Polymorphism Of MICA Gene And Its Association With Recurrent Spontaneous Abortion And Infertility

Objective:(1)To investigate the polymorphism of MICA gene in Han nationality population of Hunan province and Hubei province.(2) To investigate the associations of polymorphism of MICA gene with Recurrent Spontaneous Abortion (RSA) and infertility, and to explore further the possible molecular mechanisms on the basis of results of associations of MICA gene.Methods:(1)Samples of 162 random individuals in Hunan province and 193 random ones in Hubei province with Han nationality were genotyped by PCR-SSP to analyze the frequencies of MICA exon 2-4 alleles, which were cmoparied with the frequencies of MICA alleles of other nationalities.(2) MICA exon 2-4 alleles were genotyped in 56 RSA patients and 78 healthy controls through PCR-SSP method, and the association was analyzed at the same time.(3)MICA exon 2-4 alleles were genotyped in 214 infertile women recruited through PCR-SSP method. Anti-Chlamydia trachomatis IgG antibodies were determined by ELISA method. The associations between prevalence of Anti-Chlamydia trachomatis IgG antibodies,tubal pathology and MICA allele polymorphism were further analyzed. (4) The genotypes of HeLa cell and U373 cells were determined by sequencing method. MICA mRNA, total MICA protein and cell surface MICA protein of HeLa and U373 cells were analyzed at different time points (0h,12h,24h,36h) after Chlamydia trachomatis infection through quantity realt-ime PCR, Western blotting and flow cytometry, respectively. Cytocity of NK cell to HeLa and U373 cells was measured at different time points (0h,12h,24h,36h) after Chlamydia trachomatis infection through in vitro cytocity method.Results:(1)11 alleles were found in Han nationality population of Hunan province, among which MICA*00801/02 (40.4%) was the dominant allele. The others were MICA*00201/020 (20.1%)and MICA*010(17.3%),etc.The rarest alleles were MICA*019 (0.3%)and MICA*031 (0.3%).In Han nationality population of Hubei province,13 alleles were determined, among which MICA*00801/02 was the dominant MICA allele, accounted for 33.7%.The others were MICA*010 (18.4%), MICA*00201/020(13.9%), MICA*01201/02(9.6%), respectively. The rarest alleles were MICA*005 (0.5%)and MICA*027 (0.5%).MICA allele frequencies were significantly different between Hunan and Hubei Han nationality population and ones from Korean, Thais,Caucasian and African American populations (P<0.05).(2) 8 MICA alleles were detected in RSA group.MICA*00801/02 is the dominant MICA allele, and the rarest allele is MICA*018.9 MICA alleles were found in normal control group.MICA*00801/02 is also the dominant MICA allele, and the rarest allele is MICA*005 and MICA*01201/02.Allele frequencies of MICA exon 2-4 in RSA patients was not significantly different from ones in control group (P>0.05).(3) Women with tubal infertility more often had antibodies to Chlamydia trachomatis(66.7% versus 39.1%;OR 3.12,95% CI 1.68-5.78,P=0.004) than infertile women without tubal pathology. High level of anti-Chlamydia trachomatis IgG antibodies increased the risk of severe tubal pathology (OR 6.88,95% CI 3.34-14.20, P=0.001).The prevalence of MICA alleles were assessed and compared between infertile women with and without anti-Chlamydia trachomatis IgG antibodies.There were 9 MICA alleles found in two groups.Among them, The frequency of the MICA*008 allele was significantly higher in infertile patients without anti-Chlamydia trachomatis IgG antibodies than ones with anti-Chlamydia trachomatis IgG antibodies (38.1% versus 22.3%,P=0.0004,Pc=0.00367,OR=2.14,95%CI:1.4-3.28).The comparison of MICA alleles was also made between infertile women with or without tubal pathology.The allele distribution in infertile women with tubal pathology was similar to the distribution in women without tubal pathology. No statistical significant differences were found in the MICA allele frequencies for tubal pathology (P>0.05).Though a significant association was clearly observed between high levels of antibodies against anti-Chlamydia trachomatis antibodies and tubal pathology, no significant differences were found in the MICA allele frequencies between anti-Chlamydia trachomatis IgG antibody positive infertile women with or without tubal pathology (P>0.05).(4) HeLa cells were MICA*008 homozygote, and U373 cells MICA*001 homozygote. After Chlamydia trachomatis infection, the changes of MICA mRNA in HeLa and U373 cells were similar, which was expressed at low level at Oh time point, increased significantly highly at 12h time point, and slowly decreased during the period from 24h to 36h. After Chlamydia trachomatis infection, total MICA protein of HeLa cells never obviously decreased during the whole infection period. However, in Chlamydia trachomatis infected U373 cells, total MICA protein ascended only a little during the period from Oh to 12h time point, then began to decrease from 24h to 36h time point. After infection, MICA molecules on the HeLa cell membrane surface never obviously decreased. MICA molecules on the U373 cell membrane surface were changed unobviously during the period from Oh to 24h, but they were obviously down-regulated during 24h to 36h time points.Cytocity of NK cell to HeLa cells infected by Chlamydia trachomatis was improved gradually during the whole infection period. But this is not true in infected U373 cells.At 12h time point after infection, the cytocity of NK cell to U373 cell increased a little, then began to step down from 24h to 36h time point. Conclusions:(1)MICA exon 2-4 allele frequencies were significantly different between Han nationality population of Hunan and Hubei provinces and ones from other races.MICA*00801/02 was the dominant allele in Han nationalty population in both Hunan and Hubei province.(2) The polymorphism of MICA exon 2～4 was not associated with RSA. (3)Anti-Chlamydia trachomatis IgG antibodies were associated with tubal pathology of infertile women. (4) MICA*008 allele was significantly reverse associated with the anti-Chlamydia trachomatis IgG antibodies in infertile women.(5)No statistical significant differences were found between polymorphism of MICA allele frequencies,tubal pathology and tubal pathology induced by Chlamydia trachomatis infection.(6) The effects of Chlamydia trachomatis infection on MICA mRNA of HeLa and U373 cells were similar, while its effects on total MICA proteins and membrane MICA proteins of two cells different. (7) The discrepancy of cytocity of NK cell to Chlamydia trachomatis infected HeLa and U373 cells was found.