Integration of hepatitis B virus into patients' sperm genome and its clinical risks.

BACKGROUND: Like the coronavirus disease 2019, the hepatitis B virus is also wreaking havoc worldwide, which has infected over 2 billion people globally. Using an experimental animal model, our previous research observed that the hepatitis B virus genes integrated into human spermatozoa can replicate and express after being transmitted to embryos. However, as of now, this phenomenon has not been confirmed in clinical data from patients. OBJECTIVES: To explore the integration of the hepatitis B virus into patients' sperm genome and its potential clinical risks. MATERIALS AND METHODS: Forty-eight patients with chronic hepatitis B virus infection were categorized into two groups: Test Group-1 comprised 23 patients without integration of hepatitis B virus DNA within the sperm genome. Test Group-2 comprised 25 patients with integration of hepatitis B virus DNA within the sperm genome. Forty-eight healthy male donors were included as control. The standard semen parameter analysis, real-time polymerase chain reaction, quantitative real-time polymerase chain reaction, sperm chromatin structure assay, fluorescence in situ hybridization, and immunofluorescence assays were utilized. RESULTS: The difference in the median copy number of hepatitis B virus DNA per mL of sera between Test Group-1 and Group-2 was not statistically significant. In Test Group-2, the integration rate of hepatitis B virus DNA was 0.109%, which showed a significant correlation with the median copy number of hepatitis B virus DNA in motile spermatozoa (1.18 × 103 /mL). Abnormal semen parameters were found in almost all these 25 patients. The integrated hepatitis B virus S, C, X, and P genes were detected to be introduced into sperm-derived embryos through fertilization and retained their function in replication, transcription, and translation. CONCLUSION: Our findings suggest that hepatitis B virus infection can lead to sperm quality deterioration and reduced fertilization capacity. Furthermore, viral integration causes instability in the sperm genome, increasing the potential risk of termination, miscarriage, and stillbirth. This study identified an unconventional mode of hepatitis B virus transmission through genes rather than virions. The presence of viral sequences in the embryonic genome poses a risk of liver inflammation and cancer.

Host microRNAs regulate expression of hepatitis B virus genes during transmission from patients' sperm to embryo.

Human sperm nucleus contains diverse RNA populations. This study aimed to screen and identify host microRNAs (miRs) that regulate gene expression of hepatitis B virus (HBV) during transmission from patients' sperm to sperm-derived embryos. Using microarrays, 336 miRs were found to be differentially expressed. After validation using real-time quantitative RT-PCR (RT-qPCR), four miRs were selected as targets. Using RT-qPCR and enzyme-linked immunosorbent assays, when patients' sperm were treated with mimics (or inhibitors) specific for hsa-miR-19a-3p and hsa-miR-29c-3p, the S gene transcription in sperm and translation in sperm-derived embryos was downregulated (or upregulated). There were significant differences in transcriptional and translational levels of the S gene between the test and control groups. These findings suggest that hsa-miR-19a-3p and hsa-miR-29c-3p significantly suppressed expression of the S gene, offering potential therapeutic targets for treating patients with HBV infection, and further reducing the negative impact of HBV infection on sperm fertilizing capacity.

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

BACKGROUND: Hepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase-dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase-independent apoptosis has not been investigated. OBJECTIVES: To evaluate the effects of HBs exposure on sperm dysfunction by activating caspase-independent apoptosis. MATERIALS AND METHODS: Spermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 µg/mL for 3 h. Flow cytometry, qRT-PCR, immunofluorescence assay, ELISA, and zona-free hamster oocyte penetration assays were performed. RESULTS: With increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2-positive cells declined and that of both Bax-positive cells and Apaf-1-positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf-1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant. CONCLUSION: HBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G-mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV-infected patients.

Transcription and regulation of hepatitis B virus genes in host sperm cells.

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo.

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.

Clinical effect of 25G+ vitrectomy combined with intravitreal injection of Conbercept in the treatment of severe proliferative diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To clinical effect of 25G+ vitrectomy combined with intravitreal injection of Conbercept for severe proliferative diabetic retinopathy ( PDR) .·METHODS: A clinical case control study. A total of 35 patients (42 eyes) with severe PDR who underwent 25G+vitrectomy in our hospital from October 2014 to August 2016 were randomly divided into two groups: A and B. Among them, 18 cases (22 eyes) was given conbercept intravitreal injection combined with vitrectomy as Group A;17 cases (20 eyes) was only given vitrectomy without conbercept injection were Group B. Observation of operation duration, intraoperative complications, the incidence of vitreous hemorrhage ( RVH) , macular foveal thickness ( CFT) at 3mo after operation were observed, best corrected visual acuity ( logMAR BCVA ) , and macular foveal thickness ( CFT ) at 6mo after operation were observed too.·RESULTS: The operative time of Group A and B was 58. 23± 8. 18min and 72. 41 ± 10. 31min, the difference was statistically significant ( t = 2. 9, P = 0. 002 ). During the operation, the main complications were iatrogenic hiatus and intraoperative bleeding, Group A of 2 eyes and 1 cases, Group B of 7 eyes and 6 eyes, the difference was statistically significant (P=0. 041, 0. 027). The incidence of vitreous hemorrhage (RVH):at 3mo after operation, that in Group A was 2 eyes, and in Group B was 8 eyes, the incidence of vitreous hemorrhage was statistically significant between the two Groups (P=0. 030). The best corrected visual acuity was 0. 92 ± 0. 35 in Group A and 1. 04±0. 43 in Group B at 6mo postoperatively, but there was no significant difference between the two groups ( t=0. 241, P= 0. 212), but compared with the preoperative visual acuity improved obviously, the difference was statistically significant (t=4. 614, t=7. 355; P<0. 01). CFT:at 3mo after operation, that of Group A was 273. 42 ± 25. 21μm, Group B was 284. 58 ± 27. 44μm, there was no significant difference between the two groups ( t=0. 488, P= 0. 179 ), but there were significantly decrease, the difference was statistically significant( t=3. 152, t=4. 933;P<0. 01 ); at 6mo after operation, CFT of Group A was 238. 16 ± 16. 35μm, Group B was 247. 04 ± 17. 43μm, there was no significant difference between the two groups ( t=0. 571, P=0. 133), but there were significantly decrease, the difference was statistically significant ( t= 2. 474, t=4. 802;P<0. 01).·CONCLUSION: The 25G+ vitrectomy combined with preoperative conbercept intravitreal injection in patients with severe proliferative diabetic retinopathy can effectively improve vision and reduce macular edema, compared with simple vitrectomy, the operation time can be shortened, the incidence of complications can be reduced, and the incidence of vitreous hemorrhage in 3mo after operation can be significantly reduced.