Title: Effect of the functional VEGFA −583C/T variant on vascular endothelial growth factor levels and the risk of recurrent spontaneous miscarriage
Abstract: The association of vascular endothelial growth factor (VEGF) −583C/T variant with recurrent miscarriage (RSM) was investigated in 173 RSM cases and 248 control women. Increased minor allele and genotype frequencies of −583C/T, and reduced serum VEGF concentrations were associated with increased risk of RSM. The association of vascular endothelial growth factor (VEGF) −583C/T variant with recurrent miscarriage (RSM) was investigated in 173 RSM cases and 248 control women. Increased minor allele and genotype frequencies of −583C/T, and reduced serum VEGF concentrations were associated with increased risk of RSM. Recurrent spontaneous miscarriage (RSM), defined as three or more consecutive miscarriages before the 20th weeks of gestation, is a significant reproductive problem that affects an estimated 1%–2% of healthy women. Although abnormal regulation of immunologic, vascular, metabolic, and endocrine events have been linked to its increased risk (1Baek K.H. Choi B.C. Lee J.H. Choi H.K. Lee S.H. Kim J.W. et al.Comparison of gene expression at the feto-maternal interface between normal and recurrent pregnancy loss patients.Reprod Fertil Dev. 2002; 14: 235-240Crossref PubMed Google Scholar, 2Sierra S. Stephenson M. Genetics of recurrent pregnancy loss.Med Semin Reprod. 2006; 24: 17-24Crossref PubMed Scopus (121) Google Scholar), the majority of RSM cases remain idiopathic. It was previously shown that angiogenesis (formation of new blood vessels from preexisting blood vessels) is central to normal pregnancy (3Vuorela P. Carpen O. Tulppala M. Halmesmaki E. VEGF, its receptors and the tie receptors in recurrent miscarriage.Mol Hum Reprod. 2000; 6: 276-282Crossref PubMed Scopus (106) Google Scholar), because is it is associated with increased uterine and umbilical blood flow (4Galazios G. Papazoglou D. Tsikouras P. Kolios G. Vascular endothelial growth factor gene polymorphisms and pregnancy.J Matern Fetal Neonatal Med. 2009; 22: 371-378Crossref PubMed Scopus (50) Google Scholar). As such, reduced angiogenesis was linked to poor placental vascular development and thereby early fetal loss. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic glycoprotein produced by many tissues, including the endometrium and ovary, with angiogenic and mitogenic activities, and is a key regulator of endothelial cell proliferation. VEGF is required for the proliferation of trophoblasts, and for the development of maternal and fetal vasculature in the uterus during pregnancy (3Vuorela P. Carpen O. Tulppala M. Halmesmaki E. VEGF, its receptors and the tie receptors in recurrent miscarriage.Mol Hum Reprod. 2000; 6: 276-282Crossref PubMed Scopus (106) Google Scholar). Reduced VEGF secretion was reportedly linked to poor pregnancy outcomes, including fetal loss (3Vuorela P. Carpen O. Tulppala M. Halmesmaki E. VEGF, its receptors and the tie receptors in recurrent miscarriage.Mol Hum Reprod. 2000; 6: 276-282Crossref PubMed Scopus (106) Google Scholar, 4Galazios G. Papazoglou D. Tsikouras P. Kolios G. Vascular endothelial growth factor gene polymorphisms and pregnancy.J Matern Fetal Neonatal Med. 2009; 22: 371-378Crossref PubMed Scopus (50) Google Scholar). VEGF expression is genetically controlled, and polymorphisms in the VEGF gene were identified, some of which [+405C>G (rs2010963) and −2578C>A (rs699947)] were associated with altered VEGF production (5Lin T.H. Wang C.L. Su H.M. Hsu P.C. Juo S.H. Voon W.C. et al.Functional vascular endothelial growth factor gene polymorphisms and diabetes: effect on coronary collaterals in patients with significant coronary artery disease.Clin Chim Acta. 2010; 411: 1688-1693Crossref PubMed Scopus (40) Google Scholar, 6Watson C.J. Webb N.J.A. Bottomley M.J. Brenchley P.E.C. Identification of polymorphisms within the vascular endothelial growth factor gene: correlation with variation in VEGF protein production.Cytokine. 2000; 12: 1232-1235Crossref PubMed Scopus (640) Google Scholar). The human VEGF gene (VEGFA, OMIM 192240) is located on chromosome 6p12. Several functional polymorphisms within the VEGF promoter and 3′-UTR regions were reported to influence the risk of RSM (6Watson C.J. Webb N.J.A. Bottomley M.J. Brenchley P.E.C. Identification of polymorphisms within the vascular endothelial growth factor gene: correlation with variation in VEGF protein production.Cytokine. 2000; 12: 1232-1235Crossref PubMed Scopus (640) Google Scholar). This was highlighted by the association of the −1154G/A (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 8Coulam C.B. Jeyendran R.S. Vascular endothelial growth factor gene polymorphisms and recurrent pregnancy loss.Am J Reprod Immunol. 2008; 59: 301-305Crossref PubMed Scopus (48) Google Scholar, 9Papazoglou D. Galazios G. Koukourakis M.I. Kontomanolis E.N. Maltezos E. Association of −634G/C and 936C/T polymorphisms of the vascular endothelial growth factor with spontaneous preterm delivery.Acta Obstet Gynecol Scand. 2004; 83: 461-465PubMed Google Scholar), and +963C/T (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) polymorphisms with RSM in some, but not all (10Traina E. Daher S. Moron A.F. Sun S.Y. Franchim C.S. Mattar R. Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion.J Reprod Immunol. 2011; 88: 53-57Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), populations. In addition, the contribution of the −583C/T (rs3025020) variant to altered hippocampus VEGF concentration was previously investigated (11Blumberg H.P. Wang F. Chepenik L.G. Kalmar J.H. Edmiston E. Duman R.S. et al.Influence of vascular endothelial growth factor variation on human hippocampus morphology.Biol Psychiatry. 2008; 64: 901-903Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), although results were inconclusive. Herein we assess the association of VEGF functional polymorphism −583T/C (rs3025020) with changes in VEGF production and risk of RSM in Bahraini cases and age-matched control women. This extends the list of VEGFA variants associated with increased risk of RSM. The study was approved by Arabian Gulf University Research and Ethics Committee, and signed informed consent was obtained from each of the subjects. RSM cases comprised 173 consecutive unrelated women with three or more unexplained consecutive pregnancy losses with the same partner that occurred up to the end of the 12th week of gestation (estimated from the first day of the last normal menstrual period to the appearance of clinical symptom of pregnancy loss (confirmed by ultrasound examination). Women were excluded in case of chromosomal and anatomic abnormalities, Rh incompatibility, preclinical miscarriages, biochemical pregnancy, diabetes (glycated hemoglobin >7%), systemic or autoimmune disease, liver function abnormalities, presence of thyroid antibodies (even with normal thyroid function), hyperprolactinemia before luteal phase defects, or Rh disease. The control group consisted of 248 multiparous women who were examined as a routine check-up after uncomplicated pregnancy from the same outpatient clinics and were of Bahraini Arab ethnicity. Data collection procedures were identical for case and control patients. Control subjects were matched to cases according to age (control 31.8 ± 4.6 years, case 31.6 ± 5.3 years; P=.676) and a number of risk factors (smoking, hypertension, previous oral contraceptive use). Higher body mass index (BMI;24.8 ± 3.9 vs. 26.7 ± 5.7 kg/m2; P<.001) and lower menarche (12.7 ± 1.2 vs. 12.1 ± 0.9 years; P=.027) were seen in control versus case subjects, respectively. VEGFA −583T/C genotyping was investigated by using the allelic (VIC- and FAM-labeled) discrimination method. TaqMan assays, as assay-on-demand, were ordered from Applied Biosystems (Foster City, NJ): C_1647366_10 (rs3025020). The reaction was performed in 10 μL volume on a StepOne real-time polymerase chain reaction system, as recommended by the manufacturer (Applied Biosystems). Replicate blinded quality control samples were included to assess reproducibility of the genotyping procedure; concordance was >99%. Serum was prepared by centrifugation of coagulated blood tubes at 2000g for 10 minutes at room temperature, and it was stored in 200-μL aliquots at ≤−40°C. Samples were tested for VEGF by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems Europe, Abingdon, U.K.). Results are given as pg/mL, with assay sensitivity of 5 pg/mL. Statistical analysis was performed on SPSS v. 17.0 statistics software (SPSS, Chicago, IL). Data were expressed as mean ± SD (continuous variables), or percentages of total (categoric variables). Pearson χ2 or Fisher exact test were used to assess intergroup significance, and Student t test was used to determine differences in means. Allele frequencies were calculated by the gene-counting method, and the −583C/T polymorphism was tested for Hardy-Weinberg equilibrium by using χ2 goodness-of-fit test using HPlus 2.5 software (http://qge.fhcrc.org/hplus). Unconditional logistic regression analysis, followed by multivariate logistic regression analyses, was performed to determine the odds ratios (ORs) and 95% confidence intervals (95% CIs) associated with the risk of RSM; ORs were adjusted (aOR) for BMI and menarche. The distribution of rs3025020 VEGFA genotypes was in Hardy-Weinberg equilibrium among study participants (P=.931; χ2 = 0.008). The allele frequency of rs3025020 minor T allele [P<.001; OR 1.84 (95% CI 1.38–2.44)] was higher in RSM cases than in control subjects (Table 1). Significant differences were noted in the distribution of rs3025020 genotype between case and control subjects (Table 1), with T/T genotype being significantly enriched in RSM cases (28.3% vs. 9.3%). Taking the C/C genotype as reference, univariate analysis, carried out under assumption of additive model, showed increased risk regarding the association of rs30250202 with RSM (Table 1). This association remained significant after adjusting for BMI and menarche. There was a significant reduction in serum VEGF concentrations in C/T (242.5 ± 122.9 pg/mL) and T/T (119.7 ± 94.0 pg/mL) compared with C/C (529.8 ± 387.5 pg/mL) genotype carriers.Table 1VEGF −583C/T allele and genotype distribution, and sVEGF levels in study subjects.aStudy subjects comprised 173 RSM cases and 248 control women.FrequencybNumber (percentage of total within group).Univariate analysisMultivariate analysissVEGFdMeasured by enzyme-linked immunosorbent assay, concentrations in pg/mL.ControlRSMPOR (95% CI)PaORcAdjusted for BMI and menarche. (95% CI)MAF152 (30.6)155 (44.8)<.0011.84 (1.38–2.44)C/C119 (48.0)67 (38.7).0031.00 (reference).0011.00 (reference)529.8 ± 387.5C/T106 (42.7)57 (32.9)<.0013.78 (2.12–6.75).0013.33 (1.68–6.58)242.5 ± 122.9T/T23 (9.3)49 (28.3)<.0013.96 (2.19–7.15)<.0014.69 (2.29–9.60)119.7 ± 94.0Note: RSM = recurrent spontaneous miscarriage; VEGF = vascular endothelial growth factor; MAF = minor allele frequency; OR = odds ratio; CI = confidence interval; aOR = adjusted odds ratio.a Study subjects comprised 173 RSM cases and 248 control women.b Number (percentage of total within group).c Adjusted for BMI and menarche.d Measured by enzyme-linked immunosorbent assay, concentrations in pg/mL. Open table in a new tab Note: RSM = recurrent spontaneous miscarriage; VEGF = vascular endothelial growth factor; MAF = minor allele frequency; OR = odds ratio; CI = confidence interval; aOR = adjusted odds ratio. We have investigated whether the association between VEGFA functional variant −583C/T of the maternal genome with RSM. Results obtained demonstrated that the −583C/T variant is associated with lower circulating levels of VEGF, and increased risk of RSM in the Bahraini population examined; homozygotes had the greatest risk, followed by heterozygotes, thereby establishing a dose relation. This was the first study linking the −583C/T variant with decreased VEGF levels and increased RSM risk, which adds to growing list of VEGFA polymorphisms linked with altered VEGF secretion and risk of RSM. VEGF is an angiogenic factor involved in the maintenance of maternal and fetal functional vasculature, and decreased expression of VEGF levels precipitated by specific VEGFA variants results in defective embryogenesis and significant tissue damage in placental samples from RSM compared with normal placenta (3Vuorela P. Carpen O. Tulppala M. Halmesmaki E. VEGF, its receptors and the tie receptors in recurrent miscarriage.Mol Hum Reprod. 2000; 6: 276-282Crossref PubMed Scopus (106) Google Scholar), leading to placental detachment and thereby spontaneous abortion. Earlier studies on the association of VEGFA variants and RSM focused on −2578C/A (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 12Lee H.H. Hong S.H. Shin S.J. Ko J.J. Oh D. Kim N.K. Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertil Steril. 2010; 93: 1244-1247Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), −1154G/A (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 8Coulam C.B. Jeyendran R.S. Vascular endothelial growth factor gene polymorphisms and recurrent pregnancy loss.Am J Reprod Immunol. 2008; 59: 301-305Crossref PubMed Scopus (48) Google Scholar, 9Papazoglou D. Galazios G. Koukourakis M.I. Kontomanolis E.N. Maltezos E. Association of −634G/C and 936C/T polymorphisms of the vascular endothelial growth factor with spontaneous preterm delivery.Acta Obstet Gynecol Scand. 2004; 83: 461-465PubMed Google Scholar), −634G/C (9Papazoglou D. Galazios G. Koukourakis M.I. Kontomanolis E.N. Maltezos E. Association of −634G/C and 936C/T polymorphisms of the vascular endothelial growth factor with spontaneous preterm delivery.Acta Obstet Gynecol Scand. 2004; 83: 461-465PubMed Google Scholar, 10Traina E. Daher S. Moron A.F. Sun S.Y. Franchim C.S. Mattar R. Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion.J Reprod Immunol. 2011; 88: 53-57Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Lee H.H. Hong S.H. Shin S.J. Ko J.J. Oh D. Kim N.K. Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertil Steril. 2010; 93: 1244-1247Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and +963C/T (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 8Coulam C.B. Jeyendran R.S. Vascular endothelial growth factor gene polymorphisms and recurrent pregnancy loss.Am J Reprod Immunol. 2008; 59: 301-305Crossref PubMed Scopus (48) Google Scholar, 9Papazoglou D. Galazios G. Koukourakis M.I. Kontomanolis E.N. Maltezos E. Association of −634G/C and 936C/T polymorphisms of the vascular endothelial growth factor with spontaneous preterm delivery.Acta Obstet Gynecol Scand. 2004; 83: 461-465PubMed Google Scholar, 10Traina E. Daher S. Moron A.F. Sun S.Y. Franchim C.S. Mattar R. Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion.J Reprod Immunol. 2011; 88: 53-57Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Lee H.H. Hong S.H. Shin S.J. Ko J.J. Oh D. Kim N.K. Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertil Steril. 2010; 93: 1244-1247Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) polymorphisms. An ethnic heterogeneity in the association of specific VEGFA variant with RSM was evident from these studies, exemplified by the association of +963C/T with increased RSM risk in North Indians (7Aggarwal S. Parveen F. Faridi R.M. Phadke S. Borkar M. Agrawal S. Vascular endothelial growth factor gene polymorphisms in North Indian patients with recurrent miscarriages.Reprod Biomed Online. 2011; 22: 59-64Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and Greeks (9Papazoglou D. Galazios G. Koukourakis M.I. Kontomanolis E.N. Maltezos E. Association of −634G/C and 936C/T polymorphisms of the vascular endothelial growth factor with spontaneous preterm delivery.Acta Obstet Gynecol Scand. 2004; 83: 461-465PubMed Google Scholar), but not Brazilians (10Traina E. Daher S. Moron A.F. Sun S.Y. Franchim C.S. Mattar R. Polymorphisms in VEGF, progesterone receptor and IL-1 receptor genes in women with recurrent spontaneous abortion.J Reprod Immunol. 2011; 88: 53-57Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) or Koreans (12Lee H.H. Hong S.H. Shin S.J. Ko J.J. Oh D. Kim N.K. Association study of vascular endothelial growth factor polymorphisms with the risk of recurrent spontaneous abortion.Fertil Steril. 2010; 93: 1244-1247Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Interestingly, the −1154G/A variant was uniformly associated with RSM in Asians and Europeans. Variability within the VEGFA gene was shown to influence VEGF protein expression (6Watson C.J. Webb N.J.A. Bottomley M.J. Brenchley P.E.C. Identification of polymorphisms within the vascular endothelial growth factor gene: correlation with variation in VEGF protein production.Cytokine. 2000; 12: 1232-1235Crossref PubMed Scopus (640) Google Scholar). Apart from a lone report investigating its effect on hippocampus VEGF concentration (11Blumberg H.P. Wang F. Chepenik L.G. Kalmar J.H. Edmiston E. Duman R.S. et al.Influence of vascular endothelial growth factor variation on human hippocampus morphology.Biol Psychiatry. 2008; 64: 901-903Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the functionality of the −583C/T polymorphism not been previously reported. Although the present study describes only the association between the −583 polymorphism and VEGF levels and altered RSM risk, it is tempting to speculate that reduced circulating levels of VEGF seen in homozygous −583T/T more so than heterozygous −583C/T genotype carriers may be attributed to the possibility that the −583 variants negatively affect transcription rate, in part by abolishing a DNA binding site (containing −583 site) required for optimal VEGF transcription (6Watson C.J. Webb N.J.A. Bottomley M.J. Brenchley P.E.C. Identification of polymorphisms within the vascular endothelial growth factor gene: correlation with variation in VEGF protein production.Cytokine. 2000; 12: 1232-1235Crossref PubMed Scopus (640) Google Scholar). This in turn results in reduced VEGF production, leading to increased risk of RSM. In conclusion, the VEGFA −583C/T polymorphism modulates VEGF expression and influences the risk of RSM, thereby confirming earlier studies linking altered VEGF levels with RSM and extending the list of VEGF variants associated with RSM. The strengths of this study lie in the number of subjects involved, in correlating VEGFA genotype with VEGF production, and in using regression analysis models to assess the contribution of the −583C/T variant to RSM. A limitation of our study is that we relied on serum VEGF measurements, and could not ascertain their expression in decidual and placental tissues. Parallel studies on other populations of related and distant ethnic origin are needed to confirm (or alternatively rule out) the association of −583C/T VEGFA variant with RSM.