| Objective: This study aims to explore the effect of the paternally expressed gene 10 (PEG10) in early pregnancy, in order to provide a new mentality for the etiopathogenisis research of inevitable abortion. The expression and histology localization of PEG10, which originated from father and imprinted by mother, was tested in deciduas from the normal early pregnancy and inevitable abortion respectively.Method: The deciduas of normal early pregnancy and inevitable abortion were obtained from the first affiliated hospital Chongqing Medical University (All patients recurited were informed before their deciduas were used to the study). Sixty normal pregnant samples were collected and the gestational age was limited to 6~11 weeks (10 cases in each pregnant week). Their age varied from 20~32 years old and intrauterine pregnancy was confirmed by the gynecological examination and the ultrasound, which was consistent with the pregnant week. In addition, 40 the inevitable abortion patients were recruited in this study. the gestational age is limited to 6~9 weeks(10 cases in each pregnant week). The age covered a wide range from 20 to 35 years old. The fact that the embryo has been stopped to growth, which means that embryo sac is smaller than its pregnant weeks or corcule or heart tube beat is not observed, was confirmed by the gynecological examination and ultrasound diagnosis. Total RNA was extracted from the pregnancy decidual tissues gained under the aseptic condition and remained a part of fresh organization fixed by 4﹪of paraformaldehyde. The expression and distribution of the imprinted gene PEG10 in decidual tissues of were examined by RT-PCR, immunohistochemistry, in situ hybridization, Western blot and laser scanning confocal miscroscopy.Result:1. RT-PCR demonstrated that the expression of PEG10 had a gradual increase with the pregnant weeks elapsing. The expression of PEG10 was low in pregnant 6th week (0.6743±0.114). Subsequently, the expression was observed increase (7th weeks 0.7349±0.0083) and reached a peak in pregnant 8th week (0.7354±0.0074). After that, it emerged a decline tendency (9 weeks 0.6340±0.0084, 10 weeks 0.5901±0.0089, 11 weeks 0.5261±0.0112). While the expression in the inevitable abortion deciduas dropped obviously during pregnant 6~9 weeks. There are significant difference between the normal early pregnancy and the inevitable abortion in every pregnant week (P<0.05).2. The ISH analysis demonstrated that the characteristic of PEG10 mRNA expression was identical with the rules of RT-PCR. Its masculine signal located in mesenchymal, cytoplasm and the glandular epithelium and showed dispersed, focus stained or suffusion distribution. The expression in glandular epithelium was strongest, which resembled hyacinthine fine sand or dark purple coarse grain in appearance; the expression in cytoplasm was second to the former and was weakest in mesenchymal. But during pregnant 6~9 weeks the glandular epithelium showed the thin blue color in inevitable abortion deciduas. The masculine signal almost was not observed in the mesenchymal and cytoplasm.3. The IHC, the LSCM and WB demonstrated that the characteristic of PEG10 protein expression was matched with the mRNA expression. IHC found that the masculine signal located in deciduas mesenchymal cell and glandular epithelium cell, while it did not show in the nucleus. In normal pregnant deciduas, the expression density of PEG10 was (+++)-(++++)-(+++) during pregnant 6~11 weeks. but the expression density of PEG10 was very low in inevitable abortion deciduas during pregnant 6~9 weeks. The LSCM discoveried that the masculine cell located in decidual mesenchymal cell and epithelioglandular kytoplasm. WB demonstrated that every case presented the interest protein and the internal reference protein in 80 kDa and 43 kDa place, of which the size of interest protein was consistent with the ORF1/ORF2 protein of PEG10 announced by the gene bank.Conclusion:1. Heredity imprinted gene PEG10 that maintained certain high level expression in the normal early pregnant decidual tissues played an important role to ensure successful pregnancy. PEG10 might have significant effect on promoting and maintaining the decidua and the embryo growth. The expression of PEG10 mRNA and protein maintained the high level at the pregnancy early time in deciduas, which possibly participated in the establishment and maintenance immunological tolerance in the fetal-maternal interface.2. The expression of PEG10 mRNA and protein in inevitable abortion deciduas is far lower than that of in the normal early pregnant decidual tissues. This research showed that lower levels of PEG10 might be one of reasons of inevitable abortion during early pregnancy. At the same time, the results further suggested that the level of PEG10 be closely associated with embryonic development, and the low expression of PEG10 might be become a new target spot for diagnosis and prevention of abortion.3. The experimental technologies used in the research built the experiment foundation for exploring the new diagnostic approach with high sensitiveness and specificity. |
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