Study On KIR And HLA-C Genes In Patients With Unexplained Recurrent Spontaneous Abortion

Background and ObjectiveRecurrent spontaneous abortion(RSA)is one of the common complications of pregnancy,which causes severe health and emotional trauma to patients and families.The rate of recurrent spontaneous abortion is about 2-5% for couples of childbearing age and a higher proportion among women aged above 35.Known causes of RSA include cytogenetic abnormalities,anatomic irregularities,endocrine disorders,infection,atypical blood clotting,autoimmunity,sperm quality,systemic diseases and environmental factors.However,~50% of RSA cases have no clearly defined etiology.During normal pregnancy,fetal trophoblast cells infiltrate the maternal uterine vessels and release blood into the interstitial region.The outer layer of chorionic villus(combined trophoblast)is directly immersed in the maternal blood so that the fetus can easily obtain nutrition without causing the mother to produce a potential allogeneic immune response.Under normal circumstances,the uterus immune response can obtain the maternal supply of the placenta and prevent excessive invasion.The disruption of the normal balance between fluid trophoblast cells and uterine tissue flowing during the placenta can lead to various clinical problems.It has been reported that dysfunction of maternal-fetal interface immune cells and abnormal secretion of cytokines may lead to miscarriage.The unique combinations and diverse functions of the killer cell immunoglobulin-like receptors(KIRs)gene,which widely exists in the decidual Natural Killer cells(natural killer cell,NKC),and its ligand—human leukocyte antigen(HLA)encoding gene play an important role in immune rejection of pregnancy.More and more researches have reported that the KIR gene family and HLA-C gene are related to RSA.However,there are disagreements over the underlying mechanisms.We did the association study on maternal KIR and HLA-C genes in RSA women of a Han Chinese ethnicity of Henan province,which aimed to provide theoretical basis of the genetic pathogenesis of RSA,to lay the foundation for risk assessment of miscarriage recurrence,for therapeutic guidance and procreation.Materials and methods:1.Study subjects and samples: Couples with two or more than two recurrent spontaneous miscarriages prior to 28 weeks of pregnancy were recruited from Henan Provincial People's Hospital(Henan,China)between September 2015 and September 2016.We excluded patients with parental chromosomal abnormalities,fetal genetic abnormality,infections,and autoimmunity that caused miscarriage..Consequently,there were 110 couples of Han ethnicity that met the aforementioned conditions and were recruited into the experimental(RSA)group(age,20-39 years).The control group consisted of 105 healthy females(age,19-39 years old)recruited from the Henan Red Cross Blood Center between November 2015 and September 2016,who had given birth to a minimum of 1 healthy child and had no history of miscarriage.All participants were informed about the study and written informed consent was obtained.2.Experimental methods: Blood samples(5 ml)taken from all female participants were preserved in tubes containing EDTA at 4? prior to genetic evaluation.Maternal genomic DNA was isolated from 5 ml of blood using the TIANamp Genomic DNA kit(Tiangen Biotech Co.,Ltd.,Beijing,China).The quantity and quality of the DNA samples was detected with a Nano Drop? 2000 spectrophotometer at wavelengths of 230,260 and 280 nm(Thermo Fisher Scientific,Inc.,Waltham,MA,USA).KIR genotyping was performed on maternal genomic DNA using a KIR Genotyping kit(cat.no.54410D;Invitrogen;Thermo Fisher Scientific,Inc.),which detected the presence or absence of KIR genes using polymerase chain reaction sequence specific primers(PCR-SSP).Formulations of locus specific primers from the kit were used to amplify genomic DNA.When the thermocycling process was complete,5 ?l PCR products were loaded onto a 2% agarose gel using a pipette.The gel was run at a voltage of 150 V for 20 min.Following electrophoresis,the gel was placed under UV light to observe and record the results.For HLA-C1 and-C2 genotyping,the same primers were used as previously reported by Tajik.2% agarose gel preparation was performed same as the above method and ethidium bromide was used as a DNA stain.The gel was run at a voltage of 150 V for 25 min.Following electrophoresis,the gel was placed under UV light(WD?9403C;Beijing Liuyi Biotechnology Co.,Ltd.,Beijing,China)to observe and record the results.3.Statistical analysis: All data were statistically analyzed using Chi-square analysis,Pearson Chi-square continuity correction and Fisher's exact test,on SPSS version 17 software(SPSS,Inc.,Chicago,IL,USA).P?0.05 was considered to indicate a statistically significant difference.Results1.Frequency of KIR genes in the RSA and the control group.A total of 16 KIR genes were genotyped in all participants: 2DL1,2DL2,2DL3,2DL4,2DL5 A,2DL5B,2DS1,2DS2,2DS3,2DS4 FUL,2DS4DEL(2DS4 allele with a 22 base pair deletion),2DS5,3DL1,3DL2,3DL3,3DS1,2DP1,3DP1 FUL and 3DP1DEL(absence of exon 2 and its flanking intron sequences).The 4 framework genes(conservative gene positions fixed and present in each person's KIR gene),KIR2DL4,3DL2,3DL3 and 3DP1 FUL or 3DP1 DEL were present in all of the samples.The KIR pseudogene KIR2DP1 was present in all samples from the normal group,but only 99.1% of the samples from the experimental RSA group.In all participants,it was identified that compared with the activating KIR genes;the inhibitory KIR genes had a higher frequency.Among the inhibitory KIR genes,KIR2DL1 demonstrated the highest frequency,as it was present in 99.1 and 100% of the experimental and control groups,respectively.The other most frequent inhibitory KIR genes were KIR2DL3 and KIR3DL1,which were present in ?91.8% in each group.Apart from KIR2DS4 positive genotypes(KIR2DS4FUL,KIR2DS4 DEL or combined genotypes),the frequency of the activating genes was <40%.KIR2DS2 demonstrated the lowest observed carrier frequency of any activating gene,as it was present in 18.2% of the experimental group and 16.2% of the control group.No statistically significant differences were identified between the frequency of each KIR gene in the experimental group and the control group.Additionally,no significant differences were identified in the frequency of the inhibitory and activated KIR genes in each group.2.Frequency of KIR genotypes in the RSA and the control group.In the present study,the RSA group was revealed as having 19 different genotypes,whereas the control group only had 17.The frequency of KIR genotypes was similar in the RSA and control group.The most frequent genotype identified in all participants(50.9% of the RSA group and 53.3% of the control group)consisted of the 3DL1,2DL1,2DL3,2DS4,2DL4,3DL2,3DL3,2DP1 and 3DP1 genes,which corresponds with an AA genotype(genotype ID 1).The KIR AB genotypes accounted for 40.0 and 42.9% of patients in the RSA and control groups,respectively.The frequency of KIR BB genotypes was revealed as 9.1% of patients in the RSA group,compared with 3.8% of patients in the control group(P=0.127;OR,2.534;CI,0.740?8.679).The haplotypes may also be subdivided into centromeric and telomeric contents: Cen-A(2DL3 and 2DL1),Tel-A(3DL1 and 2DS4),Cen?B(2DS2,2DL2,2DL5 B and 2DS3)and Tel-B(3DS1,2DL5 A,2DS5 and 2DS1).Comparing the control group with the RSA group,no significant differences3.Frequency of HLA?C alleles in the RSA patients and the controls.The frequencies of the HLA-C alleles C1 and C2,in the RSA and control groups were analyzed.No significant differences were identified between the two groups.The patients in the RSA group who had a homozygous HLA-C2C2 had a higher frequency of the 2DS1 gene compared with the control group(5/11 vs.0/7,45.5 vs.0.0%,respectively;P=0.101).4.Stratified analysis of number of miscarriageThe experimental group was divided into two groups based on the number of abortions experienced by each participant.The frequency of KIR3DL1 gene was significantly reduced in women who had ?3 miscarriage compared to the control group(33/40 vs.101/105,82.5% vs.96.2%,P=0,015;OR,0.187;CI,0.051-0.678).However,there was no significant difference in the frequency of inhibition(KIR2DL1,2DL2,2DL3)and KIR gene activation between the two groups.When the women who had miscarried ?3 times were compared with the control group,the rate of the BB haplotype was significantly higher(P=0.025;OR,3.617;CI,1.107?11.818).Additionally,the frequency of the Tel-BB haplotype was also significantly higher in the women who have aborted ?3 times compared with the control group(P=0.025).In addition,the coexist frequency of HLA-C2C2 genotype and KIR2DS1 gene in the case group(abortion greater than 3 times)was higher than that in the control group,which was statistically significant(60.0% vs.0.0%,P=0.045).Conclusion1.The frequency of inhibitory KIR gene decreased in women with recurrent spontaneous abortion.2.The combination frequency of KIR and HLA-C activating genes in women with recurrent spontaneous abortion increased.3.This study shows that the correlation between RSA and genetic susceptibility factors,especially the KIR and HLA-C genes,was more significant in patients with recurrent spontaneous abortion?3 times.