45%
52%
Clinical pregnancy/ 2/18
total embryo transfer
13/27
19/38
Clinical pregnancy/
total no. of cases
13(32.5%)
19(47.5%)
0.001*
11
15
0.012*
2 (5%)
Number of live births 2
significantly higher in HCV PCR positive females compared with the
two control groups. Five out of seven women, who underwent previous ICSI trials, mentioned that they had been HCV PCR positive
during those trials, whereas the remaining two females could not be
contacted. Although data concerning the HCV status during previous
ICSI trials were not available for all the patients, this observation
appears to be consistent with our current results from ICSI cycles.
0.0035*
Conclusion
0.038*
*Significant difference (ANOVA test).
detection of high HCV RNA levels in the follicular fluid to the vascular
injury induced by ovarian puncture (Sifer et al., 2002). A group of
researchers (Marcellin et al., 1993; Poiraud et al., 2001) showed that
hepatitis C had no impact on pregnancy and fetal development.
Devaux et al. (2003) demonstrated that an HCV RNA titre
.106 IU/ml was detected in 4 out of 16 serum samples of females
undergoing IVF. This virus titre was never detected in follicular fluid,
and a 25% pregnancy rate was achieved for this subgroup of patients
(Devaux et al., 2003). The same research group conducted another
national follow-up case –control study and reported 25 pregnant
females out of 140 IVF procedures in HCV positive cases (Devaux
and Roudot-Thoraval, 2002). Interestingly, it had been observed
that HCV antigens and products encoded by the HCV genome
could affect intracellular transduction and phosphorylation processes
(Schulze zur Wiesch et al., 2003). Furthermore, studies have shown
that other viral infections may affect fertility and ovarian function in
animals (Fray et al., 2000a,b; McGowan et al., 2003). In agreement
with our results, Englert et al. (2007) found an association between
HCV sero-positivity and poor ovarian response to stimulation. The
authors reported a high number of cancelled cycles resulting from
the absence of ovarian response to stimulation in this group of patients
and concluded that chronic infection might alter both the reserve of
small pre-antral follicles and granulosa cell function. They also noted
the need for higher amounts of FSH to obtain a similar peak level of
E2 at the time of injection of HCG. In addition, no difference in implantation and delivery rates between HCV sero-positive and seronegative females was observed (Englert et al., 2007). In an earlier
study by Englert et al. (2004) carried out on a smaller number of
cases, the authors noted similar embryo quality, implantation and
delivery rates in sero-positive females compared with controls.
However, they found that sero-positive females might have alterations
in ovarian stimulation response and follicular maturation, as indicated
by an increased requirement for FSH, lower number of oocytes and
decreased number of fertilized oocytes (Englert et al., 2004). Our findings confirm those of previous studies that HCV may cause higher cancellation rates of cycles and poor ovarian response in HCV female
carriers. The number of cases with previous failed ICSI trials was
Impaired ovarian response to stimulation and lower pregnancy rates
were found in sero-positive females, with or without viraemia, with
a higher magnitude in the latter group. We also showed that the
embryo quality was not affected by the presence of active viral replication. Our results suggest that HCV infection in females undergoing
ICSI has a negative impact on the outcome, and this is higher in the
presence of HCV viraemia (PCR positive). This result might be attributed to a hormonal disturbance associated with viral liver cirrhosis,
which occurs in the context of active viral replication, but more
detailed studies of the hormonal profile all through the menstrual
cycle in this group of cases are required.
Authors’ roles
N.F.H. was the main contributor to the study concept and design.
A.H.A.A. was involved in the acquisition of data N.F.H. and
A.H.A.A. were contributors in the lab work and assays, the data of
which were analysed in this study. N.F.H. drafted the manuscript
and performed the statistical analysis. All authors critically revised
the manuscript and read and approved the final manuscript.
Acknowledgements
We gratefully acknowledge Dr A. Zalat and Dr E. Saleh for their continuing participation.
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