A Preliminary Study Of ADAM 12 As A Marker In Prenatal Screening

Objective(1) Measure the level of ADAM 12-S in the first-trimester maternal serum by Time-Resolved Fluorescence Immunoassay(TR-FIA) and preliminarily assess the value of measuring ADAM 12 in the first-trimester(2) Synthesize full length ADAM 12-S protein by gene synthesis technology,which helps to build basis of developing test kits using full length ADAM 12 as antigen.Materials and methods(1) ADAM 12 concentrations were measured by TR-FIA in a set of serum samples collected in the first-trimester(6⁺⁰-13⁺⁶ gestation weeks) from cases of pregnant women in Peking Union Medical College Hospital from July 2007 to January 2008.The relationship between different pregnancy outcomes and the levels of serum ADAM 12 were analyzed.(2) Using Oligo gene synthesis technique,full length ADAM 12 nucleotides were synthesized and sequenced which were then inserted into the prokaryotic expression vector pET-30a,the ADAM12-S vector was built and the recombinant protein was expressed in E.coli BL21;Western blot,Mass Spectrometry analysis and specific antibody detection methods were applied for protein identification.Results11.664 serum samples from 518 pregnancy cases were collected.Maternal age≥35 accounts for 138 cases,or 25.5 percent,and there are 7 cases of twin pregnancy.12.(1) Among the 518 cases of pregnancy,there were 32 cases of early embryo loss(27 cases of embryo growth arrest,4 cases of ectopic pregnancy,1 case of scar pregnancy),2 cases of induced labor due to fetal abnormality,14 cases of premature delivery,2 cases of pre-eclampsia,2 cases of fetal growth restriction(FGR),22 cases of gestational diabetes mellitus(GDM),5 cases of placenta previa.(2) 13 cases of chromosomal abnormality,of which 3 cases of 21-trisomy.13.Multiple of Medians(MoM) values of ADAM 12 were 14.63ng/ml, 35.08ng/ml,88.9ng/ml,186.51 ng/ml,370.62ng/ml,537.71 ng/ml, 632.55ng/ml,769.42ng/ml from 6 to 13 weeks,respectively.The level of serum ADAM 12 increased with gestation weeks,exhibiting a linear relationship with a correlation coefficient 0.992(P＜0.01).14.There were statistical significant differences between MoMs of aneupolyhaploid(0.34),embryo growth arrest(0.24) and normal pregnancy (1.008).(P＜0.05)15.The MoMs of ADM12 in the euchromosome aneupolyhaploid patients (0.29) were lower than normal pregnant women,and the MoMs of ADAM 12(1.48,0.39,0.23)reduced gradually in the DS cases with gestational week.16.The maternal serum concentration of ADAM 12 was significantly increased in the 5 cases of placenta previa.The median MoM was 1.45.(P＜0.05)17.There was no obvious correlation between fetal weight and early serum ADAM 12 level,r=-0.15(P＜0.05).18.The relationship with GDM and premature delivery was not significant(P＞0.05).19.The synthesized nucleotides for full length ADAM 12 expression were consistent with expected sequence.20.The ADAM12-S plasmid was built.21.The molecular weight of the recombinant protein was in accordance with the predicted protein.22.The expressed protein can be detected by antibody specific for ADAM 12 but the amount of synthesized protein is low.Conclusions 11.The level of ADAM 12 in first-trimester maternal serum is increased with gestation weeks.12.ADAM 12 can be a potential marker of aneupolyhaploid in the first-trimester prenatal screening,the suggested application time is between 9 to 12 gestation weeks.13.ADAM 12 in early first trimester may predict early pregnancy loss such as embryo growth arrest and ectopic pregnancy.14.There is no correlation between early maternal serum ADAM 12 level and fetal weight,and the relationship between early ADAM 12 level and GDM was not significant.15.Escherichia coli expression system can express intact ADAM 12-S,but the expressed protein is different from natural protein,probably due to the wrong modification.16.Replacement of expression system such as yeast expression system is the next choice.