Biochemical Detection And MRNA Differential Expression In Peripheral Blood Of Patients With Intrahepatic Cholestasis During Pregnancy

ObjectiveRetrospective analysis was conducted to investigate the blood biochemical indexes and the incidence of adverse outcomes in both mother and child of patients with intrahepatic cholestasis of pregnancy(ICP)at different diagnostic time.Meanwhile,through interdisciplinary cooperation,RNA sequencing(RNA-seq)will be adopted to analyze the m RNA differential expression in the peripheral blood of ICP pregnant women and healthy pregnant women in early pregnancy,in order to guide the early treatment and nursing intervention of the disease.Methods1.Clinical data of pregnant women with complete records in the Department of Obstetrics of Longgang District Maternal and Child Health Hospital of Shenzhen from September 2017 to September 2018 were collected for retrospective analysis.Pregnant women who met the inclusion and exclusion criteria were divided into group A(ICP group,n=200 cases)and group B(healthy group,n=100 cases).Group A was divided into subgroups according to different gestational weeks of ICP diagnosis: Group A1(diagnosis gestational age ≤28 weeks,set as early diagnosis ICP group,n=20 cases)and group A2(diagnosis gestational age≥28weeks +1,set as late diagnosis ICP group,n=180 cases).Baseline data(age,gravidity,systolic blood pressure,diastolic blood pressure,etc.)between group A and B were compared by t-test;hospitalization days and hospitalization costs between subgroups A1 and A2 were compared;serum biochemical indexes(total bile acid,glycocholic acid,alanine transaminase,glutamic-oxalacetic transaminase,total bilirubin,etc.)before and after diagnosis in group A2 were compared.Chi-square test was also used to compare the incidence of adverse outcomes such as cesarean section,premature delivery,macrosomia,umbilical cord around the neck,premature rupture of membranes and other adverse outcomes between group A and B and A1 and A2 subgroups.The rate of counting data was indicated,and P<0.05 was considered statistically significant.2.Peripheral blood samples of 7 ICP pregnant women with gestation≥ 12 weeks from March 2018 to August 2020 in the Department of Obstetrics of Longgang District Maternal and Child Health Hospital of Shenzhen were collected as the ICP group,and peripheral blood samples of 5 healthy pregnant women during the same period were collected as the control group.Baseline data(age,gestational age,systolic and diastolic blood pressure,etc.)were compared between ICP group(n=7cases)and normal control group(n=5 cases)using t test.RNA was extracted from 12 samples respectively,and the purity and concentration of RNA were determined by spectrophotometry.The absorbance A260/A280 of RNA samples was ≥1.6,indicating that the samples had good concentration and purity.Reaching the machine standard;The expression level of mRNAs in ICP pregnant women(n=7)and healthy pregnant women(n=5)was analyzed by RNA-seq technique.The differential expression of deseq2 in peripheral blood of ICP pregnant women and healthy pregnant women in early pregnancy was analyzed by R corrected deseq2.To P < 0.001,|log2 FC|>2 defined as the threshold of mRNAs differentially expressed,combined with the GO,KEGG,STRING database key screening differentially expressed genes related to ICP,comments by biological process,biological signal pathway enrichment,text mining and comprehensive bioinformatics methods,such as protein and gene interactions,differentially expressed genes that may be associated with disease were screened out.Results1.Comparison of baseline dataThe results showed that there were no significant differences in age,gravidity,systolic blood pressure and diastolic blood pressure between group A and group B(P > 0.05),and they were comparable.2.Influence of Group A and Group B on maternal and infant outcomesThe rate of premature delivery in group A and B was 40%(80/200),14%(14/100),the rate of cesarean section was 55%(110/200),25%(25/100),and the incidence of umbilical cord around the neck was 41%(82/200)and 2%(2/100).The difference between group A and B was statistically significant(P<0.05).There was no significant difference in the incidence of macrosomia and premature rupture of membranes between group A and B(P>0.05).3.The influence of different gestational age diagnosis on maternal and infant outcomesThe rates of premature delivery were 15%(3/20)and 42.8%(77/180),the rates of cesarean section were 30%(6/20)and 57.8%(104/180),and the incidence of umbilical cord around the neck was 20%(4/20)and 43.3%(78/180)in group A1 and A2.The difference between A1 and A2 groups was statistically significant(P<0.05).There was no significant difference in the incidence of macrosomia and premature rupture of membranes between A1 and A2 groups.(P>0.05).4.The influence of different gestational age on the hospitalization days and hospitalization costsThe length of hospital stay and cost of A1 group were(5.88±2.73)days and(5387.62±1546.47)yuan,respectively,and the length of hospital stay and cost of A2 group were(7.25±4.71)days and(6548.38±5207.97)yuan,respectively,with statistical significance(P<0.05).5.Serum biochemical indexes before and after diagnosis in group A2In A2 group,the serum total bile acid(26.51±18.60)μmol/L,glycocholic acid(10.03±9.47)mg/L,alanine aminotransferase(31.26±69.99)U/L,glutamic-oxalacetic aminotransferase(30.62±48.35)U/L after diagnosis were significantly higher than those before diagnosis.Were(3.05±1.39)μmol/L,(0.83±0.51)mg/L,(11.87±5.29)U/L,(17.74±4.62)U/L,respectively,and the differences were statistically significant(P<0.05).There was no significant difference in total bilirubin level between the two groups before and after diagnosis(P>0.05).6.Comparison of baseline data of sequencing samplesThe results showed that there were no statistically significant differences in age,gestational age,systolic blood pressure and diastolic blood pressure between ICP pregnant women(n=7)and healthy pregnant women(n=5)in basic conditions(P>0.05),and they were comparable.7.RNA quality inspection results and differentially expressed genes analysisThe RNA concentration and purity of all samples were qualified,and all of them met the requirements of computer sequencing.A total of 25 differentially expressed genes were obtained by RNA-seq analysis.There were 8 up-regulated genes: FOSB,ATF3,S100 P,TAP2,UTS2,SPSB2,CCR5 and HLA-DQB1.There were 17 down-regulated genes: JUND,H19,HCG27,GSTM3,PSPHP1,ARL17 B,JUN-MUC16,THNSL2,TUBBP5,GSTM3,LRRC37 A,KAZN,KLRC2,Tas2R40,Zn F501 and KLF6.8.GO,KEGG and STRING databases were used to analyze the differentially expressed genes related to ICPIt was found that the up-regulated and down-regulated genes had statistically significant differences in biological behavior control,immune system,inflammatory factors,signal transduction,cell grouping morphology and mutual function among biological processes.Conclusions1.Early diagnosis of ICP is beneficial to reduce the incidence of adverse pregnancy outcomes.Early monitoring,early treatment and nursing intervention should be carried out for ICP pregnant women,in order to effectively reduce and reduce the occurrence of maternal and infant complications,shorten the number of days of hospitalization of pregnant women,reduce the cost of hospitalization,and reduce the economic burden of family and society.2.Key genes FOSB,JUND,JUN,ATF3 and KLF6 may be potential early diagnostic markers of ICP.Early detection of biomarkers through interdisciplinary cooperation is convenient for clinical nursing workers to carry out long-term dynamic follow-up of high-risk population,and provide an important reference for the implementation of nursing intervention.