Effect of hepatitis B virus infection in males on pregnancy outcomes of intrauterine insemination.

This retrospective study was conducted at the Assisted Reproductive Center of Zhongshan City People’s Hospital. Data were collected from couples who underwent IUI between January 2015 and September 2023. All couples were tested for HBV serological markers within 6 months prior to their IUI treatment. Based on these results, HBV infection status was classified into three categories: (1) HBV-uninfected status was defined as hepatitis B surface antigen (HBsAg)-negative, hepatitis B surface antibody (HBsAb)-positive or HBsAb-negative, hepatitis B e antigen (HBeAg)-negative, hepatitis B e antibody (HBeAb)-negative, and hepatitis B core antibody (HBcAb)-negative. (2) HBV-convalescent status was defined as HBsAg-negative, HBeAg-negative, and either HBeAb-positive or HBcAb-positive. (3) HBV-infected status was defined as HBsAg-positive or HBeAg-positive. Inclusion criteria are as follows: (1) Couples diagnosed with infertility and eligible for IUI. (2) Undergoing IUI treatment. (3) Female partner in HBV-uninfected status. (4) Male partner with either HBV-infected or HBV-uninfected status. (5) HBV-infected males must have received standard anti-HBV treatment until liver function normalized. Exclusion criteria included: (1) Presence of other infections (e.g., hepatitis C virus, human immunodeficiency virus, syphilis, gonorrhea, mycoplasma, and chlamydia). (2) Ectopic pregnancy after IUI. (3) Induced abortion after IUI. (4) Either partner is classified as HBV-convalescent. (5) Two IUI procedures performed in the same treatment cycle. (6) Use of donor semen. (7) Chromosomal abnormalities, varicocele, prior surgeries or congenital anomalies of the urological or reproductive systems, long-term medication use, or exposure to toxins or radiation. Couples in which the male partner was HBV-infected were assigned to the study group (HBV-infective group). Those in which the male partner was HBV-uninfected were assigned to the control group (HBV-uninfective group). A 1:4 matching ratio was applied using propensity scores, with a caliper value of 0.03. The nearest neighbor matching method was employed, without replacements, to ensure the most accurate comparison between groups. Female patients underwent either a natural cycle or an ovulation-inducing cycle as part of their IUI treatment. Common medications used for ovulation induction included clomiphene (Fertilan; Codal-Synto Ltd., France), letrozole (Fu Rui, Jiangsu Hengrui Medicine Co., China), recombinant follicle-stimulating hormone (rFSH; Puregon, N.V.Organon, The Netherlands), and human menopausal gonadotropin (HMG; Menotropins for Injection, Livzon Biochemistry Co., Zhuhai, China). Follicular development was meticulously tracked using B-ultrasound. When the dominant follicle was deemed fully developed and matured, an intramuscular injection of 5000–10,000 IU human chorionic gonadotropin (hCG, Chorionic Gonadotrophin for Injection, Livzon Biochemistry Co., Zhuhai, China) was administered. The IUI procedure was scheduled within 24–36 h post-injection. In cases of spontaneous luteinizing hormone (LH) surge, typically when the average follicle diameter was approximately 18 mm, no hCG injection was given, and natural ovulation ensured. For these patients, IUI was conducted either on the day of the LH surge or the following day. Male participants were advised to abstain from sexual activity for 3–7 days before semen collection. Samples were obtained via masturbation directly into a sterile container at the laboratory, within 2 h prior to the IUI procedure. Following liquefaction, routine semen analysis was performed in accordance with the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th edition) . Sperm preparation was performed using the density gradient centrifugation method. Semen was gently layered over the gradient medium using a pipette or syringe, then centrifuged at 500 × g for 15 min. After centrifugation, all layers except the bottom ~ 0.3 mL were removed. This bottom layer was then mixed with 4 mL of Fertilization Medium (G-IVF, Vitrolife, Sweden), and the mixture was resuspended. A subsequent centrifugation was performed at 300 ×g for 5 min. The supernatant was discarded, and the pellet was resuspended in 0.5–1 mL of the same Fertilization Medium. The final step involved assessing sperm concentration and viability before performing the IUI procedure. IUI was performed using a soft catheter (Intrauterine Insemination Catheter, COOK) connected to a 1 mL syringe. A volume of 0.5–1 mL of the previously prepared sperm suspension was aspirated into the syringe. The IUI catheter was delicately inserted through the cervical canal, with the tip extending 1–2 cm beyond the internal cervical os. The sperm suspension was then gradually released into the uterine cavity. After the insemination catheter was removed, the patient was advised to remain in a supine position for 30 min to enhance the chances of successful fertilization. Luteal phase support was tailored to the patient’s clinical needs and determined by the attending physician. Progestogen supplementation was initiated within 4 days post-ovulation and could be administered orally, intramuscularly, or vaginally. Oral dydrogesterone (Duphaston, Abbott Biologicals, USA) was prescribed at 20–40 mg per day. Alternatively, micronized progesterone (Utrogestan, Besins Healthcare, Utrecht, the Netherlands) was administered orally or vaginally at 200–400 mg per day. Intramuscular progesterone injections (Progesterone Injection, Xianju Pharmaceutical, Zhejiang, China) were administered at a dosage of 20 mg per day. If pregnancy was confirmed via serum hCG testing 14 days after initiating progestogen, luteal phase support was continued until 35 days post-ovulation. Upon confirmation of an intrauterine pregnancy, gradual dose reduction was recommended, with the goal of discontinuing luteal phase support between the 10th and 12th weeks of gestation. hCG positive: Serum hCG was detected 14 days after IUI. A result of hCG ≥ 30 U/L was defined as hCG positive. HCG positivity rate = (Number of hCG positive cases/Number of IUI treatment cycles) ×100%. Clinical pregnancy: Clinical pregnancy was confirmed when serum hCG ≥ 30 U/L was detected 14 days post-IUI, and a gestational sac was observed in the uterine cavity via vaginal ultrasound examination 4 weeks post-IUI. Clinical pregnancy rate = (Number of clinical pregnancy cases/Number of IUI treatment cycles) ×100%. Biochemical pregnancy: This condition was identified when serum hCG ≥ 30 U/L was detected 14 days post-IUI, but no gestational sac was observed in the uterine cavity upon vaginal ultrasound examination 4 weeks post-IUI, followed by a subsequent decrease in hCG levels. Early abortion: Early abortion was defined as pregnancy loss occurring within 12 weeks of gestation after a clinical pregnancy had been established. Early pregnancy loss: This category included both biochemical pregnancies and early abortions. Early pregnancy loss rate = (Number of early pregnancy loss cases/Number of hCG positive cases) ×100%. Singleton pregnancy: A clinical pregnancy confirmed 14 days post-IUI, with a fetal heart visualized in the uterine cavity via ultrasound examination 6 weeks post-IUI. Preterm birth: Preterm birth was defined as delivery occurring between 28 and 36 weeks of gestation. Preterm birth rate (singleton deliveries) = (Number of preterm singleton deliveries/Number of singleton deliveries) ×100%. Low birth weight: Low birth weight was defined as a birth weight less than 2,500 g. Low birth weight rate (singleton deliveries) = Number of low birth weight singleton deliveries/Number of singleton deliveries) ×100%. All data used in this retrospective analysis were obtained from the electronic medical record system. Propensity score matching was executed using the “Matchit” package in R software (code is provided in Supplementary Material 1). Statistical analyses were conducted using SPSS version 26.0. The test method used for the inter-group balance test was consistent before and after propensity score matching. Data conforming to a normal distribution were expressed as mean ± standard deviation and analyzed using t-tests. Non-normally distributed data were presented as median (interquartile range) and compared using the two independent sample rank sum test. Count data were presented as proportions or rates (%) and analyzed using the chi-square test. Multivariate analysis was performed using generalized estimating equation (GEE). Results were presented as odds ratios (OR) with 95% confidence intervals (CI). The assigned values of each factor are shown in Supplementary Material 2 . P  < 0.05 was considered statistically significant. All procedures in this study were conducted in accordance with the guidelines and regulations stipulated in the Declaration of Helsinki. This study was approved by Zhongshan City People’s Hospital clinical research and animal experiment Ethics Committee (No. 2024-029). Informed consent was obtained from all participants.

A total of 1,686 IUI cycles were performed at the Assisted Reproductive Center of Zhongshan City People’s Hospital from January 2015 to September 2023. Several cycles were excluded from analysis for the following reasons: 117 cycles due to the female partner’s HBV-infected status, 412 cycles due to the female partner’s HBV-convalescent status, 382 cycles due to the male partner’s HBV-convalescent status, and 4 cycles due to ectopic pregnancy. After applying these exclusion criteria, 771 cycles remained eligible for analysis. Within this refined cohort, the study group (HBV-infective group) comprised 95 cycles in which the male partner was HBV-infected and the female partner was HBV-uninfected. The control group (HBV-uninfective group) consisted of 676 cycles in which both partners were HBV-uninfected, as depicted in Fig.  1 . Fig. 1 The flow chart for the screening and grouping methodology. The flow chart for the screening and grouping methodology. Before the matching process, there were no significant differences between the two groups in several key characteristics, including D-dimer levels, thyroid-stimulating hormone levels, number of IUI treatments, stimulation protocols, body mass index, basal FSH (bFSH), basal LH (bLH), bFSH/bLH ratio, endometrial thickness on the day of IUI, number of follicles ≥ 14 mm on IUI day, luteal phase support, presence of polycystic ovary syndrome, endometriosis, male infertility factor, sperm concentration, percentage of progressive motile sperm, and total count of progressive motile sperm after semen processing ( P  > 0.05). However, significant differences were observed in female age, male age, duration of infertility, and type of infertility between the two groups ( P  < 0.05). To control for these significant differences and important IUI treatment factors, the two groups were matched for female age, duration of infertility, and type of infertility. After matching, the study group (HBV-infective group) included 88 cycles and the control group (HBV-uninfective group) included 311 cycles, as illustrated in Fig.  1 . Following matching, a statistically significant difference was observed in male age between the two groups ( P   0.05), as shown in Table  1 . Table 1 Comparison of characteristics of two groups of couples before and after matching. Variable Before matching After matching HBV-infective group HBV-uninfective group p-value HBV-infective group HBV-uninfective group p-value Cycles(n) 95 676 88 311 Female age (y) 31.00(29.00, 33.50) 30.00(27.00, 33.00) 0.001 31.00(29.00, 33.00) 31.00(28.00, 32.00) 0.348 Male age (y) 33.00(20.00, 38.00) 31.00(29.00, 34.00) 0.001 33.00(30.00, 37.00) 31.00(29.00, 34.00) 0.010 The number of IUI treatments 0.881 0.685 1 49 339 47 159 2 28 213 24 87 ≥ 3 18 124 17 65 Stimulation protocol(n) 0.341 Natural cycle 41 327 36 155 0.139 Ovulation- inducing cycle 54 349 52 156 D-dimer (mg/L) 0.27(0.26, 0.34) 0.27(0.22, 0.35) 0.445 0.27(0.25, 0.34) 0.27(0.22, 0.35) 0.526 TSH (mIU/L) 1.72(1.32, 2.45) 1.73(1.24, 2.37) 0.178 1.69(1.32, 2.51) 1.72(1.27, 2.34) 0.134 BMI (kg/m 2 ) 21.50(19.10, 23.65) 20.70(19.09, 22.64) 0.111 20.30(19.10, 23.60) 20.96(19.02, 22.50) 0.259 bFSH (mIU/mL) 6.40(5.60, 7.60) 6.50(5.54, 7.77) 0.75 6.40(5.60, 7.55) 6.30(5.20, 7.52) 0.459 bLH (mIU/mL) 5.10(3.15, 6.90) 5.00(3.60, 7.50) 0.378 5.10(3.15, 7.08) 5.10(3.40, 7.33) 0.764 bFSH/bLH ratio 1.27(0.95, 2.29) 1.30(0.86, 1.85) 0.257 1.27(0.94, 2.22) 1.29(0.86, 1.92) 0.566 Duration of infertility (y) 3.00(2.00, 3.50) 2.00(1.00, 3.00) 0.002 3.00(2.00, 3.00) 2.00(1.00, 3.00) 0.058 Type of infertility(n) 0.020 0.551 Primary 52 452 49 162 Secondary 43 224 39 149 Male factor (n) 0.097 0.447 No 20 98 19 56 Yes 75 578 69 255 PCOS(n) 0.572 0.494 No 84 529 77 280 Yes 11 84 11 31 EMT(n) 0.346 0.168 No 87 597 80 265 Yes 8 79 8 46 Endometrial thickness (mm) 10.00(9.00, 0.75) 10.00(9.00, 11.30) 0.199 10.00(9.00, 10.85) 10.00(9.00, 11.00) 0.838 ≥ 14 mm follicles(n) 0.295 0.275 1 82 554 76 253 ≥ 2 13 122 12 58 Sperm concentration (×10 6 /mL) 54.00(38.00, 69.00) 50.00(36.00, 69.00) 0.275 56.30 ± 28.25 52.00(39.00, 70.00) 0.985 Progressive motile sperm (%) 45.00(35.00, 50.00) 45.00(36.00, 53.00) 0.290 44.00(35.00, 50.00) 45.00(37.50, 52.00) 0.325 Total progressive motile sperm count after semen processing (×10 6 ) 19.11(11.38, 27.75) 20.09(11.77, 30.85) 0.353 19.32(11.25, 27.75) 21.39(12.60, 32.12) 0.125 Luteal phase support(n) 0.449 0.863 No 22 134 21 77 Yes 73 542 67 234 bFSH, basal follicle-stimulating hormone; bLH, basal luteinizing hormone; BMI, body mass index; EMT, endometriosis; HBV, hepatitis B virus; IUI, intrauterine insemination; PCOS, polycystic ovary syndrome; TSH, thyroid-stimulating hormone. Comparison of characteristics of two groups of couples before and after matching. TSH (mIU/L) bFSH (mIU/mL) bLH (mIU/mL) bFSH, basal follicle-stimulating hormone; bLH, basal luteinizing hormone; BMI, body mass index; EMT, endometriosis; HBV, hepatitis B virus; IUI, intrauterine insemination; PCOS, polycystic ovary syndrome; TSH, thyroid-stimulating hormone. Before the matching process, there were no significant differences between the two groups in terms of hCG positivity rate, clinical pregnancy rate, early pregnancy loss rate, preterm birth rate, low birth weight rate, mean birth weight, or gender ratio ( P  > 0.05). These results indicate that the initial outcomes following IUI treatments were comparable, regardless of the male partner’s HBV infection status. After matching based on relevant characteristics, hCG positivity rate, clinical pregnancy rate, preterm birth rate, low birth weight rate, mean birth weight, and gender ratio continued to show no significant differences between the groups ( P  > 0.05). However, a significant difference was observed in early pregnancy loss rate, which was notably higher in the HBV-infective group compared to the HBV-uninfective group (43.75% vs. 14.00%, P  < 0.05), as depicted in Table  2 . Table 2 Comparison of pregnancy outcomes of IUI between the two groups before and after matching. Before matching After matching HBV-infective group HBV-uninfective group p-value HBV-infective group HBV-uninfective group p-value hCG positive rate(%) 18.95(18/95) 15.98(108/676) 0.463 18.18(16/88) 16.08(50/311) 0.639 Clinical pregnancy rate(%) 13.68(13/95) 15.24(103/676) 0.692 12.50(11/88) 15.11(47/311) 0.244 Early pregnancy loss rate(%) 38.89(7/18) 17.59(19/108) 0.080 43.75(7/16) 14.00(7/50) 0.029 Preterm birth rate in delivery of singleton pregnancies(%) 18.18(2/11) 6.31(5/79) 0.203 11.11(1/9) 2.63(1/38) 0.350 Low birth weight rate in delivery of singleton pregnancies (%) 18.18(2/11) 15.19(12/79) 1 11.11(1/9) 13.15(5/38) 1 Mean birth weight in delivery of singleton pregnancies (g) 3400(3100,3550) 3200(2835,3440) 0.339 3400(3200,3450) 3200(2850,3430) 0.304 The gender ratio of males to females in the delivery of singleton pregnancies 0.84(5/6) 1.26(44/35) 0.523 0.8(4/5) 1.38(22/16) 0.721 HBV, hepatitis B virus; hCG, human chorionic gonadotropin; IUI, intrauterine insemination. Comparison of pregnancy outcomes of IUI between the two groups before and after matching. HBV, hepatitis B virus; hCG, human chorionic gonadotropin; IUI, intrauterine insemination. GEE was used for data analysis before and after propensity score matching. After adjusting for potential confounding factors, including female age, male age, type of infertility, bFSH/bLH ratio, endometrial thickness on IUI day, number of follicles ≥ 14 mm on IUI day, luteal phase support, and total progressive motile sperm count post-semen processing, the male partner’s HBV infection status was not significantly associated with hCG positivity or clinical pregnancy in IUI treatment ( P  > 0.05), both before and after matching. However, after adjusting for female age, male age, type of infertility, bFSH/bLH ratio, and luteal phase support, male HBV-infection status was significantly associated with an increased risk of early pregnancy loss in IUI treatment both before and after matching ( P  < 0.05). Specifically, after matching, the likelihood of experiencing early pregnancy loss was 7.02 times higher in the HBV-infective group compared to the HBV-uninfective group (adjusted OR = 7.022; 95% CI 1.889–26.100; P  < 0.05), as shown in Table  3 . Table 3 GEE analysis of pregnancy outcome of IUI in male with HBV-infection. Before matching After matching OR 95%CI p-value OR 95%CI p-value Lower Upper Lower Upper hCG positive 1.256 0.702 2.250 0.442 1.080 0.567 2.059 0.814 Clinical pregnancy 0.926 0.500 1.715 0.812 0.749 0.379 1.477 0.404 Early pregnancy loss 3.695 1.145 11.925 0.029 7.022 1.889 26.100 0.004 HBV, hepatitis B virus;hCG, human chorionic gonadotropin; IUI, intrauterine insemination; OR, odds ratios. GEE analysis of pregnancy outcome of IUI in male with HBV-infection. HBV, hepatitis B virus;hCG, human chorionic gonadotropin; IUI, intrauterine insemination; OR, odds ratios. China is recognized as a high-prevalence region for HBV, with approximately 5–7% of the population testing positive for HBsAg 4 . Studies conducted across multiple reproductive centers in China have revealed that the prevalence of HBsAg among male patients ranges from 7.34% to 10% 5 , 6 . At our center, the prevalence rate of HBsAg in male patients undergoing IUI treatment is 11.3%, which is marginally higher than that in the general population. HBV is known not only for its potential to cause liver damage but also for its ability to affect male genitourinary tract tissues, thereby reducing male fertility. Moreover, studies have demonstrated that HBV can be detected in the testes, sperm, and semen 7 , 8 , and it may even be transmitted vertically via sperm 9 . Given these potential effects on male fertility, many infected individuals seek ART to achieve conception. Considerable research has examined how male HBV infection influences the outcomes of ART, such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) 10 – 13 . In most of these studies, patients were divided into two groups based on HBsAg status (positive vs. negative). The results showed that HBV infection is associated with a lower fertilization rate in IVF cycles, although the overall pregnancy rate appears unaffected 10 , 11 . In ICSI cycles, however, HBV infection has been linked to reduced normal fertilization rates, lower high-quality embryo formation rates, decreased implantation rates, and lower clinical pregnancy rates 13 . Despite these findings for IVF and ICSI, there is limited research on the relationship between male HBV infection and the pregnancy outcomes of IUI treatments. Consequently, it remains unclear whether the effects observed in IVF and ICSI also apply to IUI. The grouping criteria in this study differed from those of previous research by focusing specifically on patients’ HBV infection status, categorizing them as either HBV-infected or HBV-uninfected, while intentionally excluding patients in the HBV-convalescent stage. This decision was based on several considerations: First, HBV can damage hepatic function and impair the oxidative breakdown of estrogen, leading to elevated estrogen levels and reduced androgen levels in the bloodstream 14 , 15 . Such a hormonal imbalance may result in testicular atrophy and sexual dysfunction in males 16 , 17 . In the context of IUI treatment, the period of HBV convalescence and liver function recovery can influence the levels of these hormones. This is particularly relevant for patients who achieve HBsAg-negative status through anti-HBV therapy, as their hormonal balance during the recovery phase may still affect the success of fertility treatments. Second, HBV infection increases oxidative stress 18 , including in semen 19 , while reducing antioxidant levels in the body. Administering appropriate antioxidants can support recovery. However, it is important to carefully assess the potential effects of different dosages and treatment durations on oxidative stress, especially during the HBV-convalescent stage. Elevated oxidative stress may negatively affect sperm motility 19 . Third, interferon and lamivudine, commonly used in anti-HBV treatments, have been associated with reproductive toxicity, including reduced sperm motility and structural damage to testicular tissue 20 – 22 . In the HBV-convalescent stage, the duration of drug administration and the time since discontinuation can vary widely between patients, potentially affecting reproductive health. Given these considerations, along with the fact that patients with serum HBsAg-negative status may include those in the HBV-convalescent phase, in whom the effects of HBV on physical function may not be entirely absent, it was crucial to select subjects distinctly categorized as HBV-infected (fully affected by HBV) and HBV-uninfected (completely unaffected by HBV). This approach ensured a more reliable assessment of the impact of HBV infection on fertility treatments. This study demonstrated that male HBV infection does not affect semen parameters, hCG positive rate, clinical pregnancy rate, preterm birth rate, low birth weight rate, mean birth weight, or infant gender ratio in IUI treatment. However, it significantly increased the risk of early pregnancy loss, with HBV-infected males being 7.02 times more likely to experience early pregnancy loss than HBV-uninfected males. These findings offer important insights for male patients with HBV infection considering IUI treatment, highlighting the need for careful consideration and management of HBV infection in the context of reproductive health. The impact of HBV on male reproductive health has garnered significant attention in recent research. Evidence shows that HBV can be detected at various stages of spermatogenesis, as well as in the sperm and semen of carriers, in both free HBV-DNA and integrated forms 7 , 8 . Specifically, free HBV-DNA has been found in the seminal plasma and the cytoplasm of the sperm head, but not in the acrosome 1 , 7 . In contrast, integrated HBV-DNA has been detected within the chromosomes of sperm cells, indicating a deeper level of interaction between the virus and male gametes 23 , 24 . Moretti et al. reported that HBV significantly increases the proportion of apoptosis and necrosis in sperm 18 . Similarly, Huang’s study revealed that HBV-DNA integrates into sperm chromosomes in a non-specific manner through multilocus recombination, resulting in elevated sperm chromosomal aberrations and DNA damage, potentially compromising the fertilization capability of the sperm and the health of the offspring 24 . Other studies have demonstrated that merely incubating sperm with HBV S protein can trigger apoptosis and DNA damage, with the severity of effects depending on exposure dose and duration 25 , 26 . Damage to sperm DNA can negatively affect the development of embryos. In addition, as previously mentioned, male patients with HBV infection may experience reproductive impairment from abnormal sex hormone levels, oxidative stress, and adverse effects of anti-HBV medications, all of which disrupt sperm production and development. In the context of IUI treatment, it is essential to use density gradient centrifugation to remove apoptotic or necrotic sperm while retaining healthy ones. However, research has found that the DNA fragmentation index (DFI) in motile sperm does not significantly decrease after this procedure, and in approximately 60% of patients, it may even increase 27 , 28 . Based on these evidence, an elevated DFI in motile sperm could potentially contribute to early pregnancy loss. Nevertheless, such sperm DNA damage does not affect the hCG positive rate, clinical pregnancy rate, preterm birth rate, and low birth weight rate. Furthermore, it is important to consider two key issues: whether processed sperm retains HBV and whether this could result in vertical transmission of HBV from father to offspring. Semen processing via density gradient centrifugation and washing can effectively remove HBV present on the sperm surface and in the seminal plasma 29 , 30 . However, HBV may persist within the plasma of the sperm head and in sperm chromosomes 31 , 32 . Although complete elimination of HBV from the semen of every patient is unlikely, processing markedly reduces the viral load 31 . After processing, only 0.3%–1.1% of sperm are found to carry HBV 32 , greatly reducing the likelihood of vertical transmission to the embryo. Despite this low probability, transmission remains possible. HBV can be transferred into the oocyte via sperm, leading to embryonic infection. Research has shown that mRNA transcription of HBV genes occurs in discarded embryos from IVF cycles involving HBsAg-positive male partners and HBsAg-negative female partners 33 , 34 , supporting the evidence that HBV can be vertically transmitted from a father to their offspring. The transcription of HBV genes in embryos is regulated by DNA methylation 35 , microRNA 36 , and host genes 37 . Experimental evidence shows that HBV S protein reduces the yield of two-cell embryos from human–hamster fertilization. In contrast, HBV X protein decreases mitochondrial membrane potential in human embryonic stem cells and increases DNA damage 34 . These effects may hinder embryonic development. Epidemiological surveys of fetal HBV serology showed that the transmission of HBV from the sperm of a carrier father to the fetus is rare 38 . Furthermore, although high-throughput sequencing of peripheral blood cells is now common in clinical diagnostics, no report has documented integration of HBV-DNA into human chromosomes. This absence of evidence implies that embryos infected via paternal transmission may fail to develop to term, being lost through implantation failure, biochemical pregnancy, or miscarriage. The results of this study support this view, suggesting that early pregnancy loss may serve as a natural mechanism for eliminating embryos infected through paternal vertical transmission. Another factor that may have influenced this study’s outcome is the effect of male HBV infection on the reproductive system of healthy female partners. HBV is present in both semen and sperm, and its protein antigens can be expressed within sperm 35 , 39 . Consequently, when semen or sperm carrying HBV is introduced into the female reproductive tract during sexual intercourse, it may trigger an immune response. Since the 1980 s, China has provided free HBV vaccinations for children 40 , which has resulted in the majority of individuals of reproductive age possessing an immune defense against HBV. Repeated exposure to HBV antigens through sexual intercourse could therefore provoke a sustained anti-HBV immune reaction. The immune environment of the uterine cavity is thought to lean towards a Th1 state 41 . We speculate that this specific immune profile may not interfere with fertilization or implantation but could hinder the maintenance of pregnancy. As a result, this study found that while the hCG positive rate and clinical pregnancy rate remained unchanged, there was an increase in the rate of early pregnancy loss. Sperm also engage in complex interactions with the female reproductive tract, influencing early embryo development 42 . Notably, Lao et al. reported that male HBV infection may increase the risk of fallopian tube damage and infertility in female partners 43 . Similarly, Zandieh et al. demonstrated that sperm DNA damage can activate the toll-like receptor signaling pathway in human fallopian tubes, thereby resulting in the upregulation of inflammatory cytokines and chemokines 44 . Based on this evidence, we hypothesize that inflammation in the fallopian tubes and uterine cavity induced by HBV-related sperm DNA damage may be another mechanism that negatively affects pregnancy. Furthermore, sexual intercourse might cause inflammation in the female reproductive tract and alter its microbiota 45 , 46 , which plays a critical role in reproductive health and infertility treatment outcomes 47 , 48 . Disruption of this microbiota may therefore contribute to the higher rate of early pregnancy loss observed in this study among female partners of HBV-infected males. This study had certain limitations. First, the sample size was modest, which may lead to biased parameter estimates, overfitting, and potential model convergence issues. Second, the study was a retrospective single-center analysis, which may have introduced selection bias and influenced patient characteristics. The retrospective design also posed challenges in sample accessibility and data supplementation, resulting in the absence of certain clinical data, such as sperm DFI, HBV viral load, and specific antiviral treatments. These gaps may have introduced unmeasured confounding variables. Third, some proposed mechanisms underlying early pregnancy loss associated with male HBV infection were speculative, based on literature, rather than directly supported by the study’s data. Therefore, the results should be interpreted with caution. To obtain more comprehensive insights and confirm the validity of these findings, future research should adopt prospective, multicenter designs with larger sample sizes. Despite these limitations, the results of this study provide important insights and serve as a valuable reference for male patients with HBV infection considering IUI treatment.

Based on the direct verification from the present study’s data, we identified a novel finding that HBV infection in males is associated with an increased rate of early pregnancy loss in IUI treatments. Based on the reasonable speculations of the existing literature, we propose that the underlying mechanisms may include the following: Sperm DNA damage induced by HBV may be one of the potential mechanisms contributing to early pregnancy loss. Second, alterations in the immune status and microbiota of the female reproductive tract, potentially influenced by sexual intercourse with an HBV-infected partner, may also play a role. Third, the possibility of vertical transmission of HBV from the father to the fetus may further contribute to this outcome. Prospective studies are warranted to clarify these mechanisms.

Hepatitis B virus (HBV) is a DNA virus characterized by its hepatotropism and is the smallest known double-stranded DNA virus that infects humans. In addition to targeting hepatocytes, HBV can also infect extrahepatic tissues, including those of the male genitourinary system. Several studies have shown that HBV can cross the blood-testis barrier and invade testicular tissue and semen 1 , 2 , potentially impairing semen quality in infected males. Consequently, men with HBV infection are at a heightened risk of infertility, prompting many couples, where the male partner is infected, to turn to assisted reproductive technologies (ART) to achieve conception. Intrauterine insemination (IUI) is widely favored for its simplicity, minimal invasiveness, short treatment duration, and relatively low cost. However, limited research has explored the impact of male HBV infection on IUI outcomes. One prior study reported that male HBV infection does not affect IUI success rates 3 . However, it is important to highlight that the classification of patients in previous research is debatable. Therefore, the primary objective of this study was to modify the methodology for patient categorization. Propensity score matching and multivariate generalized estimating equations (GEE) were employed to comprehensively assess the potential effects of male HBV infection on IUI outcomes.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1 Supplementary Material 2 Supplementary Material 2