The Role Of HLA-G In Human Early Embryo Cleavage, Implantation And Immunotolerance

Background: HLA-G is a major histocompatibility complex (MHC) class Ib molecule with a selective expression at the fetal-maternal interface. Studies have exposed an increasing diversity of HLA-G's functional aspects in human reproduction. The preimplantation embryo development (Ped) gene product Qa-2 is mouse MHC class Ib protein which influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. Data from many human in vitro fertilization (IVF) clinics suggest that the mouse Ped phenomenon exists in human because embryos fertilized at the same time have different cleavage rates and consequent IVF outcome. And HLA-G is proved being expressed in human early embryos and believed to be the functional homolog of Qa-2. Many studies have found embryo secretes solubole HLA-G is associated with a higher implantation, ongoing gestation and live birth rates in IVF cycles. And successful pregnancy can be created only when well developed early embryo implants to decidua and is not rejected by the maternal immune system. Implantation is initiated by the trophoblast of the blastocyst establishing contacts to the surface of the uterine epithelium, followed by firm adhesion and penetration. For this process, a state of adhesion competence of the trophoblast as well as a state of receptivity of the uterine epithelium is required. This process is partly controlled by the trophoblasts and the maternal microenvironment. The dNK cells which are the main maternal decidual immune cells, can directly lyse trophoblast cells. HLA-G has been implicated in protecting susceptible target cells ( trophoblasts and some tumor cells) from lysis by NK cells. And many studies suggest that the occurrence of some pregnancy-associated diseases due to abnormal trophoblasts proliferation and invasion is associated with abnormal HLA-G expression. Thus HLA-G may have the function of regulating trophoblasts proliferation and differentiation, simultaneously protecting them from attacking by maternal immune cells. HLA-G research is still a relatively new field. And to date, the precise role of HLA-G in the placenta remains unclear. More extensive research is required to achieve a more wholesome understanding of HLA-G. That could shed light on the immunoregulatory states of pregnancy, pregnancy-associated diseases and also processes of graft rejection and tumour invasiveness. And our study may have potential clinical value for in vitro fertilization.Objective: Based on previous reports indicating that HLA-G is essential for successful pregnancy, we hypothesis that trophoblasts lacking HLA-G are vulnerable to attacke by the maternal immune cells and are incompetent to adhere or invade the decidua. These defective trophoblasts result in implantation failure and deseases associated with pooly decidua invading. And trophoblast and embryo have deficient HLA-G expression may result in proliferation abnormal and thus poor placentation and embryonic development. The purpose of the study is to elucidate: (1) whether the HLA-G expression level change of trophoblast derived cells can influence NK cytolysis to them; (2) whether HLA-G has the function to improve human early embryo cleavage and human trophoblasts proliferation; (3) whether HLA-G can regulate trophoblast derived cells adhesion and invasion.Methods: Using RNAi technology, we novelly constructed adenovirus vector which can transcribe siRNAs specially targeting HLA-G mRNA to knock-down HLA-G expression in cells and embryos. The HLA-G high expression cell line JEG-3(derived from human chorionic carcinoma) was used for trophoblasts function studies. To construct HLA-G low expression trophoblasts model, expression of HLA-G in Ad vector transfected JEG-3 cells was checked by RT-PCR and Western blot. Then NK cell cytotoxicity to such HLA-G deficient JEG-3 cell was studied by non-radioactive cytotoxicity assay. We also detected the proliferation and invasion changes in JEG-3 cells after treated with Ad vecter or HLA-G blocking mAb, using XTT method and a commercial invasion kit respectively. Mice embryos were transfected with Ad vectors and detected the infection efficiency by X-Gal stain to determine the exact MOI for human embryo transfection. Then we transfected human early embryos from patients undergoing in vitro fertilization by siRNA expression Ad vector to interfere HLA-G expression of embryo and observed the following cleavage rate. We also detected HLA-G isoforms expression by RT-PCR in several trophoblast derived cell lines, including JEG-3, JAR and TEV-1.Results: (1) The Ad vectors we have constructed can transfect JEG-3 cells with the efficiency of 100% at the MOI of 100 and induce significant HLA-G RNAi in these cells. HLA-G expression in JEG-3 cells was successfully knocked-down on both mRNA and protein level, decreased 23.73 % and 48.36 % respectively; (2) The decreased HLA-G expression level resulted in a significant NK cells-mediated lysis in transfected JEG-3 cells. At E:T ratios of 10:1, 5:1 and 2.5:1, the percentages of cytotoxicity in the HLA-G siRNA group vs that in the scrambled siRNA transfected control group were 58.82 % vs 67.65 %, 51.47 % vs 60.29 % and 39.71 % vs 48.53 % respectively which were significantly differed; (3) The Ad vectors are efficient to transfect mouse and human early embryos. The 3PN day 3 human embryos cleavage were significant slowed down after infected with HLA-G RNAi induced Ad vectors at the titre of 106 pfu/ml. 92.31% (12 of 13) HLA-G deficient human embryos stoped cleavage during the following two days'culture. And the average blastomeres number per embryo was only 17.71±6.57 at day 5; (4) The JEG-3 cells proliferation rate decreased after transfecting with the HLA-G RNAi induced Ad vectors or incubating with anti-HLA-G blocking mAb. But the differences are not significant. And the HLA-G expression down-regulated JEG-3 cells invasion capability decreased significantly. In Ad scrambled control group and Ad HLA-G RNAi group, the mean OD592 value are 0.790±0.048 and 0.605±0.133 respectively. And in anti HLA-G MAb 4H84 1ug/ml or 0ug/ml added groups, the mean OD592 value are 0.626±0.106 and 0.923±0.025 respectively.Conclusions: These results indicated that the recombinant Ad vectors we designed were efficient tools to create HLA-G expression down-regulated cells and embryos models for HLA-G function research. To our knowledge, this is the first report using siRNA expression Ad vector transfected human early embryo in HLA-G function study. Our results directly demonstrated that the HLA-G could protect JEG-3 cells against human NK cells lysis. And our findings suggested HLA-G could have special non-immune functions in human reproduction. We found the HLA-G deficient embryos had a significant slow cleavage rate. The correlation of HLA-G expression and cleavage rate suggests that this molecule may play an important role in human early embryo development. By HLA-G deficient JEG-3 cells'function studing, we found HLA-G may regulate human trophoblasts proliferation and invasion behaviors during implantation and placentation.