Expression Of Imprinted Genes PEG10,L3MBTL1in The Placenta Of Offspring From Assisted Reproductive Technology

Background and ObjectiveSince the birth of Louise Brownin in1978， the application of in vitrofertilization (IVF) has more than30years.about4,000,000babies overe bornthrough assisted reproductive technology (ART) worldwide.>200,000ART babiesare born worldwide each year. As IVF was introduced into Practice without formalevaluation of its effects on the health of children concedure with ARTtechnology.There are mounting concerns about the health of ART-conceived offspring.Increasing risks associated with ART have been reported, including spontaneousabortion, small for gestational age,low or very low birth weight, cerebral palsy, majormalformations, and so on. The imprinting disorders, including Prader-willisyndrome、 Beckwith-Wiedemann syn-drome and Angelman syndrome, haverecently attracted considerable attention regarding the safety of ART. Different caseseries and case-control studies have consistently suggested the increasing risks ofimprinting disorders in pregnancies after ART. Therefore, this issue investigates theexpression of imprinted genes--PEG10and L3MBTL1,which are closely related tothe growth and development of ART offspring. The purpose is to provide a new theoretical basis for the safety of assisted reproductive technology. One importantfunction of placenta is nutrition and substance,and exchanging between mother andfetus, which is important for the fetus. Many imprinted genes are expressed in theplacenta tissue. ART operation may lead to abnormal expression of imprinted genes.PEG10(Paternally expressed gene10) is a paternal imprinted gene. Ryuich ono foundit in HCC by the cDNA microarray in2001. It locates on the long arm with two Zone1(?) of human chromosome7(7q21.3).PEG10was expressed in the placenta, brain,kidney, liver, ovary and testis, which encodes a protein closely related to fetal growth,placental formation and differentiation of trophoblast cells. The study found thatknocking out the mouse embryos’ PEG10gene can cause placental abnormalities,And increased miscarriage rate and early embryonic mortality. PEG10showed a lowexpression status in the tissues of spontaneous abortion and inevitable abortion, but itwas highly expressed in the liver and gastric carcinoma. Paternal imprinted genesL3MBTL1(Paternally expressed gene L3MBTL1) was located on the20th long armof chromosome13districtand12band (20q13.12) and expressed in the placenta,hematopoietic progenitor cells and tumor tissues, and participation and maintainnormal cell mitosis.It encoded protein inhibited the growth of malignant braincanner.It showed low expression status In leukemia cells and abnormal proliferationof bone marrow cells and almost no expression. Knocking out mice embryo’sL3MBTL1gene lead to the obstacles of embryonic stem cells differentiation intotrophectoderm, and effect the placenta and embryo’s development.Our previous study detected the expression mRNA and changes of PEG10mRNA and,L3MBTL1mRNA on the placenta of natural pregnancy offspring andART offspring by real-time quantitative PCR (QRT-PCR). The methylation-specificPCR technique (MSP)was used to detect gene PEG10and L3MBTL1methylationstatus in the ART group and make a comparison with the control group, The resultsshowed that PEG10mRNA expression up-regulated in the ART group; but theL3MBTL1mRNA expression showed a downward trend. ART group placentalPEG10promoter methylation positive rate was lower than that in the naturalpregnancy group.But L3MBTL1promoter methylation was higher than that in thenatural pregnancy group. So we thinked that the abnormal expression of ART placental tissue PEG10, L3MBTL1may be associated with maternal and offspringborn poor pregnancy outcomes. In this study,we use the western blotting andimmunohistochemistry to examine the expression of imprinted gene PEG10andL3MBTL1,mark clearify histological localization and investigate,the effects ofassisted reproductive technologies on the expression of these proteins.Materials and methods1. We collected the placental tissue from30cases of ART and33cases of naturalpregnancy and compare their basic information and pregnancy outcomes.2. Routine HE was stained for placenta and the optical microscope was used tocompare the morphological about placental between the two groups.3. The immunohistochemical techniques was used to compare PEG10,L3MBTL1histological localization and Semi-quantitative analyze the differences inprotein expression of the two genes.4. The Western Blotting was used to between the PEG10, L3MBTL1proteinexpression of the ART and natural pregnancy.5. The SPSS17.0software was used for statistical analysis). The mean±standard deviation was used for measurement data (symmetrical distribution). T-testwas used between the two groups.Results1. There were no statistical significance differevce between the two groups inthe aspact of age, weight gain during pregnancy（P>0.05）. ART group offspring bodyweight and length, placental weight were lower than natural pregnancy group’s.2. ART and natural pregnancy placenta Routine HE staining showed that typicalvillous structures existed In the placental tissue of the two groups and the fetal hasRich blood vessels with No significant calcification and edema,little infiltration ofinflammatory cells and normal placenta structure.3. IHC showed that PEG10, L3MBTL1were all expressed in the two placentas,locating in the interstitial cells cytoplasm of the placenta with no expressing in the nucleus. The expression of PEG10seemed upregulated in placental tissue of the ARTgroup offspring Compared with the control.(P＜0.05). The expression of L3MBTL1seemed downregulated in ART offspring placentas, and the difference wassignificant(P＜0.05).4. Western Blotting showed that the expression regulation of imprint genePEG10, L3MBTL1were similar to the study previously,that is,the expression ofPEG10were upregulated in placental tissue of the ART group offspring comparedwith the control.;the expression of L3MBTL1were downregulated in ART offspringplacental tissues compared with the control.Conclusion1.PEG10, L3MBTL1in normal placentas were expressed, both involved in theformation and development of the placenta, and participate in normal pregnancy.2. Imprinted gene PEG10maintain a certain high level in the placental tissue ofART offspring, Imprinted gene L3MBTL1maintain a certain low level in theplacental tissue of ART offspring，May be the cause of poor birth outcomes inmaternal and offspring.