Association Of Killer Cell Immunoglobulin-Like Receptors And Their Specific HLA-C Ligands With Early Unexplained Recurrent Spontaneous Abortion

SECTIONⅠASSOCIATION OF KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR GENES AND THEIR SPECIFIC HLA-C LIGANDS WITH EARLY UNEXPLAINED RECURRENT SPONTANEOUS ABORTIONPART 1 Polymorphisms of Killer Cell Immunoglobulin-like Receptor Genes and their Specific HLA-C Ligands in Couples with Early Unexplained Recurrent Spontaneous AbortionOBJECTIVES Killer cell immunoglobulin-like receptors(KIR)are expressed on the surface of natural killer(NK)cells and some of T Lymphocytes.NK cell receptors have been suggested have a high-level infiltration with stromal cells and more importantly with invading trophoblast.But their function at the fetomaternal interface remain unknown.To investigate whether the outcome of pregnancy depends on women's KIRs and couples' HLA-C ligands,we studied the KIR and HLA-C gene polymorphisms in couples with early unexplained recurrent spontaneous abortions(RSA).METHODS We searched seventy-three pairs of childless couples with three or more early abortions characterized as unexplained after extended evaluation (exclusion of anatomical,chromosomal,hormonal,metabolic,infectious, autoimmune and thrombophilic etiologies)and sixty-eight pairs of healthy control couples.Peripheral blood was drawn to obtain genomic DNA which was used for a polymerase chain reaction using sequence specific primers(PCR-SSP)in order to determine whether fifteen selected KIR genes and two groups of HLA-C alleles were present.The genotypic frequencies of each KIRs and HLA-C alleles were calculated as the percentage of positive numbers among all specimens.KIR genotypic frequency differences significance between patients and control subjects were tested by the two-tailed Fisher's exact test.The different significance of average activating KIR numbers between RSA patients and control subjects were tested by t-test.RESULTS 1.Our result showed that gene frequency of KIR2DS1 was higher in patients with RSA compared to that of control subjects(P=0.029).2.Increased numbers of activating KIR genes was observed in patients(P=0.041).3.Women who possessed more than two activating KIR genes were found more frequently in patients than those in control subjects(P=0.018).4.From a cohort of husband and wife couples,couples with a decreased group 2 HLA-C allele had a tendency to fall into the RSA group(P=0.005).5.From a cohort of husband and wife couples,the women with a KIR2DL1 gene had a decreased group 2 HLA-C allele(P=0.007), while the women with a KIR2DS1 gene also had a decreased group 2 HLA-C allele(P=0.002).6.From a cohort of husband and wife couples,the women with a KIR2DS1 gene,and with a decreased group 2 HLA-C allele for the homologous inhibitory receptor KIR2DL1,had a tendency to fall into the RSA group(P=0.004).CONCLUSIONS 1.The increased frequences of KIR2DS1 may be one of the reasons of early unexplained recurrent spontaneous abortions.A genetic imbalance between activating and inhibitory KIR genes especially the increased activating KIR genes may influence the pathogenesis of RSA.2.A decreased group 2 HLA-C allele maybe related to RSA.3.The results suggest that a genetic variation at the KIR locus influences the susceptibility to unexplained RSA in the Chinese Han population.Moreover,decreased ligands for inhibitory KIRs could potentially lower the threshold for NK cell activation,mediated through activating receptors,thereby contributing to pathogenesis of RSA.PART 2 Killer Cell Immunoglobulin-like Receptors'Repertoire in Patients with Unexplained Recurrent Spontaneous AbortionsOBJECTIVES To investigate whether patients with unexplained recurrent spontaneous abortions(RSA)have specific KIR genotype.METHODS 95 childless women with two or more unexplained early abortions and 111 randomly healthy women as control subjects were genotyped for fifteen KIR gene using polymerase chain reaction of sequence specific primers(PCR-SSP).RESULTS The genotype of 2DL1~+,2DL2~+,2DL3~+,2DL4~+,2DL5~+,3DL1~+, 3DL2~+,3DL3~+,2DS1~+,2DS2~+,2DS3~-,2DS4~+,2DS5~+,3DS1~+,KIR2DP1~+ was the most frequency in the RSA group(14.74%);the genotype of2DL1~+,2DL2~-,2DL3~+, 2DL4~+,2DL5~-,3DL1~+,3DL2~+,3DL3~+,2DS1~-,2DS2~-,2DS3~-,2DS4~+,2DS5~-, 3DS1~-,KIR2DP1~+ was the most frequency in the control subjects(20.72%);The genotype of 2DL1~+,2DL2~+,2DL3~+,2DL4~+,2DL5~+,3DL1~+,3DL2~+,3DL3~+,2DS1~+, 2DS2~+,2DS3~+,2DS4~+,2DS5~+,3DS1~+,KIR2DP1~+ in the RSA patients is significantly higher than that in control subjects(11.58%in RSA vs 1.80%in controls;P=0.007). CONCLUSIONS The different of KIR genotype between patients with RSA and healthy control subjects may explain the pathogenesis of RSA.SECTIONⅡEXPRESSION OF KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTOR mRNA IN DECIDUA OF PATIENTS WITH UNEXPLAINED EARLY RECURRENT SPONTANEOUSOBJECTIVES Decidual natural killer(NK)cells express killer cell immunoglobulin-like receptors(KIRs),which bind to ligands on trophoblast cells. This interaction appears to block NK cytotoxicity against trophoblast cells.In this study,we investigated the expression of mRNA of KIRs in decidua of women with early recurrent spontaneous abortion(RSA).METHODS The decidual tissue samples taken via uterine curetting from 30 RSA patients and 30 healthy controls and divided into three groups by pregnancy days.KIR2DL1,2DL2,2DL3,2DL4,3DL1,3DL2 mRNA expression in decidual tissue were carried out by the RT-PCR and real time quantitive RT-PCR.We used two-tailed Fisher's exact test and Mann-Whitney U tests to compare variables between groups.RESULTS 1.Transcripts of KIR2DL1,2DL2,2DL3,2DL4,3DL1,3DL2 were detectable by RT-PCR in the individuals.The percentage of transcripts of KIR did not differ between women with RSA and normal controls.However,there was an decrease in transcripts of KIR2DL2 in women with RSA when compared to normal controls.2.The KIR2DL1,2DL3 mRNA was reduced in women with RSA compared with healthy controls,but the differences were not reached statistical significance.3. The KIR2DL1,2DL3 mRNA was reduced in women with RSA compared with healthy controls between different groups,but the differences did not reach statistical significance.CONCLUSIONS KIR mRNA expression may not explain the pathogenesis of RSA.SECTIONⅢEXPRESSION OF CD158a AND CD158b IN DECIDUA OF PATIENTS WITH EARLY UNEXPLAINED RECURRENT SPONTANEOUSOBJECTIVES To investigate the expression of CD158a and CD158b receptors in decidua of women with early recurrent spontaneous abortion(RSA).METHODS The decidual tissue samples taken via uterine curetting from 30 RSA patients and 30 healthy controls and divided into three groups by pregnancy days.Immunofluorescent analysis were employed to demonstrate the expression of the CD56+,CD158a and CD158b in the deciduas.We isolated mononuclear cells from deciduas by ficoll density gradient centrifugation.Expression of the receptors CD56+,CD16-,CD158a and CD158b were detected by three-colour flow-cytometric analysis.We used two-tailed Fisher's exact test and Mann-Whitney U tests to compare variables between groups.Spearman's correlation and Kruskal-Wallis tests were used for association(all two-tailed,α＜0.05).RESULTS 1.CD56+ cells were found scattered throughout the stroma and thickly clustered around glands and spiral arteries.2.CD56+ cells were significantly decreased in women with RSA when compared with normal controls(P=0.007),the CD56+cells were greatly reduced in RSA women pregnanted no more than 7 weeks compared to normal controls(P=0.004).CD56+CD16-cells were significantly decreased in women with RSA when compared with normal controls(P＜0.05).3. CD158a as inhibitory receptor expressed by mononuclear cells was significantly decreased in women with RSA in every study group,however,CD158b were significantly decreased only in RSA women pregnanted no more than 10 weeks(P＜0.05)but no differences when pregnanted more than 10 weeks.4.CD158a expressed by CD56+CD16- cells were significantly decreased in women with RSA especially in pregnanced 7-10 weeks(P=0.010).5.The expressions of CD158a and CD158b in normal deciduas were related to CD56(r=0.623 and 0.585 respectively, P＜0.01).CONCLUSIONS 1.Decreased CD56+CD16- expression are found in mononuclear cells of deciduas of women with RSA.2.CD158a expressed by CD56+CD16- cells may explain the pathogenesis of RSA.3.The expressions of CD158a and CD158b in normal deciduas may be related to CD56.