Relationship between the mechanism of hepatitis B virus father-infant transmission and pregnancy outcome.

PURPOSE: This study examined the relationship between the mechanism of hepatitis B virus (HBV) father-infant transmission via reproductive cells and pregnancy outcome. METHODS: Abandoned in vitro fertilization (IVF) embryos of fathers with chronic HBV infection were taken as study objects. HBV mRNA in embryos was detected, and successfully transplanted embryos were followed up to determine the relationship between HBV-infected embryos and pregnancy outcome. RESULTS: HBV mRNA signals were detected in one embryo in the group with HBV-positive fathers; the positive rate was 1/18 (5.5%). IVF embryos of HBV-positive fathers with HBV mRNA signals were successfully implanted, but early abortion occurred. CONCLUSIONS: HBV mRNA was found in abandoned IVF embryos of HBV-infected fathers, which confirmed that HBV could not only enter early cleavage embryos via sperm but also replicate in embryos, resulting in HBV father-infant transmission. HBV may interfere with embryonic development and thus affect pregnancy outcome.

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5.

BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.

Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos.

In previous experiments, zona pellucida (ZP)-intact in vitro-produced (IVP) embryos incubated for 1 hr with 10(6.3) TCID(50)/ml bovine herpes virus-1 (BHV-1), 10(5.3) TCID(50)/ml cytopathic (CP) bovine viral diarrhea virus (BVDV) or 10(5.3) TCID(50)/ml noncytopathic (NCP) BVDV showed no signs of virus replication or embryonic degeneration. The aims of the present study were to investigate whether a prolonged presence (24 hr or 8 days) of 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml BVDV in an in vitro embryo production system affected the rate of cleavage and embryonic development of ZP-intact embryos, and to point out eventual causes of adverse effects. When virus was present in each step of an IVP system, significantly lower rates of cleavage and blastocyst formation of virus-exposed embryos were observed, in comparison with control embryos (P < 0.01). When embryos were only exposed to virus during the in vitro fertilization (IVF), the rates of cleavage and blastocyst formation were significantly affected. The introduction of BHV-1 or BVDV during in vitro maturation (IVM) or in vitro culture (IVC) resulted only in significantly lower rates of blastocyst (P < 0.01). In all experiments, virus replication was not detected in the embryonic cells. On the other hand, virus replication was clearly demonstrated in oviductal cells in the co-culture system, resulting in a degeneration of these cells. In an additional experiment, synthetic oviduct fluid (SOF) without somatic cells was used as an alternative culture system. Even when SOF-embryos were exposed to 10(6.3) TCID(50)/ml BHV-1 or 10(5.3) TCID(50)/ml CP, and NCP BVDV, the rates of blastocyst formation of the BHV-1-, CP-, and NCP BVDV-exposed embryos were not different from the unexposed control embryos, 23%, 24%, and 24%, respectively, vs. 27%. Taken together, it can be concluded that the virus-induced adverse effects on embryonic development in conventional co-cultures were due to changes in the embryonic environment caused by infection of oviductal cells.

Expression of human immunodeficiency virus long terminal repeat-coupled genes in early cleaving embryos.

Early studies suggested that exogenous retroviral genes are not expressed by mouse embryos infected at early cleavage stages. The general view has therefore been that, since human eggs are non-dividing cells, they could not be infected with HIV before fertilization, nor would HIV be expressed if infection occurred during early cleavage stages following fertilization. In contrast with this view, more recent work has shown that genes coupled to the long terminal repeat (LTR) promotor elements in Rous Sarcoma Virus are readily expressed in early cleaving mouse embryos; in addition, pHIV-LTR, a plasmid construct with HIV-LTR coupled to the reporter gene, lacZ (which encodes beta-galactosidase), is expressed when transfected into a human embryonic carcinoma cell line. To determine if HIV LTR coupled genes would be expressed in early cleaving embryos, one nucleus of mouse zygotes were microinjected with approximately 5000 copies of the pHIV-LTR plasmid. Following microinjection, zygotes were cultured for 48 h to the four cell stage and stained for expression of beta-galactosidase. A plasmid construct containing lacZ without HIV LTR was microinjected as control. A total of 25 of the 111 mouse zygotes microinjected with pHIV-LTR stained positively for beta-galactosidase activity. Blastomeres within individual embryos were not uniformly stained, suggesting a mosaic pattern of distribution of the microinjected plasmids among the cleaving blastomeres. None of the 22 mouse zygotes microinjected with control plasmid stained positively. A total of six polypronuclear human eggs were obtained as discarded by-products of a human IVF program. All four human eggs microinjected with approximately 6000 copies of HIV-LTR reporter gene construct and cultured for 48 h stained highly positive for beta-galactasidase. Staining intensity varied among blastomeres, suggesting a mosaic pattern similar to the mouse embryos. The two human eggs microinjected with control plasmid were negative for beta-galactosidase activity. These results suggest that HIV-LTR is active in both early cleaving mouse embryos and in early cleaving polypronuclear human eggs. These findings indicate that human eggs infected at the time of fertilization with HIV could express viral proteins during early embryonic development.