Abstract
This study aimed to investigate the effects of male hepatitis B virus (HBV) infection on male fertility, embryonic development, and in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcomes. We performed a retrospective cohort study that included 3965 infertile couples who received fresh embryo transfer cycles for the first time at the Fujian Maternity and Child Health Hospital (Fuzhou, China) from January 2018 to January 2021. Infertile couples were categorized based on their HBV infection status into the HBV group (HBV-positive men and HBV-negative women) and the control group (HBV-negative couples). A 1:1 propensity score matching was performed with relatively balanced covariates. Baseline characteristics, semen parameters, laboratory outcomes, clinical outcomes, and obstetric and neonatal outcomes were compared between groups. After propensity score matching, 821 couples were included in each group. Both groups had similar semen parameters and obstetric and neonatal outcomes. The HBV group showed a significantly lower live birth rate than the control group (P < 0.05). The HBV group had a significantly higher abortion rate than the control group (P < 0.05). The rates of high-quality embryos and blastocyst formation were significantly lower in the HBV group than those in the control group (both P < 0.05). In conclusion, in couples who undergo IVF/ICSI, male HBV infection reduces the live birth rate and increases the risk of miscarriage. However, the incidence of low birth weight in women with IVF/ICSI does not increase with male HBV infection.
Keywords: hepatitis B virus, in vitro fertilization, intracytoplasmic sperm injection, live birth rate, propensity score matching
INTRODUCTION
Although considerable advances in treating hepatitis B virus (HBV) infection have been achieved, public health continues to be affected by HBV worldwide.1 In 2019, the global prevalence of HBV infection was estimated to be 296 million, and 1.5 million new infections are being diagnosed every year,2,3 HBV infection is the main contributor to liver diseases and liver cancer, and can cause a massive economic and social burden in patients.4
Bacterial and viral infections play an important role in the function of the male reproductive system.5 Bacteria and viruses (e.g., Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, human immunodeficiency virus, and hepatitis C virus [HCV]) can not only cause infections of the male reproductive system but also induce male infertility through spermatogenesis disorders, sperm DNA damage, and orchitis.6,7,8 HBV has partially double-stranded DNA. Therefore, this hepatotropic virus can cross the blood–testis barrier and enter male germ cells to integrate itself into their genomes and chromosomes.9,10 HBV also can fracture, fragment, and distort the sperm because it increases genome instability.11,12
HBV mRNA was found in in vitro fertilization (IVF)-discarded embryos of HBV-infected fathers.13 This finding suggests that HBV not only enters early cleavage embryos via sperm but also replicates in the embryos, leading to HBV parent–child transmission. Therefore, HBV may interfere with embryonic development and affect pregnancy outcomes. A retrospective study investigated 277 infertile couples who underwent oocyte donation cycles.14 Compared to HBV-negative couples, the incidence of implantation was lower in couples with an HBV-positive husband and an HBV-negative wife (26.7% vs 40.6%) and the clinical pregnancy rate was lower (42.1% vs 63.8%), but these differences were not significant. Therefore, the authors concluded that male HBV infection was not associated with IVF clinical pregnancy. Another retrospective case–control study examined the effects of HBV on sperm parameters and embryo transfer outcomes.15 A logistic regression analysis showed that HBV infection was negatively correlated with the fertilization rate (odds ratio: 0.410, 95% confidence interval: 0.186–0.906, P < 0.05). No significant difference in the clinical pregnancy rate was found between the hepatitis B surface antigen-seropositive and hepatitis B surface antigen-seronegative groups.
The association between HBV infection and IVF/intracytoplasmic sperm injection (ICSI) outcomes is unclear. Therefore, in this study, we examined this association and investigated the effects of male HBV infection on male fertility, embryonic development, and pregnancy outcomes.
PARTICIPANTS AND METHODS
Study design and participants
A cohort study was conducted using propensity score matching (PSM). We recruited infertile couples who underwent IVF/ICSI treatment for the first time at the Fujian Maternity and Child Health Hospital (Fuzhou, China) between January 2018 and January 2021. The infertile couples were categorized based on their HBV infection status into the HBV group (HBV-positive men and HBV-negative women) and the control group (HBV-negative couples). The inclusion criteria were as follows: (1) female age <40 years, (2) body mass index ≤30 kg m−2, and (3) a fresh embryo transfer cycle was performed. The exclusion criteria were as follows: (1) uterine abnormalities (e.g., uterine malformations, moderate to severe uterine adhesions, uterine effusion, and untreated endometrial polyps), (2) recurrent abortion, (3) endocrine diseases (e.g., thyroid diseases, hyperprolactinemia, and diabetes), (4) polycystic ovary syndrome,16 (5) chromosomal abnormalities in either/both men and women, (6) autoimmune diseases, (7) one of the couples was infected with human immunodeficiency virus, HCV, or Treponema pallidum, or (8) women with HBV infection. This study was performed in accordance with the Declaration of Helsinki and approved by the ethics approval committee of Fujian Maternity and Child Health Hospital (Approval No. 2021KLRD09036). Informed consent was obtained from all participants after providing them with a detailed description of the study and its effects.
IVF procedures
A gonadotropin-releasing hormone agonist long protocol was performed on days 2–5 of menstruation in all women. If the pituitary downregulation criteria were achieved (estradiol concentrations <50 pg ml−1, follicle-stimulating hormone concentrations <5 IU l−1, luteinizing hormone concentrations <5 IU l−1, and an endometrial thickness <5 mm), gonadotropin (Gonal-F; Merck Serono, Aubonne, Switzerland) was administered at a dose of 75–300 IU until the day of human chorionic gonadotropin (hCG) administration. The women were then injected with 250 μg of recombinant hCG (OVIDEREL; Merck Serono, Modugno, Italy) if there were at least two follicles with a diameter >18 mm. A gonadotropin-releasing hormone antagonist protocol was initiated on days 2–3 of menstruation, and gonadotropin was injected to stimulate the ovaries. If the diameter of the dominant follicle reached 14 mm, 0.25 mg of a gonadotropin-releasing hormone antagonist (Cetrotide; Merck Serono, Fareva Pau, France) was injected until the trigger day. If there were at least two follicles of 18 mm diameter each, 250 μg of recombinant hCG was injected. After 36–38 h, a transvaginal ultrasound-guided egg extraction was performed, and routine IVF or ICSI was performed after egg collection. The embryos were divided into four grades: (1) grade I embryos: 6 to 9 blastomeres, uniform size, fragmentation degree 0 to 5%; (2) grade II embryos: the size is basically uniform, and the degree of fragmentation is 10% to 25%; (3) grade III embryos: uneven blastomere, fragmentation degree of 25% to 50%; and (4) grade IV embryos: blastomere is very uneven, fragmentation degree >50%. Grade I and II embryos were classified as high-quality embryos. The observation indexes included the size of blastocyst cavity, inner cell mass, and trophoblast cells. Blastocysts with day 5 or day 6 ≥4BB were considered high-quality blastocyst embryos.17
Semen collection and analysis
All men who were included in this study were asked to abstain from sex for 3–7 days. Their semen specimens were collected from masturbation, transferred to a disposable sperm collection tube, and tested in an incubator at 37°C. After complete liquefaction, the semen samples were mixed, and 10 μl of each sample was run on a computer-assisted semen analysis system (Microptic; MicropticSL, Barcelona, Spain) to detect semen parameters. The sperm cells were stained with Diff-Quik stains (Shenzhen Huakang Biomedical Engineering Co., Ltd., Shenzhen, China) and their morphology was visualized under an optical microscope. According to the World Health Organization criteria, a sperm was considered normal if its concentration was ≥15×106 ml−1, the percentage of forward motile sperm was ≥32%, and the percentage of normal morphological sperm was ≥4%.18 The DNA fragmentation index (DFI) was determined by flow cytometry (Anke Biotechnology Co., Ltd., Hefei, China) and the sperm chromatin structure assay.
Outcome measures
Pregnancy outcomes were measured 14 days after the embryo transfer cycles, and a positive hCG test was defined as blood or urine hCG concentrations ≥10 IU l−1. Clinical pregnancy was defined as the presence of gestational cysts and fetal heartbeats observed by B-ultrasound monitoring 30 days after embryo transfer. The number of gestational cysts observed indicated the number of implanted embryos. Abortion was defined as the termination of pregnancy before 28 weeks. Preterm birth was defined as a live birth before 37 weeks of gestation, and low birth weight was defined as a birth weight <2500 g.
The primary outcome measure was the live birth rate. Secondary outcome measures were male semen parameters, and the rates of prime embryos, blastocyst formation, clinical pregnancy, abortion, perinatal complications, preterm birth, and low birth weight.
Statistical analyses
All statistical analyses were conducted using the IBM SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The data of normally distributed samples were expressed as the mean ± standard deviation, and differences between means were tested for significance with independent sample Student’s t-test. Continuous variables were shown as the median (interquartile range) and categorical variables were shown as proportions. Group means were compared using the Mann–Whitney U rank sum test (for continuous variables) or the Chi-square test (for categorical variables). P < 0.05 was considered statistically significant for two-tailed tests. We used PSM to balance baseline characteristics and sample sizes between the groups. Matched variables included the patient’s age, female body mass index, basal follicle-stimulating hormone, basal luteinizing hormone, basal estradiol, anti-Müllerian hormone, the duration of infertility, the type of infertility, the number of embryos transferred, and the type of embryos transferred. Propensity scores were estimated by characteristics that were considered potential confounding variables, which could affect assisted reproductive technology (ART) outcomes. These scores were included in the model and were matched in a 1:1 manner using substitute-free nearest neighbor matching (random order, caliper value of 0.05).
RESULTS
Baseline characteristics
Of 5728 couples who were initially screened and received IVF/ICSI-assisted pregnancy, only 3965 couples were finally enrolled in the study. Of these, 1110 couples were in the HBV group, and 2855 couples were in the control group (Supplementary Figure 1 (324.1KB, tif) ). Figure 1 shows the propensity scores before and after PSM. There were 821 matched couples in each group after PSM. No significant differences in baseline characteristics were found between the matched cohorts (Table 1).
Figure 1.
Propensity scores (a) before and (b) after PSM. PSM: propensity score matching.
Table 1.
Baseline characteristics of the participants before and after propensity score matching
| Characteristic | Before PSM | After PSM | ||||||
|---|---|---|---|---|---|---|---|---|
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| Control group (n=2855) | HBV group (n=1110) | Z/χ2 | P | Control group (n=821) | HBV group (n=821) | Z/χ2 | P | |
| Age (year), median (IQR) | ||||||||
| Male | 32.0 (29.0–35.0) | 32.0 (29.0–35.0) | −0.163 | 0.870 | 31.0 (29.0–35.0) | 32.0 (29.0–35.0) | −0.296 | 0.767 |
| Female | 33.0 (30.0–37.0) | 34.0 (30.0–37.0) | −0.665 | 0.506 | 33.0 (30.0–37.0) | 33.0 (31.0–37.0) | −0.424 | 0.671 |
| BMI (kg m−2), median (IQR) | 21.3 (19.6–23.4) | 21.4 (19.6–23.4) | −0.212 | 0.832 | 21.5 (19.6–23.56) | 21.5 (19.8–23.4) | −0.027 | 0.979 |
| Baseline FSH (IU ml−1), median (IQR) | 6.3 (5.3–7.6) | 6.6 (5.4–8.0) | −2.748 | 0.006* | 6.2 (5.3–7.3) | 6.3 (5.3–7.5) | −1.733 | 0.083 |
| Baseline LH (IU ml−1), median (IQR) |
3.3 (2.4–4.4) | 3.5 (2.6–4.7) | −2.880 | 0.004* | 3.4 (2.5–4.5) | 3.4 (2.5–4.6) | −0.630 | 0.529 |
| Baseline estradiol (pg ml−1), median (IQR) | 37.0 (28.0–50.0) | 40.9 (30.0–53.0) | −0.422 | 0.673 | 37.0 (27.0–50.0) | 39.0 (29.0–51.0) | −0.194 | 0.846 |
| AMH (ng ml−1), median (IQR) | 2.8 (1.7–4.6) | 3.0 (1.8–4.9) | −1.245 | 0.213 | 3.1 (1.7–4.8) | 3.0 (1.8–4.9) | −0.160 | 0.873 |
| AFC (n), median (IQR) | 10.0 (6.0–12.0) | 9.0 (7.0–12.0) | 1.490 | 0.223 | 10.0 (7.0–12.0) | 9.0 (6.0–12.0) | 0.291 | 0.777 |
| Infertility duration (year), median (IQR) | 3.0 (2.0–5.0) | 3.0 (2.0–5.0) | −1.091 | 0.276 | 3.0 (2.0–5.0) | 3.0 (2.0–5.0) | 0.805 | 0.421 |
| Infertility type, n (%) | 3.036 | 0.081 | 0.088 | 0.766 | ||||
| Primary | 1469 (51.5) | 606 (54.6) | 445 (54.2) | 452 (55.1) | ||||
| Secondary | 1386 (48.5) | 504 (45.4) | 376 (45.8) | 369 (44.9) | ||||
| Infertility causes, n (%) | 3.604 | 0.307 | 0.476 | 0.924 | ||||
| Tubal factor | 1208 (42.3) | 456 (41.1) | 368 (44.8) | 372 (45.3) | ||||
| Male factor | 760 (26.6) | 295 (26.6) | 235 (28.6) | 237 (28.9) | ||||
| Unexplained | 430 (15.1) | 193 (17.4) | 114 (13.9) | 117 (14.3) | ||||
| Others | 457 (16.0) | 166 (15.0) | 104 (12.7) | 95 (11.6) | ||||
| COH protocol, n (%) | 1.842 | 0.398 | 2.145 | 0.342 | ||||
| GnRH-agonist | 1954 (68.4) | 750 (67.6) | 571 (69.5) | 569 (69.3) | ||||
| GnRH-antagonist | 837 (29.3) | 327 (29.5) | 230 (28.0) | 222 (27.0) | ||||
| Others | 64 (2.2) | 33 (3.0) | 20 (2.4) | 30 (3.7) | ||||
| Gn duration (day), median (IQR) | 12.0 (9.0–13.0) | 12.0 (10.0–13.0) | 0.917 | 0.359 | 12.0 (10.0–13.0) | 12.0 (10.0–13.0) | 0.713 | 0.476 |
| Gn dosage (IU), median (IQR) | 2550.0 (2025.0–3159.4) | 2550.0 (2025.0–3225.0) | −0.365 | 0.715 | 2550.0 (2025.0–3150.0) | 2475.0 (2025.0–3150.0) | 0.279 | 0.780 |
| Estradiol on hCG day (pg ml−1), median (IQR) | 2222.0 (1335.8–3284.0) | 2427.0 (1452.0–3709.0) | −4.782 | <0.001* | 2516.0 (1477.0–3518.0) | 2343.0 (1437.0–3626.0) | 0.363 | 0.717 |
| Progesterone on hCG day (ng ml−1), median (IQR) | 0.6 (0.4–0.9) | 0.7 (0.5–1.0) | −6.216 | <0.001* | 0.7 (0.4–1.0) | 0.7 (0.4–1.0) | −0.221 | 0.825 |
| Endometrium thickness on hCG day (mm), median (IQR) | 11.0 (10.0–12.5) | 11.0 (10.0–12.0) | 0.812 | 0.417 | 11.0 (10.0–12.5) | 11.0 (10.0–12.3) | 0.529 | 0.597 |
| Fertilization proportion, n (%) | 1.982 | 0.159 | 0.413 | 0.413 | ||||
| IVF | 2160 (75.7) | 864 (77.8) | 624 (76.0) | 636 (77.5) | ||||
| ICSI | 695 (24.3) | 246 (22.2) | 197 (24.0) | 185 (22.5) | ||||
| Number of embryos transferred, median (IQR) | 2.0 (2.0–2.0) | 2.0 (2.0–2.0) | −1.332 | 0.183 | 2.0 (2.0–2.0) | 2.0 (2.0–2.0) | −0.972 | 0.331 |
| Cleavage embryo or blastocyst transfer, n (%) | 3.993 | 0.046* | 1.159 | 0.282 | ||||
| Cleavage embryo | 2741 (96.0) | 1081 (97.4) | 759 (92.5) | 771 (93.9) | ||||
| Blastocyst | 114 (4.0) | 29 (2.6) | 62 (7.6) | 50 (6.1) | ||||
*P<0.05 was considered statistically significant. PSM: propensity score matching; IQR: interquartile range; BMI: body mass index; FSH: follicle-stimulating hormone; LH: luteinizing hormone; AMH: anti-Müllerian hormone; AFC: antral follicle count; COH: controlled ovarian hyperstimulation; Gn: gonadotropin; hCG: human chorionic gonadotropin; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; HBV: hepatitis B virus; GnRH: gonadotropin-releasing hormone
Semen parameters and laboratory outcomes
We found no significant differences in semen quality parameters between the groups before and after PSM (Table 2). Additionally, laboratory outcomes, such as the number of eggs retrieved, proportion of normal fertilization, and proportion of normal cleavage, were not significantly different between the groups (Table 3).
Table 2.
Sperm parameters before and after propensity score matching
| Characteristic | Before PSM | After PSM | ||||||
|---|---|---|---|---|---|---|---|---|
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| Control group (n=2855) | HBV group (n=1110) | Z | P | Control group (n=821) | HBV group (n=821) | Z | P | |
| Semen volume (ml) | 3.4 (2.8–4.6) | 3.4 (2.8–4.6) | 0.539 | 0.590 | 3.4 (2.8–4.6) | 3.4 (2.8–4.6) | 0.528 | 0.573 |
| Sperm concentration (×106 ml−1) | 45.2 (34.0–107.9) | 44.4 (34.1–103.5) | 0.196 | 0.844 | 44.5 (33.6–105.5) | 44.6 (34.1–104.2) | 1.519 | 0.129 |
| Sperm PR (%) | 41.2 (36.8–58.4) | 39.9 (35.5–56.5) | 0.405 | 0.525 | 40.0 (36.0–57.5) | 40.2 (36.4–57.0) | 0.087 | 0.764 |
| Normal form rate (%) | 12.5 (11.6–13.6) | 12.5 (11.3–13.6) | 0.565 | 0.572 | 12.5 (11.4–13.2) | 12.5 (11.5–13.3) | 0.511 | 0.589 |
| Sperm DFI (%) | 17.6 (16.0–20.6) | 17.2 (15.6–20.4) | 1.519 | 0.129 | 17.3 (15.4–20.2) | 17.3 (116.0–20.8) | 0.412 | 0.675 |
Continuous variables are presented as the median (IQR). PR: progressive motility percentage; DFI: DNA fragmentation index; PSM: propensity score matching; HBV: hepatitis B virus; IQR: interquartile range
Table 3.
Laboratory outcomes before and after propensity score matching
| Characteristic | Before PSM | After PSM | ||||||
|---|---|---|---|---|---|---|---|---|
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| Control group (n=2855) | HBV group (n=1110) | Z/χ2 | P | Control group (n=821) | HBV group (n=821) | Z/χ2 | P | |
| Number of oocytes retrieved, median (IQR) | 8.0 (5.0–12.0) | 8.0 (5.0–12.0) | −1.410 | 0.159 | 9.0 (6.0–12.0) | 9.0 (6.0–12.0) | −0.412 | 0.807 |
| Mature oocyte rate, n/total (%) | 12 799/24 926 (51.3) | 4846/9947 (48.7) | 19.566 | <0.001* | 3983/7495 (53.1) | 3989/7572 (52.7) | 0.303 | 0.582 |
| Normal fertilization rate, n/total (%) | 20 494/24 926 (82.2) | 8174/9947 (82.2) | 0.007 | 0.935 | 6179/7495 (82.4) | 6108/7448 (82.0) | 0.450 | 0.502 |
| Normal cleavage rate, n/total (%) | 19 237/20 494 (93.9) | 7637/8174 (93.4) | 1.821 | 0.177 | 5789/6179 (93.7) | 5687/6108 (93.1) | 1.588 | 0.208 |
| High-quality embryo rate, n/total (%) | 11 317/19 237 (58.8) | 4253/7637 (55.7) | 22.116 | <0.001* | 3304/5789 (57.1) | 3103/5687 (54.6) | 7.333 | 0.007* |
| Blastocyst formation rate, n/total (%) | 5276/7980 (66.1) | 1939/3112 (62.3) | 13.661 | <0.001* | 1539/2426 (63.4) | 1482/2473 (59.9) | 6.237 | 0.013* |
*P<0.05 was considered statistically significant. IQR: interquartile range; PSM: propensity score matching; HBV: hepatitis B virus
Clinical outcomes
We observed a similar implantation rate and CPR between the HBV and control groups before and after PSM. The HBV group had a significantly higher abortion rate before PSM (P=0.003) and after PSM (P=0.015) than the control group. The HBV group also had a lower live birth rate before PSM (P=0.022) and after PSM (P=0.029) than the control group. Both groups had similar multipregnancy and ectopic pregnancy rates before and after PSM (Table 4).
Table 4.
Clinical outcomes before and after propensity score matching
| Characteristic | Before PSM | After PSM | ||||||
|---|---|---|---|---|---|---|---|---|
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| Control group (n=2855) | HBV group (n=1110) | χ2 | P | Control group (n=821) | HBV group (n=821) | χ2 | P | |
| Implantation rate, n/total (%) | 1672/5074 (33.0) | 607/1954 (31.1) | 2.294 | 0.130 | 495/1463 (33.8) | 462/1479 (31.2) | 2.144 | 0.143 |
| Clinical pregnancy rate, n/total (%) | 1308/2855 (45.8) | 487/1110 (43.9) | 1.138 | 0.286 | 380/821 (46.3) | 368/821 (44.8) | 0.297 | 0.586 |
| Multiple pregnancy rate, n/total (%) | 389/1308 (29.7) | 142/487 (29.2) | 0.033 | 0.856 | 126/380 (33.2) | 115/368 (31.3) | 0.230 | 0.631 |
| Abortion rate, n/total (%) | 139/1308 (10.6) | 77/487 (15.8) | 8.527 | 0.003* | 37/380 (9.7) | 58/368 (15.8) | 6.119 | 0.015* |
| Ectopic pregnancy rate, n/total (%) | 75/1308 (5.7) | 28/487 (5.8) | <0.001 | 1.000 | 19/380 (5.0) | 15/368 (4.1) | 0.186 | 0.667 |
| Live birth rate, n/total (%) | 1094/2855 (38.3) | 382/1110 (34.4) | 5.214 | 0.022* | 338/821 (41.2) | 295/821 (35.9) | 4.754 | 0.029* |
*P<0.05 was considered statistically significant. PSM: propensity score matching; HBV: hepatitis B virus
Maternal complications and neonatal outcomes
There were 1094 and 338 live births after fresh embryo transfer cycles in the control group before and after PSM, respectively. There were 382 and 295 live births after fresh embryo transfer cycles in the HBV group before and after PSM, respectively. No significant differences in the rates of preterm birth, maternal complications (gestational hypertension, gestational diabetes, gestational hypertension, and premature rupture of membranes), or cesarean section were found between the groups before and after PSM (all P>0.05; Table 5).
Table 5.
Obstetric and neonatal outcomes before and after propensity score matching
| Characteristic | Before PSM | After PSM | ||||||
|---|---|---|---|---|---|---|---|---|
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| Control group (n=1094) | HBV group (n=382) | Z/χ2 | P | Control group (n=338) | HBV group (n=295) | Z/χ2 | P | |
| Premature birth rate, n (%) | 183 (16.7) | 59 (15.5) | 0.340 | 0.560 | 56 (16.6) | 47 (15.9) | 0.047 | 0.829 |
| Gestational hypertension, n (%) | 38 (3.5) | 16 (4.2) | 0.411 | 0.522 | 15 (4.4) | 11 (3.7) | 0.201 | 0.654 |
| Gestational diabetes, n (%) | 175 (16.0) | 65 (17.1) | 0.216 | 0.642 | 53 (15.7) | 46 (15.6) | 0.232 | 0.630 |
| Premature rupture of membranes, n (%) | 291 (26.6) | 89 (23.4) | 1.614 | 0.204 | 83 (24.6) | 69 (23.4) | 0.117 | 0.732 |
| Cesarean section, n (%) | 371 (33.9) | 122 (32.1) | 0.497 | 0.481 | 109 (32.3) | 94 (31.9) | 0.011 | 0.918 |
| Low birth weight rate, n (%) | 175 (16.0) | 58 (15.2) | 0.074 | 0.785 | 51 (15.1) | 49 (16.6) | 0.274 | 0.601 |
| Birth weight (kg), median (IQR) | 3.1 (2.7–3.4) | 3.1 (2.7–3.4) | −0.917 | 0.360 | 3.1 (2.7–3.4) | 3.1 (2.7–3.4) | −0.428 | 0.765 |
*P<0.05 was considered statistically significant. IQR: interquartile range; PSM: propensity score matching; HBV: hepatitis B virus
DISCUSSION
HBV infections greatly affect the IVF-embryo transfer outcomes in infertile couples with ART. However, the effect of male HBV infection on embryonic development and clinical outcomes remains controversial in the early stages of IVF/ICSI-embryo transfer cycles.11,12 On reviewing the clinical data of 3965 cycles of infertile couples who underwent fresh cycles with IVF/ICSI treatment, the couples were divided into the HBV group (HBV-positive men and HBV-negative women) and the control group (HBV-negative couples). A 1:1 PSM was then performed to match the HBV group to the control group with relatively balanced covariates. Finally, we included 821 cycles in the HBV group and 821 cycles in the control group after PSM. We found no significant differences in demographic or clinical features between the groups.
In this study, we found no significant difference in semen density, the forward motion of the sperm, the normal morphogenetic rate, or sperm DFI between the HBV and control groups before and after PSM. These results are in agreement with those from a cross-sectional study in Iran.19 However, Karamolahi et al.11 found reduced semen parameters, such as the semen count, sperm morphology, and sperm forward motion, in HBV-infected infertile men, which suggested decreased male fertility due to HBV infection. HBV infection has been reported to affect sperm forward motion, acrosomal protein activity, and sperm DFI, and cause a high DNA staining rate of male sperm, indicating a lower semen quality in HBV-infected infertile men.20 This could be due to the HBV-induced activation of the B-cell lymphoma 2 (Bcl2)/Bcl2 associated X (Bax) signaling cascade. This cascade triggers apoptosis-inducing factors or endonuclease G-mediated apoptosis, which results in sperm DNA fragmentation, sperm damage, and death, and a reduction in the sperm fertilization ability.21
Liu et al.22 reported no significant correlation between the HBV DNA viral load in semen and the sperm count. However, HBV may affect sperm motility, speed, and DFI, and there is a correlation between the load effect of the HBV DNA copy number in semen and the integrity of sperm nuclear DNA. A molecular hybridization study showed the integration of HBV DNA sequences in the sperm genome of two patients with hepatitis B, and HBV was hypothesized to be transmitted vertically through germ cells.23 Additionally, specific HBV DNA hybridization signals were observed on sperm chromosomes in patients with chronic HBV infection.24 This finding provided direct evidence of the integration of HBV DNA in human sperm chromosomes. The integration of HBV genes in the sperm genome of patients with hepatitis B might lead to instability of sperm chromosomes and induce chromosomal aberration. However, further studies are required to examine the effect of HBV infection on oocyte fertilization and embryo development during ART therapy. Previous studies mainly relied on conventional semen parameters, such as sperm concentration, sperm motility, and normal sperm morphology. The evaluation of conventional semen parameters has certain limitations because of the interaction between HBV and the male reproductive system at the molecular level.
Currently, the effects of HBV infection on pregnancy outcomes of ART, such as the fertilization rate, embryo quality, implantation rate, and pregnancy rate, remain controversial. Lee et al.25 reported no significant difference in the pregnancy or live birth rate between HBV-infected couples and HBV-negative couples, which suggested that HBV infection had no significant effect on ART outcomes. Pocate-Cheriet et al.26 found that male HBV infection affected sperm function and morphology, and had no effect on pregnancy outcomes of ART. Another study showed that the fertilization rate in male HBV-infected patients was lower than that in patients without HBV infection during IVF treatment, while the embryo quality, implantation, and pregnancy rates were similar between the groups.15 Our study showed normal fertilization and normal cleavage rates, which were similar between the HBV and control groups before and after PSM. However, significant differences were detected in the high-quality embryo and blastocyst formation rates between the groups. These findings suggest that male HBV infection reduces the embryonic developmental potential. Any variation in the effect of HBV infection on pregnancy outcomes could be present even if sperm function is affected and the fertilization rate is low/normal. ICSI technology is adopted during ART if the normal sperm formation rate is >3%, thus compensating for a decreased fertilization rate caused by sperm damage.27
In our study, we found significant differences in the live birth and abortion rates between the two groups before and after PSM. These findings suggested that male HBV infection increased the risk of abortion and reduced the rate of live births in ART-assisted pregnant women. However, we observed no significant differences in the rates of embryo implantation, clinical pregnancy, multiple pregnancies, or ectopic pregnancy between the groups. The reason for this lack of finding may be that HBV is integrated into the embryo, which mainly leads to abnormal development of the inner cell mass of the embryo, but has little effect on the outer trophoblast of the embryo. Therefore, this process has little effect on the clinical pregnancy rate and implantation rate but increases the risk of abortion and reduces the live birth rate. This finding could also be due to gradient centrifuge washing and upstream sperm treatment during IVF/ICSI embryo transferred (ET) cycles, which result in a reduced HBV DNA viral load in the culture environment. There is also a self-repair mechanism between gametes and embryos, which results in early embryonic development and embryo implantation being unaffected.28 A previous study has shown that HBV DNA is only integrated into the gametes and embryos in HBV carriers.29 Even though such integration may affect the embryo quality, we selected optimal in vitro-cultured embryos for transplantation or increased the number of transplanted embryos during fresh cycles to reduce any possibility of implantation of HBV-infected or integrated embryos in the uterine cavity. Consequently, this might have reduced any effects of HBV infection on gamete quality, embryo development, and IVF/ICSI outcomes.
There have been few follow-up studies on the maternal and infant health status of HBV-infected individuals after ART. In a study of 504 patients with single fetal delivery, female hepatitis B surface antigen, hepatitis B e antigen, and serum hepatitis B core antibody seropositivity reduced gestational age at delivery, whereas the male HBV serum status did not affect gestational age at delivery.30 In this study, we followed up 1476 live births after fresh embryo transfer cycles. We found that male HBV infection did not increase the risk of maternal or neonatal complications. This finding indicated that male HBV infection did not increase the risk of premature birth or low birth weight of newborns from women who received IVF/ICSI treatment.
The present study has some limitations. We did not take into consideration the cumulative live birth rate and the long-term effect of male HBV infection on pregnancy outcomes. Unfortunately, some patients who underwent an abortion did not undergo any chromosome or genetic testing of embryos to ascertain any effects, thus affecting further analysis of an increased abortion rate caused by male HBV infection. Additionally, because of the different immune responses of the human body after HBV infection, the serological patterns of HBV infection in the population are diverse. Therefore, attention should be paid to whether there are differences in the effects of different HBV infection statuses on male semen quality and IVF outcomes. Further studies are required to unravel the mechanisms that underlie male HBV infection’s effect on reproduction.
In summary, this study shows that male HBV infection is associated with a reduced live birth rate and can increase the risk of miscarriage in women undergoing IVF/ICSI treatment. However, male HBV infection is not significantly associated with neonatal birth weight.
AUTHOR CONTRIBUTIONS
XJC and BHZ designed the research. SQZ, HLX, and WWJ collected the data. SQZ, XHL, WWJ, and YS analyzed and interpreted the data. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
ACKNOWLEDGMENTS
This study was supported by grants from the innovation Platform Project of Science and Technology, Fujian Province (2021Y2012), the Key Project on the Integration of Industry, Education and Research Collaborative Innovation of Fujian Province (No. 2021YZ034011), the Key Project on Science and Technology Program of Fujian Health Commission (No. 2021ZD01002), the Fujian Provincial Health and Young and Middle-aged Key Personnel Training Program (No. 2022GGA035), and the Natural Science Foundation of Fujian Province (No. 2023J011221).
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
Flow chart of the study. IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; ET: embryo transferred; HBV: hepatitis B virus; PSM: propensity score matching; HIV: human immune deficiency virus.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Flow chart of the study. IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; ET: embryo transferred; HBV: hepatitis B virus; PSM: propensity score matching; HIV: human immune deficiency virus.