| Background and objectivePreeclampisa is one of Hypertensive disorders complication pregnancy (HDCP). The characteristic triad of sustained hypertension, proteinuria and edema is clinically diagnosed after20weeks’gestation. This condision resolves after delivery. Preeclampisa is the main cause of maternal and perinatal morbidity and mortality. Despite of its importance impact on materal and fetal health, its etiology is still undefined. Epidemiological studies showed that:primiparous women who first contact with the extravillous trophoblast and the women who exposure to excessive extravillous trophoblast such as twin pregnancy and hydatidiform mole are associated with increased risk of preeclampsia.compared with those who did not use barrier contraceptives or who were with the same partner for a longer period. Preeclampsia is the outcome of placental dysfunction secondary to oxidative stress and a maternal inflammatory reaction. There are seveal hypotheses of the pathogenesis of preeclampisa, included trophoblast cell dysfunction, immune regulation dysfunction of maternal interface, genetic factors, oxidative stress and the environmental impact of nutrition. Preeclampisa is like an autoimmune disease triggered by allograft rejection which is mediated by the human leukocyte antigen (HLA).The etiology of preeclampsia is still unclear. It is usually considered to be unilateral maternal. In fact, pregnancy is a successful phenomenon of semi-allograft. Both mother and fetus may contribute to the risk. An immune/immunogenetic maladaptation model for pre-eclampsia has been suggested. The maternal immune system is confronted with paternal antigens of the child.The paternal factors in the causation of preeclampsia can not be excluded. The aim of this paper is to discuss the partner’s HLA-B in the etiology of preeclampsia.Material and methodsObjectsThe studied population were from the Third Affiliated Hospital of Zhengzhou University between Oct2008and Dec2010comprised of92patients with preeclampisa and95normal pregnant women. The patients were classified according to the definition of the Santion Ministry of the People’s Republic of China on the7th edition of "Obstetric and Gynecology". We also collected37couples out of the preeclampisa patients and38couples out of the normal pregnant patients.The mean maternal age at conception was (30.62±5.80) years old and mean gestational weeks were (32.68±6.23) weeks. All the pregnant women have no consanguinity relationship. No significant difference in their ages,smoking history, body mass index(BMI)and delivery times except the gestational weeks.Sample collectionTo extract2mL peripheral blood of the case and control groups.EDTA anticoagulant,-80℃freeze for test of allele genes.Genetic analysisDNA isolated from peripheral white blood cells used DNA extracted kit supported by Shanghai Lifeng Biotechnology Company. All the operations accorded to the instructions. Adjust the concentration of the DNA to100mg/L, and stored it at-20℃for HLA testing. After amplification by polymerase chain reaction, HLA antigens were tested by reverse sequence-specific oligonucleotide (PCR-SSO) method. A large number of gene probes coated on the nano-sized beads hybridized with the amplification of DNA fragments, then reading on the Luminex.The detailed steps were as follows:1. PCR:The DNA sample was diluted with sterile distilled water20～100/ul, purity in1.65-2.0. Taking diluted DNA solution2μl, and adding Dmix buffer13.8μl, Primer4μl and Taq polymerase0.2μl.All the tubes were sticked with parafilm. Two pairs of primers were used to amplify the second and third exon of HLA-B gene, the reaction conditions were as follows: denature at96℃for3min and followed by5cycles of95℃20s,60℃20s,72℃20s,35cycles of95℃10s,60℃15s,72℃20s,and extension at72℃10min.3μl of PCR products was added into2%agarose gel contain0.5mg/L ethydium bromide by80-100V electrophoresis for20min. The fragment size of the amplification:the second exon45bp and third exon426bp.(Picture1).2. Hybridization:the amplification of DNA by added BM (beads) and HM (bead buffer), shocked,then added50μl SAPE (fluorescent fluid), incubated at60℃for6min. After repeated washing put them into the Luminex to read the sample.Statistical analysisDescriptive data were expressed as mean±standard deviation (SD) and all frequencies were directly counted. χ2and Fisher’s exact test were used to the difference of allelic distribution between the two groups. P value of0.05was used to deem statistical significance. Statistical analysis were performed with the Statistical Package for Social Science (SPSS), Version16.0.Results1Comparison of clinical informationWe analysis the age and the gestational weeks of the preeclampsia and control group. Our data shows that except the gestational weeks there is not significant.2Distribution of the HLA-B between preeclamptic patients and normal pregnant women 34kinds of alleles were obtained in all samples.The allelic frequencies of HLA-B13(9.47%),-B46(9.47%)and-B51(8.95%)were relatively high in the control group,while allelic frequencies of HLA-B13(16.30%),-B61(9.24%)and-B62(8.70%) were higher in the preeclampisa group.But all the allelic frequencies showed no significant difference(P>0.05).3Distribution of the HLA-B in partners of preeclamptic patients and normal pregnant women28kinds of alleles were obtained in all samples. The allelic frequencies of HLA-B13(15.79%),-B38(9.21%),-B35(7.89%) and-B61(7.89%) were higher in the control group, while HLA-B13(22.97%),-B51(9.46%),-B58(6.76%) and-B60(6.76%) were higher in the case group. All the allelic frequencies showed no significant difference (P>0.05).4Analysis of maternal/paternal HLA-B gene association in preeclamptic patients and normal pregnant womenWe analysed some special allelic bindings HLA-B13,-B46and-B51which were revelatively higher in the alleles. Analysis of the genetic compatibility between the couple and the results showed as follows:(1) The frequency of preeclampisa patients with HLA-B13positive and partners with HLA-B13negative was higher than that in nomal pregnant women and partners(P<0.05).(2) The frequency of preeclampisa patients with HLA-B13negative and partners with HLA-B13negative was lower than that in nomal pregnant women and partners (P<0.05).Conclusions1Paternal factors play an impotant role in preeclampsia.2Maternal/paternal special bindings of HLA-B13gene may be associated with susceptibility to protective preeclampsia and protection against the disease. |
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