Hepatitis B infection and outcomes of in vitro fertilization and embryo transfer treatment

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1 Hepatitis B infection and outcomes of in vitro fertilization and embryo transfer treatment Po Mui Lam, M.D., a Sik Hung Suen, M.R.C.O.G., a Terence Tzu Lao, M.D., b Lai Ping Cheung, M.R.C.O.G., a Tak Yeung Leung, M.D., a and Christopher Haines, M.D. b a Department of Obstetrics and Gynecology, Prince of Wales Hospital and b The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People s Republic of China Objective: To examine the prevalence of hepatitis B virus (HBV) infection, the associated causes of subfertility, and the outcomes of the first IVF and embryo transfer treatment cycles in these infertile couples. Design: A retrospective cohort study. Setting: Assisted reproduction technology (ART) unit. Patient(s): Two hundred eighty-seven couples undergoing IVF and embryo transfer cycles. Intervention(s): Analysis of data on patients characteristics, controlled ovarian hyperstimulation (COH), embryology, and pregnancy (PR) and implantation rates of IVF and embryo transfer cycles according to HBV serostatus of the infertile couples, which was routinely screened. Main Outcome Measure(s): The PRs and the implantation rates. Result(s): Twenty-nine (10.1%) women were HBV seropositive, whereas 32 (11.1%) of their husbands were HBV seropositive. Concerning the causes of infertility, there was a trend toward more tubal blockage (57.1% vs. 42.2%) in the HBV-infected group. Among the 190 women undergoing their first IVF and embryo transfer cycles, both the ongoing PR or live birth rate and implantation rate in HBV group were significantly higher than the controls (53.3% vs. 24.2% per cycle with embryo transfer; and 43.3% vs. 18.4%, respectively). Conclusion(s): Our results demonstrate for the first time significantly higher PRs and implantation rates of IVF and embryo transfer cycles for couples with at least one partner being HBV seropositive. Further studies to elucidate the underlying mechanisms are warranted. (Fertil Steril Ò 2010;93: Ó2010 by American Society for Reproductive Medicine.) Key Words: Hepatitis B, in vitro fertilization, pregnancy rate Hepatitis B (HBV) is one of the commonest serious viral infections in humans. Two billion people worldwide have evidence of hepatitis B virus exposure, and an estimated 400 million are actively infected (1). Assisted reproduction in infertile couples where at least one partner being HBV seropositive raised many concerns about transmission of the infection to the newborn, medical or laboratory staff, and cross-contamination of other virus-free gametes or embryos. However, given the acceptability of a spontaneous pregnancy in patients infected with HBV, there is no ethically sound reason for declining infertility treatment in these patients, especially in areas highly endemic of HBV infection. Screening for HBV before assisted reproductive treatment (ART) is an increasingly common practice in most centers (2). Knowledge of the serostatus of the couple allows first, for immunoprophylactic measures to be taken to reduce the risk of transmission to the partner or fetus, and second, for precautions to be taken against cross-contamination during sample handing and embryo cryostorage. It also enables couples to Received December 1, 2008; revised January 14, 2009; accepted January 24, 2009; published online March 25, P.M.L. has nothing to disclose. S.H.S. has nothing to disclose. T.T.H.L. has nothing to disclose. L.P.C. has nothing to disclose. T.Y.L. has nothing to disclose. C.H. has nothing to disclose. Reprint requests: Po Mui Lam, M.D., Department of Obstetrics & Gynecology, Prince of Wales Hospital, Shatin, New Territories, Hong Kong S.A.R., China (FAX: ; Lampomui@cuhk.edu.hk). make an informed decision regarding pursuance of treatment. Nevertheless, little is known about the reproductive performance of couples seropositive for HBV. Hepatitis B viral load has been detected in the semen of HBV-infected patients who were seeking ART (3). It has also been shown that HBV infection can increase the instability of sperm chromosomes (4). Although the semen parameters are not necessarily compromised, the sperm apoptosis and necrosis may be within normal range (5), which can be a problem in natural fertility as well as in ART. Similarly, HBV antigen and DNA can also be detected in the ovum or ovarian tissue (6), but its implications other than vertical transmission remain unclear. Pirwany et al. (7) were the only investigators who examined the reproductive performance of 13 couples discordant for HBV (10 men and 3 women) and 27 controls, after their first IVF and embryo transfer treatment. This small retrospective study showed that couples discordant for HBV have much lower pregnancy rates (PR) (7.7% vs. 40.7%; P<.01) than their age-matched controls. There were no significant differences in semen parameters or fertilization rate between groups. The investigators acknowledged the limitation of the small sample size and they postulated that the extra precautions and handling techniques of the potentially infective samples in HBV group might have contributed to the lower PRs compared with the controls (7). However, one possible 480 Fertility and Sterility â Vol. 93, No. 2, January 15, /10/$36.00 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 contributing factor is that because HBV infection can be transmitted by sexual contact, there may be more pelvic inflammation that could influence the PR. In Hong Kong and China and among the Asian immigrant population in Australia or New Zealand as well as Chinese women in the United Kingdom, the prevalence of HBV is up to 20% (8 10). It is possible among populations with a high prevalence that HBV infection may be an unrecognized cause of subfertility and also affects the outcome of IVF and embryo transfer treatment. Our unit has routinely screened for HBV before IVF and embryo transfer cycles since Given the paucity of available data on the reproductive performance of couple seropositive to HBV, we performed a retrospective study to examine the prevalence of HBV infection, the associated causes of subfertility, and the outcomes of the first IVF and embryo transfer treatment cycles in these couples. MATERIALS AND METHODS This was a retrospective cohort study on 314 IVF and embryo transfer cycles performed on 287 couples during the period from July 2007 to September 2008 in the Assisted Reproductive Unit, the tertiary referral center affiliated with the Department of Obstetrics and Gynecology, The Chinese University of Hong Kong. Ethical approval was not required for this retrospective study. The causes of infertility for women undergoing IVF and embryo transfer cycles included tubal, male, endometriosis, unexplained and mixed factors. All semen samples were prepared by density gradient method, and intracytoplasmic sperm injection (ICSI) was carried out in couples with severe semen abnormalities. The HBV serostatus for both the women and their husbands has been checked upon referral and within 2 years of the treatment cycles. Those couples with at least one partner being HBV seropositive was categorized as HBV (study) group, whereas those screened negative served as the control group. The standard approach at the time was that HBV carriers would not be given anti-hbv treatment unless active hepatitis was diagnosed. Controlled Ovarian Hyperstimulation Protocol All subjects received the routine protocol for ovulation induction. Pituitary down-regulation was achieved by the long (luteal) GnRH agonist (GnRH-a) down-regulation protocol. Buserelin nasal spray (Suprecur; Hoechst, Hørsholm, Germany) 600 mg daily has been given for at least 14 days from the midluteal phase of the preceding cycle. Complete pituitary desensitization was confirmed by low serum LH (<10 IU/L) and E 2 (<200 pmol/l) concentrations. Patients also had an ultrasound examination to exclude functional ovarian cysts and verify that endometrial thickness was less than 5 mm. Once adequate down-regulation had been achieved, ovarian stimulation started using hmg (Pergonal; Serono, Aubonne/ Switzerland) or recombinant FSH (Gonad-F; Serono; or Puregon; Organon, Skovlunde, Holland). The dose of gonadotropin was determined according to the age of the female partner and the previous responses to treatment, if any. The ovarian response was monitored by ultrasound and serum E 2 concentrations from stimulation day 6 onward. When the response was adequate, defined by the presence of at least three mature follicles R18 mm in diameter, an ovulatory dose of hcg (Profasi; Serono) 5,000 IU was given and oocyte retrieval was performed approximately 36 hours later by the ultrasound-guided transvaginal approach. For those with poor ovarian response (defined as less than three mature follicles on ultrasound), the cycles were cancelled. Embryo transfer was performed 3 days after oocyte retrieval and surplus embryos were cryopreserved. Separate storage tanks were used for couples with at least one partner being HBV seropositive. Patients undergoing embryo transfer received IM hcg or vaginal P as luteal phase support starting from the day of oocyte retrieval. For those with excessive ovarian response, all viable embryos were cryopreserved so as to minimize the risk of ovarian hyperstimulation syndrome (OHSS). Outcome Measures The outcomes of the IVF and embryo transfer cycles were assessed by urine pregnancy test performed 10 days after the last dose of hcg luteal phase support or 2 weeks after embryo transfer, whichever came later. If it was positive, vaginal ultrasound scan of pelvis would be performed 2 weeks later to assess the site, the number, and the viability of gestation. The primary outcome measures of the study were PR (defined as apositive urine pregnancy test per cycle with embryo transfer) and implantation rate (defined as number of gestational sacs per embryo transferred). Data on patients characteristics, controlled ovarian hyperstimulation (COH), and embryology were also collected. These included age of patients, type, duration, and cause of infertility, ovarian reserve assessment (cycle day 3 serum levels of FSH), number of previous trial, duration and total dose of gonadotropin treatment, numbers of mature oocytes retrieved, semen parameter, fertilization rate, total viable embryos, and embryos transferred. Statistics The sample size calculation was based on the reported differences in PRs of IVF and embryo transfer treatment cycles for couples discordant for HBV (7.7% vs. 40.7%; P<.01) compared with their age-matched controls (7). To detect such differences with approximate ratios of controls to study subjects being 5, a sample size of 20 study subjects and 99 controls gave a power of 90% and a of <0.05 (11). A larger sample size gave a higher power of the study for the same a error. Statistical analyses were performed using the Statistical Package for the Social Sciences, version 16.0 (SPSS Inc., Chicago, IL). Differences between groups were tested statistically by the use of c 2 test for categorical data and the Fertility and Sterility â 481

3 TABLE 1 Clinical characteristics of the participants or husbands who were HBV seropositive compared with the controls being HBV seronegative for both partners. Characteristics HBV group (n [ 56) Control group (n [ 231) P value Age (y) NS Type of infertility Primary 25 (44.6%) 120 (51.5%) NS Secondary 31 (55.4%) 112 (48.53%) Duration of infertility (y) Cause of infertility Tubal blockage 32 (57.1%) 98 (42.4%).06 Endometriosis 29 (51.8%) 148 (64.1%) NS Male factor 19 (33.9%) 102 (44.2%) NS Anovulatory 8 (14.3%) 32 (13.9%) NS Unexplained 3 (5.4%) 3 (1.3%) NS Elevated day 3 serum FSH level R10 IU/L 3 (5.4%) 20 (8.7%) NS Note: Values are numbers (percentages) of participants or mean SE. NS ¼ not significant; HBV ¼ hepatitis B virus. Lam. Hepatitis B and IVF. Fertil Steril independent sample t-test for continuous variables. Regression analysis was performed to test the potential predicting variables to determine the importance of each individual variable in its association with the outcome variable. Data were considered to be statistically significant with a P value <.05. RESULTS Study Population Of the 287 women, 29 (10.1%) were HBV seropositive, whereas 32 (11.1%) of their husbands were HBV seropositive. Five pairs of couples were HBV seropositive for both partners. The data on patients characteristics are summarized in Table 1. There was a trend toward longer duration of infertility (5.9 vs. 4.9 years; P¼.05) and more tubal blockage (57.1% vs. 42.2%; P¼.06) in the HBV-infected group, although statistical significance has not been reached. Otherwise, there were no significant differences in patients age, proportion of patients with elevated day 3 serum FSH levels higher than 10 IU/L, as well as the type and other causes of infertility between the HBV group and the control group. Outcomes for Those Undergoing the First IVF and Embryo Transfer Treatment Cycles One hundred ninety of 287 (66.2%) women, 34 in the HBV group and 156 in the control group, were undergoing their first IVF and embryo transfer treatment cycles during this period and therefore the outcomes of these cycles were analyzed. The data on COH and embryology are summarized in Table 2. There were no significant differences in ovarian hyperstimulation regime, endometrial thickness, embryology data including semen parameters, and fertilization rate between groups. Pregnancy rates in the HBV group were significantly higher than the controls (50.0% vs. 23.1% per cycle initiated, 50.0% vs. 23.7% per cycle with oocyte retrieval, and 56.7% vs. 28.1% per cycle with embryo transfer; all P<.01). Of 17 pregnancies in HBV group, one ended up in a spontaneous miscarriage in the first trimester of gestation, whereas the remaining 16 ended up in either live births or still being ongoing pregnancies in the second or third trimester of gestation. Of 36 pregnancies in control group, two ended up in spontaneous miscarriage in the first trimester of gestation and three ended up tubal pregnancies, whereas the remaining 31 ended up in either live births or still being ongoing pregnancies. In other words, the ongoing pregnancy or live birth rates per cycle with embryo transfer were also significantly higher in the HBV group than in the controls (53.3% vs. 24.2%; P<.01). Similarly, implantation rates in the HBV group were also significantly higher (43.3% vs. 18.4%; P<.01). In subgroup analysis, cycles of couples with wives alone being HBV seropositive, but not those with husbands being HBV seropositive, achieved significantly higher PR, ongoing pregnancy or live birth rate, and implantation rate than the control group (66.7% vs. 28.1% and 66.7% vs. 24.2% per cycle with embryo transfer, respectively; both P%.01; and implantation rate of 58.3% vs. 18.4%; P¼.01). These results are summarized in Table 3. Regression analysis was performed to test patients age, type, duration, and cause of infertility, elevated cycle day 3 serum FSH levels, duration and total dose of gonadotropin treatment, numbers of mature oocytes retrieved, semen parameter, insemination method, fertilization rate, total viable 482 Lam et al. Hepatitis B and IVF Vol. 93, No. 2, January 15, 2010

4 TABLE 2 Data on controlled ovarian hyperstimulation regime and embryology in the first IVF and embryo transfer treatment cycles for couples with at least one partner being HBV seropositive and their controls. Characteristics HBV group (n [ 34) Control group (n [ 156) P value Duration of gonadotropin stimulation (d) NS Total dose of gonadotropin used (IU) 3, , NS No. of mature follicles NS Serum E 2 level (pmol/l) on the day of 16,185 1,892 15, NS ovulatory dose of hcg Endometrial thickness (mm) on the day of NS ovulatory dose of hcg No. of oocyte retrieved NS Number of mature oocyte retrieved NS Sperm parameters Count (10 6 /L) NS Motility (%) NS Normal form (%) NS Cycle with ICSI performed 13 (38.2%) 55 (35.2%) NS Fertilization rate for conventional IVF (%) NS Fertilization rate for ICSI (%) NS Total number of viable embryo NS No. of embryos transferred NS Note: Values are numbers (percentages) of participants or mean SE. HBV ¼ hepatitis B virus; ICSI ¼ intracytoplasmic sperm injection; NS ¼ not significant. Lam. Hepatitis B and IVF. Fertil Steril embryos, and embryos transferred, as well as the HBV status to determine the importance of each individual variable in its association with the outcome of IVF and embryo transfer cycles (namely pregnancy). After correction for the confounding effects, successful pregnancy was found to be positively associated with HBV seropositive status (P<.01). Further analysis was performed on 102 controls matched with the 34 study subjects in the ratio of three, with regard to age (with the differences less than 1 year) and basal serum FSH level (with the differences less than 3 IU/L). The results were similar, with significantly higher PR, ongoing pregnancy or live birth rate, and implantation rate in the HBV group than in the matched controls (66.7% vs. 29.3% and 66.7% vs. 24.4% per cycle with embryo transfer, respectively; both P<.01; and implantation rate of 58.3% vs. 18.5%; P<.01). These results are summarized in Table 3. DISCUSSION Worldwide, the prevalence of HBV varies greatly among different ethnic groups and in different geographical areas. In Chinese and Asian women, the carrier rate is up to 20% (8 10). In our hospital, the annual obstetric statistics indicated that up to 2007, the prevalence of HBV was still 10%. One obvious but never addressed question is that in hyperendemic areas, could chronic HBV infection be associated with or contributed to subfertility among women in the reproductive age group. Although this study has only estimated the prevalence of HBV in those infertile couples undergoing IVF and embryo transfer treatment, the result indicated that the prevalence of HBV carrier status among the infertile women was comparable not only with that in their male partners, but also with that in the general obstetric population. This observation suggested that chronic HBV infection is unlikely to represent a significant cause of infertility, even in a hyperendemic area, as IVF treatment tends to be the last resort in the management of infertile couples. To the best of our knowledge this is the first study to report on the prevalence of HBV infection in infertile couples undergoing IVF and embryo transfer treatment. In addition, although retrospective in nature and confined to couples undergoing IVF treatment, our result nevertheless suggested that among these couples, the presence of HBV infection was not associated with significantly different types or causes of infertility, although there could be a trend toward more tubal blockage in the HBV-infected group. At present only one study on the effect of HBV status on fertility potential based on only 13 couples has been published, and it showed much lower PRs of IVF and embryo transfer cycles (7). On the other hand, our observation of significantly higher PRs and implantation rates of the first IVF treatment cycles in the HBV group was in direct contrast. Fertility and Sterility â 483

5 TABLE 3 Pregnancy and implantation rates of the first IVF and embryo transfer treatment cycles for couples with at least one partner being HBV seropositive and their controls. HBV group (n [ 34) Control group (n [ 156) Matched controls (n [ 102) Pregnancy rate/cycle initiated 17/34 (50.0%) a,b 36/156 (23.1%) 24/102 (23.5%) Pregnancy rate/cycle 17/34 (50.0%) a,b 36/152 (23.7%) 24/98 (24.5%) with oocyte retrieval Pregnancy rate/cycle 17/30 (56.7%) a,b 36/128 (28.1%) 24/82 (29.3%) with embryo transfer Wife alone HBVþ (n ¼ 12) Husband alone HBVþ (n ¼ 14) Both partners HBVþ (n ¼ 4) 8 (66.7%) a,b 7 (50.0%) 2 (50.0%) Ongoing pregnancy 16/30 (53.3%) a,b 31/128 (24.2%) 20/82 (24.4%) or live birth rates/cycle with Wife alone HBVþ (n ¼ 12) Husband alone HBVþ (n ¼ 14) Both partners HBVþ (n ¼ 4) embryo transfer 8 (66.7%) a,b 6 (42.9%) 2 (50.0%) Implantation rate 43.3% a,b 18.4% 18.5% Wife alone HBVþ (n ¼ 12) Husband alone HBVþ (n ¼ 14) Both partners HBVþ (n ¼ 4) 58.3% a,b 32.1% 37.5% Note: Values are numbers (percentages) of cycles. HBV ¼ hepatitis B virus. a P%01, indicate statistically significant differences from overall control group. b P%01, indicate statistically significant differences from controls matched with age and basal serum FSH level. Lam. Hepatitis B and IVF. Fertil Steril In our unit, all semen samples were prepared by density gradient centrifugation, which has been shown to reduce the viral load in hepatitis C-infected patients (12). Most important, all the HBV samples were handled by the same personnel in exactly the same way as control subjects. These standardized procedures would have eliminated the attributable cause for the lower PR in the form of extra precautions in handling potentially infective samples in the previous study (7). However, this leads to the question of why was there an improved PR. Our results were totally unexpected but probably true, and the underlying mechanisms certainly warrant further exploration. It is noteworthy that higher PRs and implantation rates were demonstrated among couples with wives being HBV seropositive but not among those with husbands being HBV seropositive. This is consistent with the observations by Zhao et al. (13), who reported comparable PRs and implantation rates of IVF and embryo transfer treatment between 102 infertile couples with the husbands (but not the wives) being HBV seropositive and another 204 couples seronegative for HBV. In addition, despite previous evidence of possible negative effects of HBVon sperms (4, 5), both our study and that by Pirwany et al. (7) did not demonstrate any significant differences in semen parameters or fertilization rate between the HBV and the control groups. Because HBV can be found in cervical and vaginal secretion (14), we would speculate that HBV infection may induce inflammatory changes in the female lower genital tract, and that IVF and embryo transfer treatment can overcome some of these inhibitory effects on sperm function and therefore, it was associated with a higher success rate. The attributed causes of subfertility might not have played such an important role as in those HBV seronegative women. Nevertheless, we did not observe any significant differences in endometrial thickness on the day of ovulatory dose of hcg between groups. The fetal transmission risk is another important impact of HBV infection on reproductive issues. The vertical transmission may occur from the HBV seropositive mother as a consequence of intrauterine exposure, transplacental transmission, or breastfeeding. Interestingly, HBV infection has been detected in newborns of HBV seronegative mother. Because HBV DNA had been detected in semen and spermatozoa (3, 15), it was inferred that there might be father neonate transmission of HBV. Indeed, HBV vertical transmission from father to fetus was consequently confirmed by direct sequencing (16). More studies should be concentrated on detection of HBV genome in gametes and embryos. With more knowledge on the vertical transmission of HBV, proper preconception counseling can be offered to HBV seropositive couples. In conclusion, this study demonstrates for the first time significantly higher PRs of IVF and embryo transfer cycles for 484 Lam et al. Hepatitis B and IVF Vol. 93, No. 2, January 15, 2010

6 couples with at least one partner being HBV seropositive. We agree that our results were totally unexpected and required additional confirmation from prospective studies with a larger sample size. In addition, the mechanisms for this improved treatment outcomes remain unclear and therefore further research on the evidence of inflammation and cytokine activation in semen, follicular fluid (FF), and reproductive tract is required. REFERENCES 1. Ocama P, Opio C, Lee W. Hepatitis B virus infection: current status. Am J Med 2005;118: Van den E. Investigation and treatment of infertile couples: ESHRE guidelines for good clinical and laboratory practice. European Society of Human Reproduction and Embryology. Hum Reprod 1995;10: Qian W, Tan Y, Chen Y, Peng Y, Li Z, Lu G, et al. Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction. World J Gastroenterol 2005;11: Huang J, Huang T, Qiu H, Fang X, Zhuang T, Liu H, et al. Effects of hepatitis B virus infection on human sperm chromosomes. World J Gastroenterol 2003;9: Moretti E, Federico M, Giannerini V, Collodel G. Sperm ultrastructure and meiotic segregation in a group of patients with chronic hepatitis B and C. Andrologia 2008;40: Ye F, Yue Y, Li S, Chen T, Bai G, Liu M, et al. Presence of HBsAg, HBcAg, and HBV DNA in ovary and ovum of the patients with chronic hepatitis B virus infection. Am J Obstet Gynecol 2006;194: Pirwany I, Phillips S, Kelly S, Buckett W, Tan S. Reproductive performance of couples discordant for hepatitis B and C following IVF treatment. J Assist Reprod Genet 2004;21: Chen C, Wang L, Yu M. Epidemiology of hepatitis B virus infection in the Asia Pacific region. J Gastroenterol Hepatol 2000;15(Suppl): E Ma J, Bauman A. Obstetric profiles and pregnancy outcomes of immigrant women in New South Wales, Aust N Z J Obstet Gynaecol 1996;36: Kawsar M, Goh B. Hepatitis B virus infection among Chinese residents in the United Kingdom. Sex Transm Infect 2002;78: Casagrande J, Pike M, Smith P. Statistical methods for rates and proportions: an improved approximate formula for calculating sample sizes for comparing two binomial distributions. In: Fleiss JL, ed. Biometrics. 2nd ed. New York: Wiley, 1981: Levy R, Tardy JC, Bourlet T, Cordonier H, Mion F, Lornage J, et al. Transmission risk of hepatitis C virus in assisted reproductive techniques. Hum Reprod 2000;15: Zhao E, Chen S, Sun L, Yin M, Xiong X, Song J, et al. Influence of chronic HBV infection in the husband on the outcome of IVF-ET treatment. Nan Fang Yi Ke Da Xue Xue Bao 2007;27: Ogunkunle M, Oni A, Odaibo G, Olaleye O. Hepatitis B surface antigen (HbsAg) in blood and genital secretions of patients with sexually transmitted diseases in Ibadan, Nigeria. West Afr J Med 2005;24: Huang J, Huang T, Qiu H, Fang X, Zhuang T, Qiu J. Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes. Asian J Androl 2002;4: Wang S, Peng G, Li M, Xiao H, Jiang P, Zeng N, et al. Identification of hepatitis B virus vertical transmission from father to fetus by direct sequencing. Southeast Asian J Trop Med Public Health 2003;34: Fertility and Sterility â 485