Impact of hepatitis B virus carrier status on IVF-ET outcomes across different ovarian reserve levels: A retrospective study.

In summary, this study has demonstrated that the HBV carrier status could adversely affect embryo quality, pregnancy rates, and miscarriage rates in women undergoing IVF-ET. Specifically, the HBV carrier status has a particularly significant impact on those with normal ovarian reserve function. Accordingly, early identification and intervention strategies should be prioritized in HBV-positive infertile women. Moreover, the ovarian reserve assessment (e.g., AMH testing) may guide individualized reproductive counseling. Further prospective studies are required to elucidate the underlying molecular mechanisms and to optimize assisted reproductive technologies outcomes in this population.

Hepatitis B virus (HBV) based infection has emerged as one of the major global health concerns, with over 250 million chronic carriers worldwide. [ 1 ] HBV primarily targets liver cells as a hepatotropic deoxyribonucleic acid (DNA) virus. [ 2 ] Moreover, it can also be detected in extrahepatic tissues, including the central nervous system, peripheral blood mononuclear cells, kidneys, spleen, and endometrium. [ 3 ] With an increase in the usage of assisted reproductive technologies, particularly IVF-ET, the reproductive potential of HBV-infected women has garnered considerable clinical attention towards its utilization. [ 4 ] HBV is predominantly transmitted through vertical (mother-to-child) transmission, raising concerns about its potential impact on oocytes, embryonic development, and reproductive outcomes. [ 5 , 6 ] Previous reports assessed the effect of HBV infection on IVF-ET success rates. [ 7 , 8 ] Li et al [ 9 ] suggested that the impact of HBV on reproductive outcomes might be masked or amplified depending on the patient’s baseline ovarian reserve, and that the lack of stratification in previous research may explain the contradictory findings. Therefore, considering ovarian reserve as a critical determinant of reproductive success, [ 10 ] the interaction between HBV carrier status and ovarian reserve levels may yield new insights into the pathophysiology underlying impaired fertility in HBV-positive women. Motivated by these considerations, this study aims to systematically investigate the impact of HBV carrier status on embryo development and pregnancy outcomes across different ovarian reserve categories, providing a more refined understanding of the reproductive implications of HBV infection.

Conceptualization: Liuming Li. Data curation: Songwei Jiang, Zushun Chen. Formal analysis: Songwei Jiang, Zushun Chen. Investigation: Songwei Jiang, Zushun Chen. Methodology: Songwei Jiang, Zushun Chen. Project administration: Liuming Li. Resources: Songwei Jiang, Zushun Chen. Software: Songwei Jiang, Zushun Chen. Supervision: Liuming Li. Validation: Songwei Jiang, Zushun Chen. Visualization: Songwei Jiang, Zushun Chen. Writing – original draft: Songwei Jiang. Writing – review & editing: Liuming Li.

The study was approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University, all methods were performed in accordance with the Declarations of Helsinki, and Written informed consents were signed by all participants before the study. This retrospective study recruited 216 infertile women undergoing IVF-ET at the Reproductive Center of the First Affiliated Hospital of Guangxi Medical University from December 2020 to December 2024. Accordingly, the HBV group comprised 112 women with positive hepatitis B surface antigen (HBsAg), while the control group consisted of 104 HBV-negative women (Fig. 1 ). Flow chart of this study. AMH = anti-müllerian hormone, HBsAg = hepatitis B surface antigen, HBV = hepatitis B virus, IVF-ET = in vitro fertilization and embryo transfer. Inclusion criteria : Patients with confirmed diagnosis of HBV carrier status based on clinical standards; patients with absence of clinical hepatitis symptoms; patients with male partners negative for HBsAg. Exclusion criteria : patients who were diagnosed with co-infections (hepatitis C virus, HCV, human immunodeficiency virus, HIV, hepatitis D virus, HDV) or other liver pathologies; patients with uncontrolled endocrine disorders (TSH of >4.5 mIU/L or hemoglobin A1C, HbA1c of >6.5%); patients who were diagnosed with severe endometriosis (rASRM grade III/IV); patients with history of poor ovarian response meeting Bologna criteria; patients with the history of using donor gametes or surrogacy arrangements; patients with cycles involving preimplantation genetic testing; or patients with incomplete medical records. Moreover, we also excluded women with psychiatric conditions requiring active treatment or any history of oncological therapy (chemotherapy/radiotherapy). All participants were subjected to serum anti-Müllerian hormone (AMH) quantification using the standardized Elecsys® AMH assay (Roche Diagnostics) during the early follicular phase (cycle days 2–4). Accordingly, the participants were grouped based on established cutoffs adapted for the Asian reproductive population. Considering the comprehensive fertility assessments, the recruited subjects were stratified into 3 distinct ovarian reserve categories: low reserve (AMH of 7 μg/L). HBV carriers were defined by persistent HBsAg positivity >6 months with normal liver function. Accordingly, patients were stratified into low (AMH 7 μg/L) ovarian reserve groups based on their AMH levels. Within each stratum, HBV carriers were matched with HBV-negative controls. The matching algorithm accounted for multiple clinically relevant covariates, including age, infertility duration (±6 months), baseline follicle-stimulating hormone levels (±2 IU/L), and previous assisted reproductive technology attempts (limited to nulliparous women only). The matching protocol was independently verified by double-blind embryologists. Any discrepancies (<3% of cases) were resolved through consensus review by the study’s endpoint adjudication committee to ensure optimal group comparability across all reserve categories. All patients were subjected to a short-acting gonadotropin-releasing hormone (GnRH) agonist protocol (Triptorelin, Germany, Ferring) followed by controlled ovarian hyperstimulation with recombinant follicle-stimulating hormone (r-FSH, Gonal-F, Merck Serono). Further, the oocyte retrieval was performed after 34 to 36 hours of r-hCG trigger (Ovitrelle, Merck Serono) under transvaginal ultrasound guidance. After 3 hours of culture, the oocytes were fertilized via conventional IVF. The intracytoplasmic sperm injection (ICSI) was applied to rescue oocytes without visible second polar body extrusion. Embryos were cultured for 3 days and evaluated based on blastomere number, symmetry, cytoplasmic granularity, and fragmentation rate. Finally, the luteal support was initiated with vaginal progesterone gel (Crinone 90 mg, Merck Serono) and oral micronized progesterone (Utrogestan 200 mg bid, Besins Healthcare) starting from the day of oocyte retrieval. All embryos were assessed on day-3 postfertilization (66 ± 2 h) by 2 independent embryologists blinded to patient groups, using standardized grading criteria adapted from the Istanbul consensus. The embryo morphology was assessed based on 3 key parameters: blastomere regularity, size symmetry, and degree of cytoplasmic fragmentation. Accordingly, the embryos with perfectly regular blastomeres with equal cell sizes and minimal fragmentation (<5%) were classified as Grade 1. Embryos with slight irregularities in blastomere shape and moderate fragmentation (6%–20%) were represented as Grade 2. Embryos with marked cellular asymmetry and substantial fragmentation (21%–50%) are presented as Grade 3. Embryos with severe morphological abnormalities and extensive fragmentation (>50%) are referred to as Grade 4. High-quality embryos were strictly defined as those meeting Grade 1 or 2 criteria with additional requirements of appropriate cell number (6–10 cells) and absence of multinucleation. The inter-observer reliability was finally confirmed through weekly quality control assessments, maintaining Cohen kappa coefficient of >0.85 for all graded parameters. The multiple reproductive outcomes defined at different stages of the IVF-ET process were rigorously investigated. Further, the cleavage rate was calculated as the percentage of fertilized oocytes that progressed to the ≥2-cell stage during day 2 post-insemination. The normal fertilization rate was determined by the proportion of oocytes displaying 2 distinct pronuclei (2PN) at 16 to 18 hours after insemination. The high-quality embryo rate included day-3 embryos meeting strict morphological criteria (Grade 1 or 2 with 6–10 cells, <20% fragmentation, and no multinucleation). Implantation rate was defined as the number of gestational sacs per transferred embryo (i.e., the denominator includes only embryos that were actually transferred). The clinical pregnancy required visualization of both the gestational sac and fetal heartbeat at 7 weeks gestation. A clinical pregnancy that failed to progress beyond 20 weeks was documented as a miscarriage. The rate of unavailable embryos represented cycles with no transferable embryos due to either fertilization failure or arrested development. All outcome assessments were performed by 2 independent clinicians with validation by a third expert in cases of disagreement (95% inter-rater concordance throughout the study period. All statistical analyses were performed using SPSS version 21.0 (Chicago). Categorical variables, including cleavage rate, normal fertilization rate, high-quality embryo rate, implantation rate, clinical pregnancy rate, miscarriage rate, and unavailable embryo rate, were expressed as counts and percentages and compared using the χ 2 test or Fisher exact test when expected cell counts were <5. Continuous variables, such as age, body mass index, baseline AMH, and FSH levels, were expressed as mean ± standard deviation and compared using independent-samples t -test or Mann–Whitney U test for non-normally distributed variables. Logistic regression analysis was used to identify factors independently associated with clinical pregnancy and miscarriage, adjusting for relevant confounders. A 2-tailed P -value <.05 was considered statistically significant.

The rates of unavailable embryos for HBV carriers and noncarriers, stratified by ovarian reserve level, are presented in Table 1 . In the low ovarian reserve group, HBV carriers demonstrated significantly higher rates of unavailable embryos compared to noncarriers (39.0% vs 22.9%, P  <.05). Similarly, in the normal ovarian reserve group, HBV carriers had a significantly higher unavailable embryo rate than noncarriers (35.1% vs 15.7%, P  <.05). In the high ovarian reserve group, HBV carriers also showed a significantly higher rate of unavailable embryos compared to noncarriers (28.6% vs 5.6%, P  <.05). Comparison of unavailable embryo rates between groups (%). HBsAg = hepatitis B surface antigen. In the normal ovarian reserve group, patients with hepatitis B virus infection had significantly lower rates of cleavage, implantation, normal fertilization, high-quality embryos, and clinical pregnancy than those without hepatitis B virus infection, and the differences were statistically significant ( P  < .05, Table 2 ). No significant differences were observed in the low and high ovarian reserve group. Comparison of embryo development and pregnancy outcomes (%). HBsAg = hepatitis B surface antigen. * P < .05 when compared with patients without hepatitis B virus infection in the same ovarian reserve group. As shown in Table 3 , the miscarriage rates were consistently higher among HBV carriers across all ovarian reserve groups compared to women without HBV carriers. In the low ovarian reserve group, the miscarriage rate was 33.3% (4/12) in HBV carriers versus 15.4% (2/13) in noncarriers ( P  <.05). In the normal ovarian reserve group, HBV carriers had a miscarriage rate of 30.0% (6/20) compared to 9.4% (3/32) in noncarriers ( P  <.05). In the high ovarian reserve group, the miscarriage rate was 37.5% (3/8) in HBV carriers versus 8.3% (1/12) in noncarriers ( P  <.05). Comparison of miscarriage rates between groups (%). HBsAg = hepatitis B surface antigen. The analysis of clinical pregnancy rate trends across various ovarian reserve levels revealed that HBV carriers consistently exhibited lower pregnancy rates than HBV noncarriers, with the most pronounced discrepancy observed in the normal ovarian reserve group (Fig. 2 ). The trend of clinical pregnancy rate by ovarian reserve. The image shows the trends in clinical pregnancy rates across low, normal, and high ovarian reserve groups, stratified by HBV carrier status. HBV carriers exhibited a consistently lower clinical pregnancy rate compared to noncarriers, with the largest discrepancy observed in the normal ovarian reserve group. *** P  < .001. HBsAg = hepatitis B surface antigen, HBV = hepatitis B virus.

In our study, we explored novel insights into the relationship between HBV carrier status and IVF-ET outcomes in women stratified by ovarian reserve function. Accordingly, it was observed that HBV infection could be associated with impaired embryo development and increased miscarriage rates, particularly in women with normal ovarian reserve. At the same time, the effect was less pronounced in women with low or high ovarian reserves. These findings were in agreement with the reported studies demonstrating that HBV DNA could exist in oocytes and embryos, potentially influencing fertilization and early embryonic development. [ 11 – 13 ] Several mechanisms could play crucial roles, involving local inflammatory responses, immune-mediated damage, or direct viral interference with oocyte and embryo quality. [ 4 , 14 , 15 ] The observation that detrimental effects were most apparent in women with normal ovarian reserve suggests that HBV may exert a more selective impact on oocyte quality rather than quantity. In women with low ovarian reserve, the severely diminished oocyte pool may mask HBV-related quality defects, as the primary limitation is oocyte quantity; in women with high ovarian reserve, the abundance of oocytes may compensate for any quality impairment. This “quantity-quality interaction” is supported by Li et al, [ 9 ] who suggested that the impact of HBV on reproductive outcomes might be masked or amplified depending on baseline ovarian reserve. These findings were consistent with the previously reported large cohort studies, indicating reduced embryo quality and implantation potential in HBV-positive women with mid-range AMH levels. [ 5 – 7 , 9 , 16 ] Moreover, the higher miscarriage rates observed among HBV carriers across all groups highlighted the persistent reproductive risks posed by HBV infection, independent of ovarian reserve status. In agreement with the reported research, these findings could be attributed to immune dysregulation, viral replication within embryonic cells, or placental dysfunction [ 4 , 8 , 9 ] Importantly, our study minimized confounding by excluding male HBV carriers, thus isolating the impact of maternal HBV status on reproductive outcomes. Previous studies indicated that paternal HBV infection could exert minimal influence on IVF success. [ 10 ] Collectively, these results suggested that HBV infection compromised both the early embryonic environment and subsequent gestational progression, warranting close reproductive monitoring and timely fertility interventions in affected women. Several limitations of this study should be acknowledged. First, due to the retrospective design, only association rather than causation could be established between HBV carrier status and the observed differences in IVF-ET outcomes. Prospective studies with long-term follow-up are needed to confirm these findings. Second, although we carefully matched HBV carriers with noncarriers by age, infertility duration, and baseline FSH levels, the possibility of residual confounding from unmeasured factors (such as detailed lifestyle factors or HBV DNA levels) cannot be completely excluded. Third, our sample size in the high ovarian reserve subgroup was relatively modest (n = 32), which may have limited the statistical power to detect smaller differences. Finally, all participants were recruited from a single center in China, which may limit the generalizability of our findings to other populations with different ethnic backgrounds or clinical practices.