The Risk Of Hepatitis B Virus Transmission During The Assisted Reproductive Technology Procedure

Objectives:1. To examine the prevalence of hepatitis B virus (HBV) infection and the reproductive performance of couples seropositive for HBV.2. To explore the outcomes of the IVF and embryo transfer treatment cycles in infertile couples seropositive for HBV.3. To define the possible vertical transmission of HBV via spermatozoa to embryos, we investigated the susceptibility of embryos from couples with hepatitis B surface antigen (HBsAg)-positive men.4. To determine whether human oocytes can carry HBV and HBV can be transmitted via oocytes to embryos, we investigated the susceptibility of oocytes and embryos from HBsAg-positive women to HBV during the Assisted reproduction technology (ART) procedure.5. To investigate the relationship between the HBV DNA levels in plasma and follicular fluid of HBsAg-positive women, quantification of HBV DNA was performed in plasma and follicular fluid samples.6. To determine whether or not an intracytoplasmic sperm injection (ICSI) procedure in chronic carriers of HBV increases the potential risk of HBV transmission, we compared the percentage of HBV-positive oocytes and embryos between ICSI and conventional IVF. 7. To define the relationship between the Serostatus of HBsAg-positive couples and HBV-positivity of oocytes and embryos, and to determine whether or not the ART increases the risk of vertical HBV transmission.Methods:1. A retrospective cohort study of 3457 infertile couples were performed during the period from January 2008 to December 2008 to examine the prevalence of HBV infection.2. This was a retrospective cohort study on IVF and embryo transfer cycles performed on couples during the period from March 2009 to December 2009 in the Assisted Reproductive Unit. The 130 couples with a HBsAg-positive male partner,102 couples with a HBsAg-positive female partner,12 couples with both HBsAg-positive partners was categorized as HBV (study) group, whereas those screened negative served as the control group. The data on patients' characteristics, controlled ovarian hyperstimulation (COH), embryology, and pregnancy and implantation rates of IVF and embryo transfer cycles were analysed.3. The HBV DNA and HBV RNA in embryos from couples with HBsAg-positive men were examined by polymerase chain reaction (PCR) analysis and reverse-transcriptase (RT)-PCR analysis, respectively.4. The HBV DNA and HBV RNA in oocytes and embryos from HBsAg-positive women were examined by PCR analysis and RT-PCR analysis, respectively. The immunofluorescence staining was used to determine the location of virus particles containing HBsAgs.5. The HBV DNA loads in the plasma and follicular fluid were analyzed quantitatively using the real-time PCR. Spearman's correlation coefficient was used to estimate the statistical relationship between two quantitative variables.6. Based on the HBV detection in oocytes and embryos from chronic HBV carriers to HBV during the ART procedure, we further compared the percentage of HBV-positive oocytes and embryos between ICSI and conventional IVF.7. HBV markers were tested by radioimmunoassay. Results:1. Among the infertile couples undergoing IVF and embryo transfer treatment, the prevalence of couples with a HBsAg-positive female partner is 7.95%(275 of 3,457), the prevalence of couples with a HBsAg-positive male partner is 12.61% (436 of 3,457), the prevalence of couples with both partners being HBsAg-positive is 1.19%(41 of 3,457), and couples with at least one partner being HBV seropositive represent 21.75%(752 of 3,457) of the total ART patients.2. Patients in the two groups (HBV and controls) had similar baseline characteristics and outcome of ovarian stimulation. There were no significant differences in the implantation (19.9% vs.23.39%), biochemical pregnancy (11.76% vs.7.46%), and clinical pregnancy rates (30.39% vs.33.56%) between the couples with a HBsAg-positive female partner and the control group, respectively. There was a lower implantation rate in the couples with a HBsAg-positive male partner compared with the control group (17.17% vs.23.39%). There were, however, no differences in biochemical pregnancy and clinical pregnancy rates between the two groups in patients(6.15% vs.7.46%,34.62% vs.33.56%). There were no significant differences in the implantation (13.04% vs.23.39%), biochemical pregnancy (0 vs.7.46%), and clinical pregnancy rates (25% vs.33.56%) between the couples with both partners being HBsAg-positive and the control group, respectively.3. Eighty-four embryos from 18 couples with HBsAg-positive males were subjected to PCR analysis, and 52 embryos from 13 couples with HBsAg-positive males were examined by RT-PCR analysis. HBV DNA and RNA were detected in 16.7%(3/18) and 69.2%(9/13) of couples with HBsAg-positive males, respectively.4. Seventy-one oocytes and embryos from 14 HBsAg-positive females were subjected to PCR analysis, and 63 oocytes and embryos from 17 HBsAg-positive females were examined by RT-PCR analysis. HBV DNA and RNA were detected in 21.4%(3/14) and 47.1%(8/17) of HBsAg-positive females, respectively. Twenty oocytes and embryos from 10 HBsAg-positive females were examined using direct immunofluorescence and laser scanning confocal microscopy. The HBsAg was detected in zona pellucida (ZP)-free oocytes and embryos from 6 HBsAg-positive females (60%). The HBsAg was located within the nuclei and/or cytoplasm of the oocytes and embryos.5. Quantification of HBV DNA was performed in 31 plasma and 30 follicular fluid samples. No relationship could be established between the HBV DNA load in the plasma and the corresponding follicular fluid (r=0.13, P=0.48).6. The percentage of HBV-positive embryos was 40%(2/5) after ICSI and 38% (10/26) after conventional IVF in male HBV carriers (P=1.00). In the cases where the females were the chronic HBV carriers, the difference between the percentage of HBV-positive oocytes and embryos in ICSI (13%) and conventional IVF (43%) cases were not statistically significant (P=0.20).7. For women who were only HBsAg positive, the HBV infection rate of oocytes and embryos was 7.7%. For women who were HBsAg positive and hepatitis B e antigen(HBeAg) or HBV DNA positive, the HBV infection rates of oocytes and embryos ranged from 50% to 100%.Conclusions:1. The prevalence of HBV infection in infertile couples is 21.75%, and is higher than that in the general population. This observation suggestes that the HBV infection may be associated with or contributed to subfertility.2. The HBV infection may not effect the outcomes of the IVF and embryo transfer treatment cycles.3. Our findings provide direct evidence supporting the hypothesis that the sperm may act as a vector for the vertical transmission of HBV to the embryos, and that the HBV can replicate itself.4. The virus particles containing HBsAgs may penetrate human oocytes and embryos.The HBV DNA, which may be present in oocytes and embryos from HBsAg-positive females, could replicate.5. There was no relationship among the HBV DNA level in the plasma, the HBV DNA load in follicular fluid and the HBV infection of oocytes and embryos. All follicular fluid should be considered as infectious, and consequently the viral risk for retrieved follicles should also be considered. 6. It seems that an ICSI procedure in chronic carriers of HBV does not increase the potential risk of HBV transmission.7. Compared with the spontaneous pregnancy, it suggests that ART does not increase the risk of vertical HBV transmission.