| ObjectiveThe expression of NK cells and NK cells related subsets in menstrual blood,decidua and peripheral blood of control group and recurrent spontaneous abortion(RSA)group were detected by flow cytometry,The results of menstrual blood,decidua and peripheral blood were compared to explore the feasibility of menstrual blood samples in predicting recurrent spontaneous abortion.MethodsTranscriptome analysis showed that NK cells in normal menstrual blood was similar to normal decidua NK(d NK)cells,then endometrium NK(u NK)cells status of RSA patients were evaluated by menstrual blood NK(MBNK)cells.Decidua samples(n=15)from normal pregnancies were obtained from women with selective pregnancy terminations,abnormal decidua samples(n=10)were obtained from women who experienced recurrent spontaneous abortion and had a normal karyotype.Menstrual blood(n=10)on the second day of the menstrual cycle was collected from healthy women who had normal reproductive history and regular menstruation,menstrual blood specimens(n=25)were obtained from patients who experienced recurrent spontaneous abortion and had a normal karyotype,meanwhile,peripheral blood was collected.All samples were collected from the First Affiliated Hospital of USTC.None of the subjects had risk factors such as chromosomal abnormalities(self or husband),uterine abnormalities,autoimmune diseases or other drug treatments that affected hormone balance.All samples obtained were analyzed by flow cytometry,mainly including NK cell surface marker molecules CD56 and CD16;Tissue resident molecule CD49a,EOMES;Inhibitory receptor CD158a,CD158b,GITR;Activated receptor NKP30,NKP44,NKG2D.The expression differences of the above molecules in the normal group and the experimental group were observed and compared.ROC curve was used to calculate the prediction efficiency of differential molecules for RSAResult(1)Transcriptomics proved that NK in normal menstrual blood was similar to decidua and different from peripheral blood.(2)Flow cytometry analysis showed the proportion of CD3~+T cells in decidual and menstrual blood of the RSA group was increased,CD56~+NK cells was decreased,while the results in peripheral blood were opposite.(3)Compared with control group,the expression of CD56~+CD16~+NK in decidua and menstrual blood of the RSA group was significantly increased,CD56~+CD49a~+NK was decreased;CD56~+CD16~+NK expression was increased in peripheral blood,and there was no different of CD56~+CD49a~+NK(4)Compared with control group,the expression of CD49a~+CD16~-and CD49a~+EOMES~+NK cells in decidua and menstrual blood of the RSA group were decreased,CD49a~-CD16~+NK cells were increased;there were no significant difference in peripheral blood.(5)Compared with control group,the inhibitory receptor CD158a in decidua NK and menstrual blood NK were decreased in the RSA group,there was no significant difference in peripheral blood NK(PBNK);CD158b was no significant difference in decidua and decreased in menstrual and peripheral blood;GITR was no significant difference of in decidua,menstrual blood and peripheral blood of RSA group.(6)Compared with control group,the activated receptor NKP30 was not significantly different in the decidua,menstrual blood and peripheral blood of the RSA group;NKP44was no significant difference in decidua and menstrual blood of the RSA group,decreased in peripheral blood;NKG2D in decidua of the RSA group was no significant difference,increased in menstrual blood and decreased in peripheral blood.(7)Menstrual blood NK cells have a high predictive effect on recurrent spontaneous abortion compared with peripheral NK cells.The AUC of CD3~-CD56~+NK,CD56~+CD16~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-NK,CD49a~+EOMES~+NK cells subsets were greater than 0.7.The accuracy more than 80%when CD49a~+EOMES~+NK,CD56~+CD49a~+NK and CD49a~+CD16~-NK for prediction,Conclusion(1)Menstrual blood NK cells are similar to decidual NK cells;(2)Compared with the control group,the expression of NK cells and subsets in menstrual blood of patients with RSA was abnormal,which was highly consistent with decidua.(3)Menstrual blood NK cell subsets CD49a~+EOMES~+NK,CD56~+CD49a~+NK,CD49a~+CD16~-,and CD56~+CD16~+can diagnose recurrent spontaneous abortion with high diagnostic efficiency. |
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