{"paper_id":"66b61b59-0862-4bf6-8a9e-f37c11c95587","body_text":"Infertility has increased in recent decades, and the estimated prevalence of\ninfertility is 10-15 percent in the general reproductive-age couple ( Chan  et al ., 2016a ;  Zeng  et al ., 2017 ). Recurrent\nImplantation Failure (RIF) is considered to be a repetitive failure to achieve\npregnancy following the transfer of high-quality embryos after several\n in-vitro  fertilization (IVF) treatment attempts ( Jung  et al ., 2016 ).\nThe causes of RIF are still poorly known. Its etiology can be divided into three\ncategories: decreased endometrial receptivity, embryonic defects, and unsynchronized\nrelation between maternal and embryonic tissue ( Vagnini  et al ., 2015 ). Uterine abnormalities, including\nthin endometrium and the altered expression of cell adhesion molecules, may lead to\na decrease in endometrial receptivity. Genetic abnormalities like chromosome\nanomalies and sperm defects play a vital role in RIF ( Boudjenah  et al ., 2012 ). It is demonstrated that low\nexpression of apoptosis and angiogenesis genes is related to implantation failure\n( Lledo  et al .,\n2014 ).\nThe vascular endothelial growth factor (VEGF) is an essential factor in the\nangiogenesis and an initial regulator of endothelial cells proliferation. In the\nearly gestation, VEGF is crucial for oocyte maturation, trophoblastic growth,\nimplantation, and fetal, placental angiogenesis in the uterus ( Xu  et al ., 2015 ). The gene which codes VEGF on\nchromosome 6p21.3 involves a 14kb coding region with eight exons and seven introns\n( Samli  et al ., 2012 ).\nUntil now, seven isoforms of human VEGF have been identified ( Galazios  et al ., 2009 ). The P53 gene has an\nessential role in fertility by regulating the expression of the leukemia inhibitory\nfactor (LIF) gene, which plays a necessary role in the implantation process ( Winship  et al ., 2018 ). Human\northologous of murine double minute 2 (MDM2) is one of the main negative regulators\nof P53 ( Chan  et al ., 2016a ).\nThe MDM2 gene contains 12 exons and three promoters, which are located on the long\narm of chromosome 12 in humans, with a length of 34 kb ( Chan  et al ., 2016b ).\nNumerous studies have identified a variety of polymorphisms in the VEGF gene, some of\nthese include -2576 A/C and -1154 G/A in the promoter region, and +405 G/C in the\n5’UTR and regions of 936 C/T in the 3’UTR, associated with the alteration of VEGF\nexpression ( Sajjadi  et al .,\n2020 ). Researchers also looked at the role of VEGF polymorphisms in\nembryonic implantation. For example, the association of -1154G/A polymorphism and\nrepeated implantation failure after IVF has been reported ( Boudjenah  et al ., 2012 ). The MDM2 SNP309\ncontains the T to G conversion in the regulatory region of the first intron. In\nSNP309, the G allele tends to increase the binding of the SP1 activator\ntranscription factor to the DNA, which increases the level of MDM2 and decreases the\np53 activity. Also, the SNP309 is located in an area that is directly regulated by\nestrogen signaling. Estrogen preferably stimulates the transcription of MDM2 with\nthe SNP309G allele ( Hu  et al .,\n2011 ). To our knowledge, there is no study about the association of VEGF\nand MDM2 polymorphisms with RIF in Iranian women.\nBecause implantation is a complex process and there are many pathways involved, the\nstudy of one pathway is not responsive to repeated implantation failure. Therefore,\ntwo genes that are involved in different mechanisms were selected for this study,\nwhich purpose was to investigate the association between rs1570360 and rs2010963\npolymorphisms in the VEGF gene and the rs2279744 polymorphism in the MDM2 gene in\nwomen with the risk of recurrent failures in IVF technique, for the first time in\nIranian women.\n\nWe had 120 women from Arab and Fars ethnicity included in this study (60 women\nwith a history of recurrent implantation failure after IVF embryo transfer and\n60 fertile women with at least one live birth served as controls). Written\ninformed consent was obtained from all the participating women, and the\nInstitutional Ethics Committee of the Ahvaz Jundishapour University of Medical\nSciences confirmed the study (Ethics code: IR.AJUMS.REC.1396.572). RIF was\ndefined in this study as a transfer of a cumulative total of four good quality\nembryos with less than 5 mIU/ml hCG serum levels 14 days after embryo transfer.\nAll transferred embryos were considered to be of good quality by the\nembryologist. Patients with a history of autoimmune disease, abnormal uterine\nanatomy - based on a laparoscopic examination, ovarian cysts, endometriosis, and\nchromosome abnormalities were excluded from the study.\nDNA was isolated from whole blood samples (5 ml collected into EDTA tubes) taken\nfrom RIF patients and control subjects. The DNA extraction was performed by a\nDNA Extraction Kit (Favorgen Biotech, Taiwan) according to the manufacturer’s\nprotocol. Genotyping was carried out by restriction fragment length polymorphism\n(RFLP) after polymerase chain reaction (PCR) and confirmed by Sanger sequencing.\n Supplementary Table 1  shows the\ndetailed information about the PCR-RFLP procedure ( Baitello  et al ., 2016 ;  Avirmed  et al ., 2017 ;  Papazoglou  et al .,\n2005 ).\nThe differences in genotype frequencies of the VEGF and MDM2 gene polymorphisms\nwere analyzed using the Chi-square test. The association between these\npolymorphisms and RIF was calculated with 95% odds ratios (OR) and confidence\nintervals (CI). A  p -value <0.05 was considered statistically\nsignificant. The Mann-Whitney U-test was used to analyze the patients’\ncharacteristics. The Hardy-Weinberg equilibrium was tested in the RIF patients\nand controls separately. All statistical analyses were performed using the SPSS\nsoftware version 22.0 (SPSS, Chicago, IL, USA). The haplotype frequencies were\nestimated using the Haploview 4.1.\n\nA total of 120 women including 60 women with a history of recurrent implantation\nfailure after IVF embryo transfer (mean age ±SD = 31.72±4.71) and 60\nfertile women with at least one live birth, served as controls (mean age ± SD\n= 31.91±6.3), were enrolled in this study. There was no significant\ndifference between the cases and controls concerning the mean age\n( p -value=0.8). Also, the mean patients’ BMIs (26.5±4.06)\nwere not significantly different ( p -value=0.6) from the controls\n(26.8±4.6). The mean of recurrent implantation failure was 1.72±0.9\n(mean±SD) in the patient group.\nThe genotype and allelic frequencies obtained in our study were consistent with the\nHardy-Weinberg Equilibrium (HWE) for rs2010963 and rs2279744 in cases\n( p -value=0.005 and  p -value=0.001,\nrespectively) and controls ( p -value=0.03 and 0.001, respectively)\n( Table 1 ). However, for the rs1570360,\nthe genotype and allelic frequency were consistent with HWE only for the case group\n( p -value=0.001), while for the controls, it was not in\nequilibrium with the Hardy-Weinberg law ( p -value=0.9). It is\nprobably due to the small sample size and high consanguinity, especially Arab\nethnicity, in our study population.\nGenotype and allelic frequencies of VEGF and MDM polymorphisms in recurrent\nimplantation failure and control subjects.\nTable 1  also represents the distribution of\nthe rs1570360, rs2010963, and rs2279744 genotypes and allelic frequencies for cases\nand controls.\nThe frequency of AA (wild type), AG and GG of VEGF rs1570360 was 8.3%, 86.7%, and 5%\nin patients, then 11.7%, 43.3% and 45% in controls, respectively. There was a\nsignificant association between the heterozygous AG genotype of VEGF rs1570360 gene\npolymorphism and RIF [OR=6.4 (1.22-33.64),  p -value=0.001].\nThe genotypic frequencies of VEGF rs2010963 showed that CC, CG, and GG was 21.7%,\n75%, and 3.3% in patients, on the other hand 16.7%, 73.3%, and 10% in controls,\nrespectively. No significant association was found between VEGF rs2010963 gene\npolymorphism and the risk of RIF. However, the variant (CC and CG) genotype\nfrequencies were more common in the cases as compared with controls. The frequency\nof the MDM2 rs2279744 TG genotype was more prevalent in the RIF group, but it was\nnot statistically different [OR=1.18 (0.3-3.7),  p -value=0.7]\nbetween controls and the RIF group.\nWe checked the genetic model of VEGF and MDM2 polymorphisms in RIF patients and\ncontrol subjects to see which model is significantly associated with the risk of RIF\n( supplementary material Supplementary Table\n2 ). The dominant model of inheritance showed significant association with\nvariant VEGF rs1570360 genotypes [OR=15.5 (4.3 - 55.2),\n p -value=0.0001]; while the recessive model genotypes did not show\nany significant association [OR= 0.6(0.2 - 2.3),  p -value=0.5]. The\ngenotype of the VEGF rs2010963 polymorphism did not associate significantly with RIF\nunder any of the inheritance models ( Table\n2 ). For the MDM2 rs2279744 TG genotype variant the dominant model of\ninheritance did not represent significant relationship with RIF [OR=0.8 (0.2-2.6),\n p -value=0.1].\nHaplotype frequencies of VEGF (rs1570360/rs2010963) polymorphisms in\nrecurrent implantation failure.\nTo check, whether there was a synergistic effect of polymorphic site interactions on\nthe risk of RIF, we analyzed the allele combination of the two VEGF polymorphisms\n( Table 2 ). Our analyses indicated that\nrs1570369/rs2010963 G-G and A-C haplotypes applied synergistic effect on an elevated\nrisk of RIF [RIF (OR=0.2 (0.075 - 0.56),  p -value=0.00089, OR=4.06\n(1.45 - 11.32),  p -value=0.004, respectively].\n\nThis investigation deciphered the possible association of MDM2 (rs2279744) and VEGF\n(rs2010963, rs1570360) polymorphisms with RIF development in Iranian women. The\ngenotype and allelic frequency, which is observed only in the polymorphism of the\n-1154 G/A VEGF gene, demonstrated a significant difference. These data suggested\nthat the presence of the A allele is associated with RIF. However, the analysis of\nthe rs2010963 and rs2279744 in VEGF and MDM2 gene polymorphisms respectively, did\nnot reveal any significant difference between genotypes in the RIF and control\ngroups.\nIn this study, we think that VEGF and MDM2 polymorphisms are confined to endometrial\nfactors for implantation failure because patients with uterine anomaly and related\nmedical diseases were excluded, and high-quality embryos were selected for\nimplantation.\nPrevious reports showed that VEGF concentration increased during the late secretory\nand pre-menstrual phases ( Sugino  et\nal ., 2002 ).  Kong  et\nal . (2008 ) reported that the rs1570360 (-1154 G/A)\npolymorphism affected the serum VEGF level marginally. Goodman  et\nal . (2008) showed that in females who are experiencing recurrent\nimplant failure VEGF rs1570360 (-1154A /A) has been observed more frequently than in\nfertile females. Therefore, a “low expression” genotype such as VEGF rs1570360\n(-1154G/A) would lead to the low concentration of the VEGF protein, which leads to\nlower angiogenesis, cytotrophoblast invasion and survival in implant failure.\nBecause the blastocyst was shown at the implantation time as a source of VEGF, the\ncorrelation between RIF and partner genotypes would be interesting to examine.\nIndeed, the reduction in endometrial VEGF could be modulated by a G/G genotype in\nthe partner because all embryos would be A/G genotype. Conversely, an increase in\nRIF might be seen with A/A genotype partners. Moreover, the authors demonstrated\nthat Polymorphisms of the VEGF gene had been correlated with variation in VEGF\nprotein production ( Goodman  et al .,\n2008 ;  Kang  et al .,\n2009 ). Taken together, these data suggest the role of VEGF in\nangiogenesis and endometrial decidualization, which is essential for a successful\npregnancy. In contrast to our finding, -1154A/G polymorphism in the VEGF gene had an\ninsignificant association with increased risk of RIF in the Brazilian population,\naccording to  Vagnini  et al .\n(2015 ).\nGenotype frequency of VEGF rs2010963 between RIF patients and controls was not\nsignificantly different ( p -value=0.7). Conversely, in a study,\n Alidadiani & Salehi (2017)  showed a\nhigher frequency of the VEGF +405 GG genotype and the G allele in a patient with a\nhistory of IVF-ET failure. Also,  Boudjenah  et\nal . (2012 ) reported a higher frequency in RIF subjects after\nICSI-ET than in controls. Inconsistent with our result, a study in the Arab\npopulation showed that the serum VEGF concentration was not affected by rs2010963\ngenotypes ( Al-Habboubi  et al .,\n2011 ). The controversy between different studies indicated that the VEGF\nserum level depends on population and ethnicity.\nBased on the effects of genotype and allele frequency of MDM2 rs2279744 on missed\nabortion and IVF failure ( Chan  et\nal ., 2016a ;  Fang  et\nal ., 2009 ), we checked to see the association of this\npolymorphism with RIF. Our analysis revealed no significant difference between RIF\nand control subjects, which is consistent with Ying Chan et al. The outcomes of some\ninvestigation about the association of VEGF rs2010963, MDM2 rs2279744 polymorphisms\nwith recurrent implantation failure risk have been contradictory. For example, two\nrecent studies indicated an association with increased risk of RIF ( Kang  et al ., 2009 ;  Alidadiani & Salehi, 2017 ), while studies in\nBulgaria and China found no association with RIF risk ( Chan  et al ., 2016a ;  Fang  et al ., 2009 ). Further studies with a\nlarge sample size across diverse ethnic communities should be performed to elucidate\nthe association between rs1570360, rs2010963, and rs2279744 (MDM2 SNP309 T/G)\npolymorphisms and RIF in the Iranian population.\n\nIn general, this is the first study, which investigates the association between\nrs1570360, rs2010963, and rs2279744 and the prevalence of RIF in the Iranian\npopulation. The findings obtained in the present study demonstrated that the VEGF\nrs1570360 polymorphism is associated with the risk of RIF in the studied population.\nIn particular, the distribution of A allele was significantly higher in RIF cases\nthan controls and it might affect the pathogenesis of recurrent implantation\nfailure. This polymorphism may be useful biomarker for predicting individual\nsusceptibility to RIF. We did not find significant association between rs2010963,\nand rs2279744 polymorphisms and RIF. Considering the limitations mentioned above,\nfurther studies are required to verify the functional significance of the VEGF -\n1154A allele and how it participates in the pathogenesis of RIF","source_license":"CC-BY-4.0","license_restricted":false}