Hepatitis B virus infection reduces fertilization ability during in vitro fertilization and embryo transfer
Abstract
Whether hepatitis B virus (HBV) infection impairs human infertility is unclear. The present retrospective case–controlled study investigated the impact of HBV on sperm parameters, ovarian stimulation, and outcomes of in vitro fertilization (IVF) and embryo transfer. A total of 224 couples with at least one partner being HBsAg-seropositive undergoing their first IVF and embryo transfer cycle were identified, which included 77 couples with female partners being HBsAg-seropositive, 136 couples with male partners being HBsAg-seropositive, and 11 couples with both partners being HBsAg-seropositive. A total of 448 both HBsAg-seronegative couples served as controls. The percentage of normal sperm morphology was significantly lower in HBsAg-seropositive male partners than that in HBsAg-seronegative male partners (11.9 ± 9.4% vs. 19.0 ± 11.9%, P < 0.01). The duration of infertility was significantly prolonged in HBV-seropositive patients compared with HBV-seronegative patients (4.9 vs. 4.1 years, P < 0.01). Couples with female partners being HBsAg-seropositive had significantly lower top-quality embryo rate than control group (22.4% vs. 31.6%, P < 0.01). In addition, the fertilization rates in groups with male or female partners being HBsAg-seropositive were both significantly lower than the matched controls (80.2% vs. 82.8%, P < 0.05; 76.6% vs. 84.3%, P < 0.01, respectively). HBV infection was also found to be associated negatively with fertilization rate by logistic regression analysis (odds ratios: 0.410, 95% confidence interval: 0.186–0.906, P < 0.05). However, there was no significant difference in clinical pregnancy rates between HBsAg-seropositive and HBsAg-seronegative group. These results suggest that chronic HBV infection is likely to represent a significant cause of infertility. J. Med. Virol. 86:1099–1104, 2014. © 2014 Wiley Periodicals, Inc.
INTRODUCTION
Hepatitis B virus (HBV) infection is a worldwide public health problem and may lead to acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [Ganem and Prince, 2004]. About two billion people worldwide have been infected with the HBV and more than 350 million have chronic infection [Liaw and Chu, 2009]. China, in particular, is a highly endemic area for HBV. Approximately 60% of the population of China has a history of HBV infection and 9.8% of the population is infected chronically with HBV [Centers for Disease Control and Prevention, 2007]. Assisted reproduction techniques (ART), including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), are increasingly being used for a variety of infertility problems. Currently, an increasing number of infertile patients infected with HBV choose ART. Because the IVF procedure mimics spontaneous conception, the risk of transmitting HBV to partner and newborn by IVF should be similar for both processes [Steyaert et al., 2000; Lutgens et al., 2009]. There is no ethical reason to reject IVF treatment in HBV-infected patients, especially in areas highly endemic for HBV infection.
Many studies have reported that bacterial and viral infections may impair human fertility [Figura et al., 2002; Englert et al., 2007; La Vignera et al., 2012]. However, no definitive answer has been given as to whether or not the HBV infection could impair human fertility [Pirwany et al., 2004; Lam et al., 2010; Lee et al., 2010]. The present retrospective case–controlled study investigated the impact of HBV on sperm parameters, ovarian stimulation, and outcomes of the first IVF and embryo transfer treatment cycles between HBV-seropositive and HBV-seronegative couples.
MATERIALS AND METHODS
Patients
Patients were scheduled for assisted reproduction procedures for their first IVF and embryo transfer cycle at the Assisted Reproduction Center of Maternal and Child Health Care Hospital of Shaanxi Province (Xian, China) from December 2008 to June 2012. Those couples with at least one partner being HBV-seropositive were categorized as HBV group, whereas those screened negative served as the control group. For each HBV-seropositive cycle, two HBV-seronegative control cycles were matched according to the following criteria: age, cause of infertility, and date of ova retrieval (±1 day). In addition, all the HBV-seropositive samples were handled by the same personnel in exactly the same way as control subjects. All women were tested for HBV, HCV, HIV, gonorrhea, and syphilis within 6 months of the treatment cycles.
Semen analysis was performed on all men to ensure the presence of sperm in the ejaculate as well as blood screening for HBV, HCV, HIV, and syphilis. Couples who were seropositive for HCV, HIV and/or syphilis were excluded from this study. None of the patients were diagnosed with acute hepatitis or received any antiviral treatment before IVF treatment. Cycles, which were cancelled in the case of no available embryo or if ovarian hyperstimulation syndrome developed, were also excluded from this study. A microparticle enzyme immunoassay was used for the qualitative detection of hepatitis B surface antigen (HBsAg) in the serum. A total of 224 couples with at least one partner being HBsAg-seropositive were identified and included. A total of 448 both HBsAg-seronegative couples served as controls. This study was approved by the Ethics Review Board of the Maternal and Child Health Care Hospital of Shaanxi Province (No. 2012–005).
Semen Analysis
Semen samples were collected by masturbation into sterile containers after 3–7 days of sexual abstinence. Semen parameters including concentration, morphology and motility were assessed according to the World Health Organization (WHO) guidelines 5th edition [WHO, 2010].
IVF and Embryo Transfer Protocols
All patients underwent a routine luteal phase down-regulation protocol with GnRH agonist (GnRH-a; Decapeptyl, Ipsen, France) and recombinant follicle stimulating hormone (FSH; Gonal-F Merck Serono, Switzerland or Puregon, N.V.Organon, The Netherlands) for controlled ovarian hyperstimulation. Human chorionic gonadotropin (hCG; Profasi Merck Serono, Coinsins, Switzerland) was administered to induce final oocyte maturation when 2/3 follicles had reached a mean diameter of 18 mm in size. Oocyte retrieval was performed 36 hr after hCG administration by transvaginal ultrasonography guided needle aspiration. Oocytes were collected into fertilization media (Vitrolife, Göteborg, Sweden) and fertilized by conventional insemination. Fertilization was assessed and confirmed 17–19 hr after insemination by the presence of two pronuclei and second polar body. Embryo quality was evaluated by the evenness and the number of blastomeres, and the percentage of fragmentation [Shi et al., 2012]. One to three embryos with the highest scores were chosen for embryo transfer on Day 3 or Day 5 depending on the female patient's age, physical status (whether the height below 1.5 m, or suffering from uterine malformation and other system diseases), and quality of embryos. Clinical pregnancy was diagnosed by presence of gestational sacs. All couples provided written informed consents prior to the start of IVF and embryo transfer treatment. In addition, the following measures were taken to prevent cross-contamination of samples and reduce the risk of transmission of HBV. The samples from viral carriers were processed in a designated space, and stored in dedicated liquid nitrogen storage tanks. Disposable supplies were used and disposed of properly. Sperm-wash procedures involving density gradient centrifugation followed by a sperm swim-up step was used to each sperm sample [ASRM, 2008, 2013].
Outcome Measures
Data on patient characteristics included age of patients; type and duration of infertility; infertility aetiology; endometrial thickness; ovarian reserve evaluation (cycle day 3 serum level of FSH); total dose of gonadotropin treatment; serum estradiol level on day of hCG injection; semen parameters on the day of oocyte retrieval; the numbers of oocytes retrieved, fertilized oocytes, two-pronuclear zygotes, cleaved embryos, top-quality embryos (grade I + II), and embryos transferred. The cleavage rate was defined as number of cleaved embryos per number of oocytes fertilized. The fertilization rate was defined as number of fertilized oocytes per number of oocytes retrieved, and two-pronuclear fertilization rate was defined as the number of confirmed two-pronuclear zygotes per number of oocytes retrieved in an IVF cycle 17–19 hr after insemination. The top-quality embryo rate was defined as the number of embryos with a grade of I plus II according to combined scoring system per number of divisive embryos on day 3 of culture. The implantation rate was defined as number of intra- or extrauterine gestational sacs per number of embryos transferred. Clinical pregnancy rate was defined as the number of women with gestational sacs per embryo transfer cycle.
Statistical Analysis
The sample size calculation was performed using the StudySize 2.0 (CreoStat HB, Frolunda, Sweden). A sample size of 76 study subjects and 152 controls would have 90% power to detect a significant difference in the clinical pregnancy rate of 10% at a significance level of 0.05.
Data were reported as mean ± standard deviation (SD) for quantitative variables or number (percentage) for categorical variables. Differences between groups were tested using two-sample Mann–Whitney U-test for quantitative variables, and all categorical data were analyzed using Chi-squared test. Logistic regression analysis was performed to assess the contribution of the potential predicting variables on fertilization rate and clinical pregnancy rate. For logistic regression analysis, fertilization rate was categorized as low-fertilization rate (≤36.4% of the oocytes fertilized, which is equivalent to the 5th percentile of the variable distribution) or high-fertilization rate (>36.4% of the oocytes fertilized). P < 0.05 was considered as statistically significant. Statistical analyses were performed by SPSS 13.0 (SPSS, Inc., Chicago, IL).
RESULTS
During the study period, 672 couples undergoing their first IVF and embryo transfer cycle were included: 77 couples with female partners being HBsAg-seropositive and 136 couples with male partners being HBsAg-seropositive. Only 11 couples were both HBsAg-seropositive. The sample size of couples with both partners being HBsAg-seropositive was too small to detect reliable significant difference.
Tables I–III summarized the baseline characteristics, ovarian stimulation variables and the IVF and embryo transfer outcomes in selected patients. Because patients were matched by age and diagnoses, there were no differences in the patients' age and causes of infertility between HBV groups and matched control groups (Table I). There were no significant difference in type of infertility, endometrial thickness, day 3 serum level of FSH, total used dose of gonadotropin between HBV groups and matched control groups (Tables I and II). The sperm concentration and progressive motility were similar among groups with male partners being HBsAg-seropositive and HBsAg-seronegative. However, the percentage of normal sperm morphology was significantly lower in group with male partners being HBsAg-seropositive than that in group with male partners being HBsAg-seronegative (11.9% vs. 19.0%, P < 0.001) (Table I). Couples with female partners being HBsAg-seropositive, but not those with male partners being HBsAg-seropositive, achieved significantly higher serum estradiol level on day of hCG injection (4722.2 vs. 3342.8 pmol/L, P = 0.002) (Table II). Otherwise, there was a trend toward prolonged duration of infertility in groups with male or female partners being HBsAg-seropositive (4.8 vs. 4.1 years, P = 0.074; 4.9 vs. 4.1 years, P = 0.112, respectively) (Table I). Congregating all the HBsAg-seropositive samples together, the duration of infertility was significantly prolonged in HBsAg-seropositive group (n = 224) than that in HBsAg-seronegative group (n = 448) (4.9 vs. 4.1 years, P = 0.009).
| Characteristics | HBV groups (n = 213) | Matched control groups (n = 426) | ||
|---|---|---|---|---|
| Male partner HBV+ (n = 136) | Female partner HBV+ (n = 77) | Male partner HBV+ (n = 272) | Female partner HBV+ (n = 154) | |
| Woman's age (y) | 30.5 ± 3.9 | 31.0 ± 4.2 | 30.4 ± 4.5 | 30.7 ± 3.7 |
| Man's age (y) | 32.9 ± 4.7 | 32.6 ± 4.6 | 32.6 ± 5.0 | 32.3 ± 3.4 |
| Type of infertility | ||||
| Primary | 73 (53.7%) | 39 (50.6%) | 126(46.3%) | 74 (48.1%) |
| Secondary | 63 (46.3%) | 38 (49.4%) | 146(53.7%) | 80 (51.9%) |
| Cause of infertility | ||||
| Female factor | 89 (65.4%) | 59 (76.6%) | 179(65.8%) | 119(77.3%) |
| Male factor | 5 (3.7%) | 3 (3.9%) | 10 (3.7%) | 6 (3.9%) |
| Combined factor and other factors | 42 (30.9%) | 15 (19.5%) | 83 (30.5%) | 29 (18.8%) |
| Duration of infertility(y) | 4.8 ± 3.2 | 4.9 ± 3.8 | 4.1 ± 2.8 | 4.1 ± 2.7 |
| Semen parameters | ||||
| Concentration (×106/ml) | 62.40 ± 29.75 | 59.67 ± 26.63 | 66.38 ± 38.19 | 66.76 ± 31.87 |
| Progressive motility (a + b) | 60.49 ± 14.84 | 61.44 ± 15.96 | 60.70 ± 14.80 | 57.86 ± 15.89 |
| Normal morphology (%) | 11.9 ± 9.4* | 16.0 ± 9.2 | 19.0 ± 11.9 | 17.7 ± 10.5 |
- HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.
- Values are expressed as numbers (percentages) of participants or mean ± SD.
- * P < 0.01, versus matched control group (Mann–Whitney U-test).
| Ovarian stimulation variables | HBV groups (n = 213) | Matched control groups (n = 426) | ||
|---|---|---|---|---|
| Male partner HBV+ (n = 136) | Female partner HBV+ (n = 77) | Male partner HBV+ (n = 272) | Female partner HBV+ (n = 154) | |
| Endometrial thickness (mm) | 10.9 ± 2.0 | 10.8 ± 1.9 | 11.0 ± 2.3 | 11.5 ± 2.1 |
| Day-3 FSH (IU/L) | 7.11 ± 2.38 | 7.03 ± 2.11 | 7.00 ± 2.16 | 7.32 ± 3.04 |
| Total dose of gonadotrophin used (IU) | 1548.6 ± 436.5 | 1528.3 ± 531.4 | 1564.4 ± 369.7 | 1490.3 ± 408.8 |
| Serum estradiol level on day of hCG injection (pmol/L) | 3591.6 ± 2017.7 | 4722.2 ± 3000.9* | 3499.5 ± 2121.5 | 3342.8 ± 1831.5 |
- HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; FSH, follicle stimulating hormone; hCG, human chorionic gonadotropin.
- Values are expressed as mean ± SD.
- * P < 0.01, versus matched control group (Mann–Whitney U-test).
| IVF and embryo transfer outcomes | HBV groups (n = 213) | Matched control groups (n = 426) | ||
|---|---|---|---|---|
| Male partner HBV+ (n = 136) | Female partner HBV+ (n = 77) | Male partner HBV+ (n = 272) | Female partner HBV+(n = 154) | |
| No. of oocytes retrieved | 13.1 ± 6.3 | 12.5 ± 7.7 | 14.0 ± 7.6 | 13.4 ± 8.6 |
| No. of fertilized oocytes | 10.6 ± 6.1 | 9.6 ± 7.2 | 11.6 ± 6.8 | 11.3 ± 8.5 |
| No. of cleaved embryos | 10.3 ± 6.1 | 9.4 ± 7.2 | 11.4 ± 6.9 | 11.2 ± 8.5 |
| No. of two-pronuclear zygotes | 7.7 ± 4.6 | 7.1 ± 5.9 | 8.7 ± 5.4 | 8.4 ± 6.3 |
| No. of top- quality embryo (grade I + II) | 2.9 ± 2.7 | 2.2 ± 2.6** | 3.2 ± 3.0 | 3.5 ± 4.3 |
| No. of embryos transferred | 2.0 ± 0.4 | 2.0 ± 0.4 | 2.0 ± 0.4 | 2.0 ± 0.4 |
| Cleavage rate (%) | 1,403/1,436 (97.7) | 704/716 (98.3) | 3,088/3,153 (97.9) | 1,702/1,722 (98.8) |
| Fertilization rate (%) | 1,436/1,791 (80.2)* | 716/935 (76.6)** | 3,153/3,808 (82.8) | 1,722/2,043 (84.3) |
| Two-pronuclear fertilization rate (%) | 1,048/1,791 (58.5)** | 529/935 (56.6)** | 2,371/3,808 (62.3) | 1,282/2,043 (62.8) |
| Top-quality embryo rate | 391/1,403 (27.9) | 158/704 (22.4)** | 882/3,088 (28.6) | 538/1,702 (31.6) |
| Implantation rate (%) | 104/270 (38.5) | 54/150 (36.0) | 206/547 (37.7) | 117/304 (38.5) |
| Clinical pregnancy rate (%) | 79/136 (58.1) | 37/77 (48.1) | 146/272 (53.7) | 78/154 (50.6) |
- HBsAg, hepatitis B surface antigen; IVF, in vitro fertilization; HBV, hepatitis B virus; No., number.
- Values are expressed as numbers (percentages) of participants or mean ± SD.
- * P < 0.05, versus matched control group (Chi-squared test).
- ** P < 0.01, versus matched control group (Mann–Whitney U-test or Chi-squared test).
For the IVF and embryo transfer outcomes, couples with female partners being HBsAg- seropositive had significantly lower number of top-quality embryos (2.2 vs. 3.5, P = 0.001) and lower top-quality embryo rate (22.4% vs. 31.6%, P < 0.001) compared with HBsAg-seronegative group. The fertilization rates in groups with male or female partners being HBsAg-seropositive were significantly lower than that in the matched controls (80.2% vs. 82.8%, P = 0.017; 76.6% vs. 84.3%, P < 0.001, respectively). The two-pronuclear fertilization rates in groups with male or female partners being HBsAg-seropositive were also significantly lower than that in the matched controls (58.5% vs. 62.3%, P = 0.007; 56.6% vs. 62.8%, P = 0.001, respectively). However, there were no significant difference in number of oocytes retrieved, number of fertilized oocytes, number of two-pronuclear zygotes, number of cleaved embryos, number of embryos transferred, cleavage rate, implantation rate, and clinical pregnancy rate between HBsAg-seropositive and HBsAg-seronegative groups (Table III).
To determine the contributions of potential predicting variables to the rates of fertilization and clinical pregnancy, binary logistic regression analysis (n = 672) was performed. For fertilization rate, which was categorized as low-fertilization rate or high-fertilization rate, logistic regression analysis was performed to test patients' age, type of infertility, cause and duration of infertility, cycle Day 3 serum FSH levels, total dose of gonadotropin treatment, serum estradiol level on day of hCG injection, semen parameters, the number of oocytes retrieved, the number of fertilized oocytes, and HBV status. For clinical pregnancy rate, more variables (the number of two-pronuclear zygotes, cleaved embryos, top-quality embryos (grade I + II) and embryos transferred) were added. In crude model, it was found that the number of fertilized oocytes and HBV status were associated with the rate of fertilization. However, the number of embryos transferred, the number of top-quality embryos (grade I + II), cause of infertility, the number of oocytes retrieved, the number of fertilized oocytes, the number of two-pronuclear zygotes, the number of cleaved embryos and the percentage of semen normal morphology were associated with the rate of clinical pregnancy. After adjustment for the confounding effects, HBV infection contributed significantly to fertilization rate (odds ratios (OR): 0.410, 95% confidence interval (CI): 0.186–0.906, P = 0.028), but was not associated with successful pregnancy (OR: 1.173, 95% CI: 0.814–1.692, P = 0.392). Whereas the number of top-quality embryos (OR: 1.192, 95% CI: 1.092–1.297, P < 0.001) and the number of embryos transferred (OR: 1.771, 95% CI: 1.145–2.738, P = 0.010) were associated with successful pregnancy.
DISCUSSION
Presently, the impact of HBV infection on the outcome of IVF and embryo transfer treatment remains controversial. One study showed that HBV-positive couples had much lower pregnancy rate than the age-matched HBV-negative controls, and no significant difference in semen parameters or fertilization rate between two groups [Pirwany et al., 2004]. Another study reported unexpected higher implantation and pregnancy rates in the HBV-seropositive group compared to those of HBV-seronegative group [Lam et al., 2010]. In both of the above studies, samples sizes were too small to draw firm conclusions and their results were in conflict with the present study. However, a study with a larger sample size demonstrated that the percentage of normal sperm morphology was significantly lower in HBsAg-seropositive male partners than in those of HBsAg-seronegative male partners. Nonetheless there was no significant difference in ongoing pregnancy rate among HbsAg-positive and -negative groups [Lee et al., 2010]. The results obtained by Lee et al. [2010] were consistent with the present study, but short of analyzing the fertilization rate, two-pronuclear fertilization rate, top-quality embryo rate, and implantation rate. In the present study, it demonstrated for the first time a lower top-quality embryo rate in couples with female partners being HBV-seropositive, and lower fertilization and two-pronuclear fertilization rates in couples with one partner being HBV-seropositive during IVF treatment.
The results of the present study reconfirmed that the percentage of normal sperm morphology was significantly lower in HBV-infected male partners. HBV was not only able to pass through the blood-testis barrier into male germ cells but also could integrate into sperm chromosomes, which inducing chromosome aberrations, leading to hereditary defects in male germinal cells and impairing spermatogenesis [Huang et al., 2003]. Other studies observed that HBV infection increased necrosis and apoptosis of the sperm cell and induced loss of sperm membrane integrity and caused sperm dysfunctions [Moretti et al., 2008; Kang et al., 2012]. In addition, research showed that HBsAg reduced the sperm motility and caused the loss of sperm mitochondrial membrane potential [Zhou et al., 2009]. The above findings would all contribute to poor sperm quality in HBV-infected men. The impaired sperm quality may be one reason of lower fertilization and two-pronuclear fertilization rates in couples with male partners being HBV-seropositive.
In the present study, significantly lower top quality embryo, fertilization and two-pronuclear fertilization rates in couples with female partners being HBV-seropositive were observed. The above findings implied that HBV infection impaired oocyte quality, whereas further research on the correlation between HBV infection and oocyte quality is required. Logistic regression analysis showed that the successful pregnancy was associated with the number of top-quality embryos and the number of embryos transferred. In this study, the embryos with highest scores were chosen for embryo transfer and more embryos were transferred when embryos scores were lower, and these operations attenuated the adverse effects of HBV on fertilization ability. It may be the reason that in spite of reduced fertilization and top quality embryo rates, the clinical pregnancy rate were not affected by HBV infection.
In the current study, it was also detected that the estradiol level on the day of hCG administration was significantly higher in HBV-infected female partners compared with HBV negative controls. Although the definite reason for the increase of estradiol concentration in HBV-positive woman is not clear. A possible explanation is that HBV infection down-regulates the expression of P450 1A2 and 3A4, and reduces both enzymes' activity [Li et al., 2006; Wang et al., 2010]. As the key enzymes for estradiol metabolism by hydroxylation [Shou et al., 1997], the reduced activity of P450 1A2 and 3A4 may be responsible for the accumulation of estradiol.
Although this study evaluated the effect of HBV infection only on the infertile couples who underwent IVF and embryo transfer treatment, the results of HBV infection impairing the quality of sperm, reducing fertilization and high-grade embryo acquisition rates, were also significant in the general obstetric population. In addition, HBV infection prolonged duration of infertility. This observation suggests that chronic HBV infection is likely to represent a significant cause of infertility.
