Alterations of DNA methylation and expression of genes related to thyroid hormone metabolism in colon epithelium of obese patients.

BACKGROUND: Colorectal cancer is common among obese individuals. The purpose of the current study was to determine changes in DNA methylation status and mRNA expression of thyroid hormone receptor beta (THRB), as a tumor suppressor, and thyroid hormone inactivating enzyme, type 3 deiodinase (DIO3) genes, in human epithelial colon tissues of healthy obese individuals. METHODS: Colon biopsies were analyzed by methylation sensitive-high resolution melting (MS-HRM) to investigate promoter methylation of DIO3 and THRB, and by quantitative real-time polymerase chain reaction to assay expression of DIO3 and THRB mRNA on eighteen obese and twenty-one normal-weight healthy men. RESULTS: There was no significant difference in mean methylation levels at the THRB promoter region between the two groups. Nevertheless, obesity decreased THRB expression levels, significantly (P < 0.05; fold change: 0.19). Furthermore, obesity attenuated DNA methylation (P < 0.001) and enhanced mRNA expression of DIO3 (P < 0.05; fold change: 3). CONCLUSIONS: Our findings suggest that obesity may alter expression of THRB and DIO3 genes through epigenetic mechanism. Alterations of THRB and DIO3 expressions may predispose colon epithelium of obese patients to neoplastic transformation.

Expression Patterns of miR181a and miR30d in Patients with Breast Cancer.

Background: One of the important molecular pathways in breast cancer is the PTEN-PI3K-AKT pathway. Any change in the activity of the PTEN gene can alter the PI3K-AKT pathway. Moreover, there are subsets of genes and pathways their expression changes by post-transcriptional regulations. For instance, gene regulation alters by non-coding RNAs such as micro-RNAs as post-transcriptional regulators that prevent the expression of the target transcript. Therefore, it is essential to assess the related alterations in micro-RNA expression patterns to find out the possible causes of conversions in related transcripts and pathways such as the PTENPI3K-AKT pathway in breast cancer. Methods: To determine the expression level of miR-181a and miR-30d in 30 breast tumor samples and 30 adjacent normal samples, the RNA extraction, and cDNA synthesis was performed by RiboEx (GeneAll, Korea). Finally, the Real-Time PCR method was used for quantitative analysis of the expression levels of these miRNAs. all the experimental part of the project in done at Islamic Azad University in 2017. Results: After analyzing comparisons in the expression level of miR-181a and miR-30d in tumor and normal tissues, there was a significant increase in the expression level of miR-181a in tumor samples compared with normal samples. Moreover, the expression level of miR-30d in tumor samples reported a significant decrease in comparison with normal samples (P<0.05). Conclusion: Upregulation of miR-181a may affect the transcription of the PTEN gene resulting in the cell progress to cancer. The Downregulation of miR-30d may also lead to cancer cell growth, due to a reduction in the affecting on the CREB gene transcript.

Co-delivery of curcumin and Bcl-2 siRNA to enhance therapeutic effect against breast cancer cells using PEI-functionalized PLGA nanoparticles.

PURPOSE: Breast cancer is the second major cause of death worldwide among women. Co-delivery of anticancer drugs and nucleic acids targeting the apoptosis pathway could be a promising new approach. METHODS: In the present study, we synthesized a novel nanostructure for the co-delivery of curcumin and siRNA to breast cancer cells. Curcumin-loaded polylactic-co-glycolic acid (PLGA) was synthesized using an O/W emulsion-solvent diffusion method. It was coated with polyethylenimine (PEI) and subsequently complexed with Bcl-2 siRNA. Also, nanoparticles were characterized such as zeta potential, size distribution and drug encapsulation. Finally, the cytotoxicity of NP and Bcl-2 expression was evaluated. RESULTS: The curcumin-loaded PLGA nanoparticles were 70 nm in size, and increased to 84 nm after incorporation of PEI plus Bcl-2 siRNA. The encapsulation ratio of the drug in our nanoparticle was 78%. Cellular internalization of PLGA-CUR-PEI/Bcl-2 siRNA NPs was confirmed by fluorescence microscopy with the broadcasting of the fluorescence in the cytoplasm and into the nucleus. The results of the cell viability assay revealed that curcumin-loaded PLGA coated with PEI and Bcl-2 siRNA exhibited the highest cytotoxicity against the T47D cell line, while the siRNA decreased the Bcl-2 expression by 90.7%. CONCLUSION: The co-delivery of curcumin plus Bcl-2 siRNA with the PLGA-PEI nanosystem could be a synergistic drug carrier against breast cancer cells.

Recent findings on the role of microRNAs in genetic kidney diseases.

BACKGROUND: MicroRNAs (miRNAs) are non-coding, endogenous, single-stranded, small (21-25 nucleotides) RNAs. Various target genes at the post-transcriptional stage are modulated by miRNAs that are involved in the regulation of a variety of biological processes such as embryonic development, differentiation, proliferation, apoptosis, inflammation, and metabolic homeostasis. Abnormal miRNA expression is strongly associated with the pathogenesis of multiple common human diseases including cardiovascular diseases, cancer, hepatitis, and metabolic diseases. METHODS AND RESULTS: Various signaling pathways including transforming growth factor-ß, apoptosis, and Wnt signaling pathways have also been characterized to play an essential role in kidney diseases. Most importantly, miRNA-targeted pharmaceutical manipulation has represented a promising new therapeutic approach against kidney diseases. Furthermore, miRNAs such as miR-30e-5p, miR-98-5p, miR-30d-5p, miR-30a-5p, miR-194-5p, and miR-192-5p may be potentially employed as biomarkers for various human kidney diseases. CONCLUSIONS: A significant correlation has also been found between some miRNAs and the clinical markers of renal function like baseline estimated glomerular filtration rate (eGFR). Classification of miRNAs in different genetic renal disorders may promote discoveries in developing innovative therapeutic interventions and treatment tools. Herein, the recent advances in miRNAs associated with renal pathogenesis, emphasizing genetic kidney diseases and development, have been summarized.

Designing and determining immunogenicity of a recombinant protein due to producing a new vaccine against Enterotoxigenic Escherichia coli containing CfaE and CotD subunits.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of mortalities in developing countries due to diarrhea. Since mucosal immune responses to CFs can prevent the disease, a chimeric protein containing ETEC's CFA/I (CfaE) tip subunits and CS2 (CotD) sub-structural units is developed to produce effective vaccine. Using bioinformatics tools, the chimeric construct was analyzed and then the optimized gene was synthesized and expressed in E. coli. The recombinant protein was expressed and purified by the Ni-NTA chromatography column and confirmed by anti-his tag antibody by western blotting. Mice were immunized with recombinant protein, and the IgG and IgA antibodies' titrations of the sera were analyzed by ELISA. In addition, the immunogenicity and protective efficacy against the live ETEC bacteria in the challenge test were determined. Western blot analysis verified the chimeric protein expression of CotD-CfaE. The outcome of ELISA was a substantial improvement in the IgG antibody titer in immunized mice. In a live ETEC challenge, the survival percentage of 30% was shown for immunized mice. The developed recombinant chimeric protein could be suggested as an effective component in producing an efficient vaccine against Enterotoxigenic E. coli with other crucial subunits, different immunization route, and other factors.

Investigation the Role of Autophagy in Non-Small Cell Lung Cancer.

OBJECTIVE: Recent studies have shown the role of autophagy in different types of cancer including lung cancer. MicroRNAs are considered as key factors in regulation of autophagy related genes. miR-30d, miR-204-5p and miR-20a are regulatory markers which can suppress the expression of beclin1, LC3, bcl2 and ULK1 as their target genes and they lead to decrement of autophagy in human cancer cells. Moreover, epigenetic modifications DNA methylation has been indicated in regulation of autophagy in different stages of cancer. METHODS: In this study, the expression levels of miR-30d, miR-204-5p and miR-20a as well as their target genes were analyzed in 30 non-small cell lung cancers (NSCLCs) patients sample and adjacent normal tissues by real-time qPCR. In addition, DNA methylation of beclin1, LC3, bcl2 and ULK1 genes were assessed by MS-HRM method. RESULTS: MiR-30d (p value= 0.01) and miR-204-5p (P=0.048) significantly down-regulated in tumor samples compared to normal adjacent tissues, while there was no significant change in expression level of miR-20a. On the other hand, target genes expression level was significantly increased in NSCLC tissues, however methylation pattern of the target gene promoters, did not show any significant alteration. CONCLUSION: These results indicate roles for miR-30d, miR-204-5p as tumor suppressor genes as well as target genes as oncogenes in NSCLC patients. Although these factors may have a significant role in NSCLC progression, further studies are necessary to investigate the implications of these findings for treatment of lung cancer. 
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PHBV nanofibers promotes insulin-producing cells differentiation of human induced pluripotent stem cells.

Tissue-engineering associated techniques have long been employed to improve the various elements of the therapeutic approaches toward the more efficient ones in diabetic states. The resultant constructs comprise of the polymeric scaffolds with proper degradation rates that produce bodily compatible components, and the pluripotent cells that are highly capable of generating islet-like cells. In this study, Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated by the Electrospinning. After validation of its 3-D structure, fibers size and non-toxicity, insulin-producing cells (IPC) differentiation potential of the induced pluripotent stem cells (iPSCs) were evaluated during growing on the PHBV nanofibers in comparison with tissue culture polystyrene (TCPS). SEM analyses confirmed the 3-D and nanofibrous structure of the fabricated scaffold. The survival rate of the iPSCs cultured on the PHBV nanofibers was increased significantly compared to the cells cultured on the TCPS, which is an evidence for the non-toxicity of the nanofibers. Insulin and C-peptide secretion levels significantly increased in the differentiated iPSCs on PHBV nanofibers compared to those cells cultured on TCPS. Moreover, levels of the gene transcription and translation results revealed that insulin, Glut-2, and Pdx-1 genes and insulin protein, in IPC-differentiated iPSCs grown on PHBV nanofibers are significantly higher than those cells grown on TCPS. Taken together, these results go beyond previous reports, showing thatiPSCs-PHBV as a promising cell-copolymer construct, could potentially be applied in the pancreatic tissue engineering applications to diabetic patient treatment.

RAR-related orphan receptor A: One gene with multiple functions related to migraine.

AIMS: RAR-related orphan receptor (RORA) involves in regulation of several biological processes including inflammation and circadian rhythm that probably are involved in migraine pathophysiology. In the current study, the association between RORA rs11639084 and rs4774388 variants and susceptibility to migraine were investigated in a sample of Iranian migraine patients for the first time. METHODS: In a case-control study including 400 participants, 200 migraineurs and 200 healthy controls, genotyping of RORA rs4774388 and rs11639084 polymorphisms was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction (TP-ARMS-PCR). RESULTS: The distribution of rs4774388 C/T and T/T genotypes differed significantly between the studied groups. Moreover, an association was observed between rs4774388 and migraine under the recessive mode of inheritance (P = 0.002; OR = 1.89.; CI = 1.25-2.87). The distribution of rs11639084 alleles and genotypes was not significantly different between migraineurs and healthy controls. CONCLUSION: Current results suggest RORA, as a molecular link, may explain inflammation and circadian rhythm dysfunction in migraine. Further studies in different ethnicities are required to confirm the function of RORA in migraine development.

Association between Coronary Artery Disease and rs10757278 and rs1333049 Polymorphisms in 9p21 Locus in Iran.

BACKGROUND: Coronary arteries disease (CAD) has been recognized as one of the most common causes of death worldwide, with an estimated seven million deaths annually. METHODS: Two hundred blood samples from Iranian CAD patients and normal healthy controls were collected. CAD and the 9p21 locus variants rs1333049 and rs10757278 were analyzed for potential associations. RESULTS: No significant differences in rs10757278 and rs1333049 polymorphisms were found between patients and controls, but a significant relationship was found between rs10757278 and rs1333049 in CAD patients at the genotype level (p= 0.0323). At the haplotype level and on the basis of diplotype analysis, a significant relationship was found between patients and controls (OR= 5.16, p= 0.047, 95% CI: 1.02-26.0). In CAD patients, rs10757278 and rs1333049 were associated at locus 9p21. CONCLUSION: The inconsistency between the results of this and other studies on different CAD populations may be due to high population, different ethnicities, low prevalence of some alleles in populations, and interactions of different genes.

Different osteogenic differentiation potential of mesenchymal stem cells on three different polymeric substrates.

The function of tissue cells is strongly depends on the extracellular matrix (ECM) that can guide and support cell structure. This support plays a crucial role in the process of cell proliferation and differentiation. Herein, three different nanofibrous scaffolds that are highly attractive for tissue engineering were selected and then osteogenic related genes and protein expression patterns of human adipose-derived mesenchymal stem cells (AT-MSCs) were investigated when grown on substrates. Polycaprolactone, Poly (L-lactic acid) and Polyvinylidene-fluoride nanofibrous scaffolds were fabricated using Electrospinning method and then AT-MSCs viability and osteogenic differentiation were evaluated while cultured on them. The highest AT-MSCs survival rate when grown on the scaffolds was detected when grown on Polyvinylidene-fluoride. In addition, the highest ALP activity and mineralization were also observed in differentiated AT-MSCs has grown on Polyvinylidene-fluoride. The expression levels of Runx2, osteonectin and osteocalcin genes and osteocalcin protein in the AT-MSCs has grown on the Polyvinylidene-fluoride were also significantly higher than the rest of the scaffolds. Based on the results, it seems that since the studied substrate have a similar structural characteristics, their nature may have an important role in the stem cell's osteogenesis process, where the Polyvinylidene-fluoride piezoelectricity was a most distinguished characteristic.

Improved osteogenic differentiation of human induced pluripotent stem cells cultured on polyvinylidene fluoride/collagen/platelet-rich plasma composite nanofibers.

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.

Comparison of human-induced pluripotent stem cells and mesenchymal stem cell differentiation potential to insulin producing cells in 2D and 3D culture systems in vitro.

Diabetes is one of the most common diseases in the world that is chronic, progressive, and costly, and causes many complications. Common drug therapies are not able to cure it, and pancreas transplantation is not responsive to the high number of patients. The production of the insulin producing cells (IPCs) from the stem cells in the laboratory and their transplantation to the patient's body is one of the most promising new approaches. In this study, the differentiation potential of the induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) into IPCs was compared to each other while cultured on poly(lactic-co-glycolic) acid (PLGA)/polyethylene glycol (PEG) nanofibrous scaffold as a 3D substrate and tissue culture polystyrene (TCPS) as a 2D substrate. Although the expression level of the insulin, Glut2 and pdx-1 genes in stem cells cultured on 3D substrate was significantly higher than the stem cells cultured on 2D substrate, the highest expression level of these genes was detected in the iPSCs cultured on PLGA-PEG. Insulin and C-peptide secretions from differentiated cells were also investigated and the results showed that secretions in cultured iPSCs on the PLGA-PEG were significantly higher than cultured iPSCs on the TCPS and cultured MSCs on both PLGA-PEG and TCPS. In addition, insulin protein was also expressed in the cultured iPSCs on the PLGA-PEG significantly higher than cultured MSCs on the PLGA-PEG. It can be concluded that differentiation potential of iPSCs into IPCs is significantly higher than human MSCs at both 2D and 3D culture systems.

In vitro osteogenic differentiation of stem cells with different sources on composite scaffold containing natural bioceramic and polycaprolactone.

Stem cells can be obtained from a variety of sources. To compare the effect of cell source on the osteogenic differentiation potential, buccal fat pad-derived mesenchymal stem cells (BFP-MSCs), bone marrow-derived MSCs (BM-MSCs) and unrestricted somatic stem cells (USSCs) with different accessibility in time and region, were cultured on bioceramic (Bio-Oss®) coated electrospun polycaprolactone (PCL) scaffold (PCL-Bio). After scaffold characterization, stem cells proliferation and osteogenic differentiation were investigated by MTT and Alizarin red staining, alkaline phosphatase activity, calcium content and gene expression assays. Proliferation rate of the stem cells was not significantly different with each other, only USSCs showed significantly lower proliferation rate while cultured on PCL-Bio; although, PCL-Bio showed better proliferation support in comparison with tissue culture plate and PCL. Mineralization of the BM-MSCs was significantly higher than others, while BFP-MSCs were close to it. Highest ALP activity was detected in BFP-MSCs cultured on PCL-Bio. USSCs demonstrated higher gene expression level in three genes, although differences were not huge compared to others. According to the results and due to the availability, facilitated preparation procedure and less patients suffering, BFP-MSCs have a better choice than BM-MSCs and USSCs for use in bone tissue engineering.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing cooD and cotD genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of E. coli in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in E. coliBL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

Phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus isolates from clinical specimens, an Atomic Force Microscopic (AFM) study.

INTRODUCTION: Staphylococcus aureus is a major cause of nosocomial infections. Biofilm formation is an important factor for bacterial pathogenesis. Its mechanisms are complex and include of many genes depends on expression of icaADBC operon involved in the synthesis of a polysaccharide intercellular adhesion. AIM: The aim of study was to investigate biofilm forming ability of Staphylococcus aureus strains by phenotypic and genotypic methods. Also Atomic Force microscope (AFM) was used to visualize biofilm formation. METHODS: 140 Isolates were collected from clinical specimens of patients in Milad Hospital, Tehran and diagnosed by biochemical tests. The ability of strains to produce slime was evaluated by CRA method. For diagnosing of bacterial EPS, Indian ink staining were used and finally biofilm surface of 3 isolates observed by AFM. The prevalence of icaA and icaD genes was determined by PCR. RESULTS: By CRA method 15% of samples considered as positive slime producers, 44.28% as intermediate and 40.71% indicative as negative slime producers. 118 staphylococcus aureus strains showed a distinct halo transparent zone but 22 strains showed no halo zone. AFM analysis of Slime positive isolates showed a distinct and complete biofilm formation. In slime negative strain, there was not observed biofilm. The prevalence of icaA, icaD genes was 44.2% and 10% of the isolates had both genes simultaneously. CONCLUSION: There is a relationship between exopolysaccharide layer and biofilm formation of Staphylococcus aureus isolates. The presence of icaAD genes among isolates is not associated with in vitro formation of biofilm. AFM is a useful tool for observation of bacterial biofilm formation.

The role of parental microRNA alleles in recurrent pregnancy loss: an association study.

The medical evaluation of recurrent pregnancy loss (RPL), the occurrence of two or more consecutive pregnancy losses prior to 20th week of gestation, is mainly focused on maternal factors. However, paternally expressed genes may also play a role in implantation and placenta quality. This study aimed to investigate the possible association between parental miR-196a2C>T and miR-499aT>C polymorphisms and RPL in a case-control study including 200 RPL couples and 400 healthy men and women. Genotyping was performed using Tetra-ARMS-PCR and PCR-RFLP for miR-196a2C>T and miR-499aT>C polymorphisms, respectively. In men, the association was observed between miR-499a and RPL under dominant (P = 0.006; odds ratio [OR] = 2.36; 95% confidence interval [CI], 1.28-4.37), recessive (P < 0.0001; OR = 2.89; 95% CI, 1.92-4.36) and additive (P < 0.001; OR = 2.77; 95% CI, 1.52-5.10) models. In women, the association was found between miR-196a2 and RPL under recessive (P = 0.02; OR = 2.19; 95% CI, 1.16-4.14) and additive (P = 0.03; OR = 1.53; 95% CI, 1.04-2.27) models. Hence, evidence was provided for association of genetic variation in parental microRNA polymorphisms with RPL. Further studies are required to validate the significance of the studied genetic variations in diverse ethnic populations.

Prevalence of occult hepatitis C virus infection in the Iranian patients with human immunodeficiency virus infection.

Occult hepatitis C virus (HCV) infection is a new form of chronic HCV infection described by the presence of the genomic HCV-RNA in liver biopsy and/or peripheral blood mononuclear cell (PBMC) samples, and undetectable levels or absence of HCV-RNA and in the absence or presence of anti HCV antibodies in the plasma specimens. The aim of the present study was to evaluate the occurrence of occult HCV infection (OCI) among Iranian subjects infected with human immunodeficiency virus (HIV) using RT-nested PCR. From March 2014 until April 2015, 109 Iranian patients with established HIV infection were enrolled in this cross-sectional study. After extraction of viral RNA from the plasma and PBMC samples, HCV-RNA status was examined by RT-nested PCR using primers from the 5'-NTR. HCV genotyping was conducted using RFLP analysis. For the confirmation of HCV genotyping by RFLP method, the PCR products were sequenced. Of the 109 patients, 50 were positive for antibodies against HCV. The HCV-RNA was detected in PBMC specimens in 6 (10.2%) out of the total 59 patients negative for anti-HCV Abs and undetectable plasma HCV-RNA and also from 4 (8.0%) out of the total 50 patients positive for anti-HCV Abs and undetectable plasma HCV-RNA. HCV genotyping analysis showed that 6 (60.0%) patients were infected with HCV subtype 3a, 3 (30.0%) were infected with HCV subtype 1a and 1 (10.0%) patient was infected with HCV subtype 1b. This study revealed the incidence of OCI (9.2%) in HIV-infected Iranian patients. Hence, designing prospective studies focusing on the detection of OCI in these patients would provide more information. J. Med. Virol. 88:1960-1966, 2016. © 2016 Wiley Periodicals, Inc.

Association of plasminogen activator inhibitor-1 and angiotensin converting enzyme polymorphisms with recurrent pregnancy loss in Iranian women.

BACKGROUND: Recurrent pregnancy loss (RPL) defined by two or more failed pregnancies before 20 weeks of gestation. Several factors play a role in RPL including thrombophilic conditions which can be influenced by gene polymorphisms. Plasminogen activator inhibitor-1 (PAI-1) and angiotensin converting enzyme (ACE) genes are closely related to fibrinolytic process, embryonic development and pregnancy success. OBJECTIVE: The aim of this study was to investigate the relationship between RPL and common polymorphisms in ACE and PAI-1 genes. MATERIALS AND METHODS: In this case control study, 100 women with recurrent abortions (at least two) were selected as cases and 100 healthy women with two or more normal term deliveries without a history of abortion as controls. Total genomic DNA was isolated from blood leukocytes. The status of the PAI-1 4G/5G and ACE (D/I) polymorphism was determined by PCR-RFLP. RESULTS: Homozygosity for PAI-1 4G polymorphism was seen in 17 cases (17%), and 5 controls (5%) (p=0.006) so patients with homozygote 4G mutation were significantly more prone to RPL in contrast to control group (OR: 4.63, % 95 CI: 1.55-13.84). In addition, 7 patients (7 %), and no one from the control group, were homozygote (I/I) for ACE polymorphism (p=0.034), suggesting no significant associations between ACE D allele or DD genotype and RPL. CONCLUSION: Considering these results, because 4G/4G polymorphism for PAI-1 gene could be a thrombophilic variant leading to abortion, analysis of this mutation and other susceptibility factors are recommended in patients with RPL.

Association of kinase insert domain-containing receptor (KDR) gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure.

BACKGROUND: Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR) gene are examined that can endanger the life of the fetus in pregnant women. OBJECTIVE: The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA). MATERIALS AND METHODS: A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H), and rs2305948 (V297I) as well as one tag SNP in the intron region (rs6838752) were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP) technique. Haplotype frequency was determined for these three SNPs' genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. RESULTS: Functional SNP (rs1870377) was highly linked to tag SNP (rs6838752) (D´ value=0. 214; χ(2) = 16.44, p<0. 001). K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. CONCLUSION: In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.