Integration Of Hepatitis B Virus In Human Oocytes And Early Embryos

BackgroundHepatitis B is a worldwide public health problem. More than 2 000 million people alive today have been infected with HBV at some time in their lives. Of these, about 350 million remain infected chronically and become carriers of the virus. Three quarters of the world's population live in areas where there are high levels of infection. Hepatitis B is estimated to cause for 60% to 80% of primary liver cancer worldwide. 1 million people a year, die from chronic active hepatitis, cirrhosis or primary liver cancer , The infection of HBV is the ninth most common cause for cancer in the world. In China, infection rates are very high, chronic carriers of hepatitis B virus (HBV) represent 10% or more of population. 500 000 to one million new cases appear every year. Chronic active hepatitis and liver cirrhosis are major causes of mortality. Therefore, studies on transmission of HBV are of substantial importance in virology as well as in public health.At present, the studies proved that most intrauterine infections could be prevented by HBVac (HBV vaccine) inoculation and HBIG (hepatitis B immunoglobulin) injection in the perinatal stage. However, 10% to 20% of the infections cannot be prevented by the combination of HBIG and HBVac. There was dispute on the mechanism for this event.The transmission routes of HBV through blood transfusion; body fluids including serum, saliva, vaginal secretions, breast milk, and semen; intrauterine infection; cell, tissue and organ transplantation and others including hemodialysis units and intravenousdrug injection have been documented. The assumption, however, that HBV vertically transmits from mother or father to offspring via affected oocyte or sperm is going to be demonstrated.Vertical transmission: HBV vertically transmits from parent to offspring via affected germ cell through fertilization. According to sex, including mother to offspring and father to offspring vertically transmission.Is vertical transmission relative to the ineffectiveness of combination of HBIG and HBVac ? Some researchers suspected that the infection of germ cell with HBV may be a factor resulting in vertical transmission. However, most present studies are about animals, more studies about human need to be executed. This was the purpose of our present study.Material and Method:35 unfertilized oocytes and 74 discarded embryos from 46 couples with positive HBsAg attended Women's hospital, School of Medicine, Zhejiang University for IVF-ET treatment from 2006.6 to 2007.1 because of infertility, including 35 unfertilized oocytes from 17 women , 27 discarded embryos from 22 women with positive HBsAg, 26 discarded embryos from 15 men with positive HBsAg, 21 discarded embryos from 8 couples both with positive HBsAg, as study group; 12 unfertilized oocytes and 34 discarded embryos from 24 healthy copules as negative controls and hepatocytes of Hepa 1175 hepatocellular carcinoma positive controls were studied. None of the patients had clinical and laboratory evidence of acquired immune deficiency syndrome. After obtaining approval from the hospital ethical committee and patient's consent, the specimens were collected after surgery. Fluorescence in situ hybridization (FISH) was carried out with biotin-labeled full length HBV DNA probe to confirm the integration of HBV in unfertilized oocytes and discarded early embryos from 46 patients with HBV infection. Dot hybridization was executed to exclude the possibility of HBV DNA from cell membrane and follicular fluid.ResultsThe specific fluorescent signal spots for HBV DNA were seen in 1 of 35 oocytesand 11 of 74 early embryos of 10 patients with positive HBsAg. The total positive rate was 11.0%(12/109),oocyte positive rate and embryo positive rate respectively is2.9%(1/35) and 14.9%(11/74).Embryo positive rate from women,men and both couples with positive HBsAg respectively is 14.8%(4/27),11.5%(3/26),19.0%(4/21).The results all exist no statistics difference .At the same time ,we detected two positive singals in one nuclear of two different eight cell embryos. 81.8% positive sample came from cases with positive preS1 and/or positive HBeAg. The negative control was negative. The positive control detects positive singal .The last three washing solutions were detected negatively by Dot blot.Conclusions(1)HBV DNA can integrate into human oocyte.(2)Human germ cell with HBV DNA is possible as a vector to deliver exogenous HBV DNA into early embryo via fertilization.(3)The integration of HBV DNA in human genome are multisites.(4)The above studies provided some preliminary evidence for the feasibility of HBV vertical transmission via oocyte and sperm, the mechanism of vertical transmission needs more study.