Human Fertility an international, multidisciplinary journal dedicated to furthering research and promoting good practice ISSN: 1464-7273 (Print) 1742-8149 (Online) Journal homepage: http://www.tandfonline.com/loi/ihuf20 The association of renin–angiotensinogen system genes polymorphisms and idiopathic recurrent pregnancy loss Mohammad Mehdi Heidari, Mozhgan Sheikholeslami, Mahdieh Yavari, Mehri Khatami & Seyed Mohammad Seyedhassani To cite this article: Mohammad Mehdi Heidari, Mozhgan Sheikholeslami, Mahdieh Yavari, Mehri Khatami & Seyed Mohammad Seyedhassani (2017): The association of renin–angiotensinogen system genes polymorphisms and idiopathic recurrent pregnancy loss, Human Fertility, DOI: 10.1080/14647273.2017.1388545 To link to this article: http://dx.doi.org/10.1080/14647273.2017.1388545 Published online: 23 Oct 2017. Submit your article to this journal Article views: 1 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ihuf20 Download by: [UAE University] Date: 25 October 2017, At: 14:18 HUMAN FERTILITY, 2017 https://doi.org/10.1080/14647273.2017.1388545 ORIGINAL ARTICLE The association of renin–angiotensinogen system genes polymorphisms and idiopathic recurrent pregnancy loss Mohammad Mehdi Heidaria, Mozhgan Sheikholeslamia, Mahdieh Yavaria, Mehri Khatamia and Seyed Mohammad Seyedhassanib Department of Biology, Faculty of Science, Yazd University, Yazd, Iran; bDr. Seyedhassani Medical Genetic Center, Yazd, Iran Downloaded by [UAE University] at 14:18 25 October 2017 a ABSTRACT ARTICLE HISTORY The most common complication of pregnancy is idiopathic recurrent pregnancy loss (RPL). To identify the contribution of gene polymorphisms to this condition, we evaluated the association between RPL and the angiotensinogen (AGT), angiotensin receptor 1 (AGTR1) and Angiotensinogen converting enzyme (ACE). In this case–control study, the frequency of AGT (rs4762 and rs699), AGTR1 (rs5186) and ACE insertion/deletion (rs4340) polymorphisms in 202 idiopathic RPL women was compared with 210 women with no history of abortion, using tetraprimer ARMS-PCR. Polymorphisms were analysed by logistic regression analysis according to inheritance models. The CT genotype of AGT rs4762, the CC genotype of AGT rs699 and the AC genotype of AGTR1 rs5186 in a co-dominant inheritance model were associated with idiopathic RPL (OR ¼ 1.63, 95% CI ¼ 1.07–2.49 of CT versus CC; OR ¼ 5.97, 95% CI ¼ 1.28–27.82 of CC versus TT; and OR ¼ 1.99, 95% CI ¼ 1.22–3.07 of AC versus AA). The allele frequency of AGT rs699 and AGTR1 rs5186 polymorphisms, but not AGT rs4762 and ACE rs4340 polymorphisms were significantly different between women with RPL patients and controls (p ¼ 0.020, p ¼ 0.003, p ¼ 0.105 and p ¼ 0.065, respectively). These results show that there is a significant relationship between AGT (rs699) and AGTR1 (rs5186) polymorphisms and idiopathic RPL in the Iranian population. Received 11 February 2017 Accepted 28 August 2017 Introduction Recurrent pregnancy loss (RPL) is defined as the loss of three or more consecutive pregnancies before 24th gestational weeks (Akolekar, Bower, Flack, Bilardo, & Nicolaides, 2011; Rull, Nagirnaja, & Laan, 2012). In another definition, two consecutive pregnancy losses are sufficient for inclusion in the RPL group (Jauniaux, Farquharson, Christiansen, & Exalto, 2006; Practice Committee of American Society for Reproductive Medicine, 2012; Su, Lin, & Chen, 2011). The prevalence of RPL has been estimated at one in 300 pregnancies (Berry et al., 1995; Branch, Gibson, & Silver, 2010). Factors that increase the risk of RPL include parental chromosomal rearrangements, uterine anatomic abnormalities, endocrine dysfunction, familial thrombophilia, immunological disorders, diabetes mellitus, lifestyle factors and maternal infection (Christiansen, Steffensen, Nielsen, & Varming, 2008; Hefler et al., 2002; Rai & Regan, 2006). Statistical studies showed that miscarriage occurs increasingly in families with a history of idiopathic RPL. At least 50% of all abortions are frequently of unknown origin and remain unexplained. These cases are considered idiopathic CONTACT Mohammad Mehdi Heidari ß 2017 The British Fertility Society Heidarimm@yazd.ac.ir KEYWORDS Recurrent pregnancy loss; renin–angiotensin system; AGT; AGTR1; ACE; polymorphism (Andersen, Wohlfahrt, Christens, Olsen, & Melbye, 2000; Kolte et al., 2011). The renin–angiotensin system (RAS) plays an important role in many functions including regulation of the cell cycle and apoptosis and its activation is vital to many common physiological conditions including growth and differentiation during very early human organogenesis (Hall, 2003; Lv & Liu, 2015; Paul, Mehr, & Kreutz, 2006; Wegman-Ostrosky, Soto-Reyes, Vidal-Millan, & Sanchez-Corona, 2015). In the RAS pathway, angiotensinogen (AGT) is cleaved by renin to produce the inactive angiotensin I, which is then converted to angiotensin II by endothelial angiotensinconverting enzyme (ACE). This circulating pathway controls blood volume, blood pressure and homeostasis (Branch et al., 2010). According to recent findings, predisposition to thrombophilia, a tendency for blood clot formation due to genetic or non-genetic causes, has been shown to play a critical role in the development of RPL (Hyde & Schust, 2015). Genetic nucleotide changes in the RAS genes, such as AGT, angiotensinogen II type-1 receptor (AGTR1) and ACE genes have been considered as candidates that increase the risk of Department of Biology, Faculty of Science, Yazd University, Yazd, Iran 2 M. M. HEIDARI ET AL. Table 1. Clinical characteristics of patients and control groups. Groups No. Age (year) RPL Controls 202 210 27.32 ± 5.46a 29.68 ± 4.74 BMI (kg/m2) Gestation age in miscarriage time (week) No. of abortion (range) No. of pregnancy (range) 24.06 ± 3.54 22.95 ± 3.25 10.42 ± 4.32 0 4.53 ± 1.63 (3–9) 0 0 2.63 ± 0.82 (2–3) Downloaded by [UAE University] at 14:18 25 October 2017 RPL: recurrent pregnancy loss. a Data are presented as mean ± SD. thrombotic events (Kusmierska-Urban, Rytlewski, & Reron, 2013; Rigat et al., 1990; Wang et al., 2013). Thus, according to this new hypothesis, patients carrying predisposing polymorphisms in AGT, AGTR1 and ACE genes may be prone to developing RPL (Alkanli, Sipahi, Kilic, & Sener, 2014; Buchholz, Lohse, Kosian, & Thaler, 2004; Choi et al., 2011). AGT is a critical regulatory system of electrolyte homeostasis and blood pressure. The rs4762 (T207M) and rs699 (M235T) polymorphisms in the AGT gene consist of nucleotide substitutions in exon 2 that lead to threonine to methionine and methionine to threonine substitutions at positions 207 and 235 in the amino acid sequence. These polymorphisms are associated with alterations in the production of angiotensin II (Abdollahi et al., 2005; Guo, Furuta, Mizukoshi, & Inagami, 1994). In the human, the AGTR1 gene comprises five exons, and spans over 55 kb of 3q21-25 (Guo et al., 1994). The rs5186 (A1166C) polymorphism is located in the 30 untranslated region, and includes an A to C transversion at nucleotide 1166. In addition, the human ACE gene on 17q23.3 contains 26 exons and 25 introns. Recent studies have also reported the presence of a 287-bp fragment (insertion, I) or its absence (deletion, D) in intron 16, affecting the plasma level and tissue specific ACE activity. The ACE DD genotype may be associated with increased expression of plasminogen activator inhibitor-1 (PAI-1), reduced levels of fibrinolysis and risk of thrombotic events, compared with the lowest ACE activity in carriers of the angiotensin II genotype. (Fatini et al., 2003; Rigat et al., 1990; Yang et al., 2012). Several studies have explored nucleotide variations of RAS genes with numerous disorders including cardiovascular disease, several types of cancers, Alzheimer disease, hypertension, diabetic nephropathy and pregnancy complications (Bagheri, Abdi Rad, Omrani, & Nanbaksh, 2010; Ding et al., 2012; Khatami et al., 2017; Xi et al., 2011; Zhang, Zhou, & Zhang, 2013). It is thought that increased angiotensinogen levels could be a result of the association of AGT, AGTR1 and ACE polymorphisms with vascular diseases and pregnancy related diseases, such as abortion, and may increase susceptibility to RPL. Despite many studies on AGT, AGTR1 and ACE gene polymorphisms, the present research is the first to evaluate the association between these gene polymorphisms with idiopathic recurrent pregnancy loss in a population of Iranian women. Materials and methods Patients We enrolled 202 women with a history of RPL; 3–9 miscarriages with fetal loss referred to Dr. Seyed Hassani at the Medical Genetics Center, Yazd, Iran. All subjects gave written informed consent. The mean ± SD age of the women with RPL and the gestational age at the time of miscarriages were 27.32 ± 5.46 years and 10.42 ± 4.32 weeks, respectively (Table 1). Exclusion criteria were the following: anatomical disorders, abnormal karyotypes and endocrine dysfunctions. Two hundred and ten healthy women without previous history of thromboembolic disease and other known causes for RPL were selected as a control group. Genetic analysis of AGT, AGTR1 and ACE polymorphisms For analysing the AGT, AGTR1 and ACE polymorphisms, genomic DNA was isolated from blood extracted DNA stored at 20 C. Tetra-primer ARMS-PCR was used for genetic screening of polymorphisms in the AGT and AGTR1 genes. All primers were designed by a web primer design program, Primer1, accessible at http:// primer1.soton.ac.uk/primer1.html. The specificity of the primers was assayed by ‘BLAST’ program at http:// www.ncbi.nlm.nih.gov/blast (Table 2). PCR was performed in a total volume of 25 mL containing 50 ng of template DNA, 5 pmol of each outer primers, 10 pmol of each inner primers, 1 Multiplex PCR Master Mix (Yekta Tajhiz Azma Co., Tehran, Iran). PCR amplification (touchdown) was carried out at 95 C for 2 min, followed by denaturation at 95 C for 20 s, first annealing at 69–60 C (10 cycles) and remaining 25 cycles of annealing at 60 C for 1 min and extension at 72 C for 1 min, followed by a final extension for 5 min. The PCR products were electrophoresed HUMAN FERTILITY 3 Table 2. Primer sequences and length of the amplified segments in tetra-primer ARMS-PCR for AGT and AGTR1 polymorphisms and PCR for ACE I/D polymorphism. Gene AGT SNP ID: rs4762 (C521T) AGT SNP ID: rs699 (T702C) AGTR1 SNP ID: rs5185 (A1166C) ACE SNP ID: rs4340 (I/D) Primer sequence Amplicon size F1: 50 -GTCCTCTCCCCAACGGCTGTCT-30 R1: 50 -GCTGTGAACACGCCCACCAACA-30 F2: 50 -GGCCCAGCTGCTGCTGTCAAC-30 R2: 50 -GCAGCCACTTCCCCACTTCTCA-30 F1: 50 -GGACTTCACAGAACTGGATGTTG-30 R1: 50 - GCTGTGAACACGCCCACCAACA -30 F2: 50 -GGAAGACTGGCTGCTCCCTTAC-30 R2: 50 -GTGCTGTCCACACTGGCTCACA-30 F1: 50 -TGAGCACGCTTTCCTACCGC-30 R1: 50 -CCTTTGGAAACTGGACAGAAC-30 F2: 50 -AGCACTTCACTACCAAATGAACA-30 R2: 50 -TTCAATTCTGAAAAGTAGCTGAG-30 F: 50 -CCCATCCTTTCTCCCATTTCTC-30 R: 50 -CATAACAGGTCTTCATATTTCCG-30 C allele (403bp) T allele (244bp) Control band (606 bp) T allele (490bp) C allele (428bp) Control band (875 bp) A allele (354 bp) C allele (188 bp) Control band (497 bp) I allele (491 bp) D allele (203 bp) Downloaded by [UAE University] at 14:18 25 October 2017 on a 1.5% Agarose gel and stained with ethidium bromide (Figures 1 and 2). Statistical analysis Distributions of continuous variables in groups were expressed as mean ± SD, and compared with unpaired Student’s t-tests. The genotype and allele frequencies, odds ratio (OR) and 95% confidence intervals (CI) between the cases and the normal controls were calculated using logistic regression. The OR and 95% CI were used as a measure of the association between genotype and allelic frequencies and RPL. Statistical analysis was performed with the Statistical Package for Social Sciences (SPSS 16.0 for Windows; SPSS Inc., Chicago, IL) and statistical significance was accepted at a p < 0.05 level. Results In the present study, we evaluated four types of polymorphisms in the AGT, AGTR1 and ACE genes in women with a history of at least three miscarriages compared with women with no history of spontaneous abortion. The genotype distributions and allelic frequencies of AGT, AGTR1 and ACE gene polymorphisms in patients and healthy women are shown in Tables 3 and 4. The genotype distributions of all polymorphisms in the cases and controls were in Hardy–Weinberg equilibrium. Of the 202 women with RPL investigated for the AGT rs4762 (C521T) polymorphism, 63.9% had the CC genotype and 37.1% the CT genotype. The variability of AGT CT genotype in the patients was higher than in the healthy women. Accordingly, the AGT CT genotype (OR ¼ 1.63, 95% CI: 1.07–2.49, p ¼ 0.022) had a significant association with RPL. We also investigated AGT rs699 (T702C) polymorphism in these patients. About Figure 1. Agarose gel electrophoresis of the tetra-primer PCR products of AGT polymorphisms. (A) The CC genotype (rs4762) (606bp and 403bp, lanes 1, 3, 4 and 6) and the CT genotype (606 bp, 403bp and 244bp), shown in lanes 2 and 5. (B) The TT genotype (rs699) (875bp and 490bp), shown in lanes 2, 3 and 6, the TC genotype (875 bp, 490 bp and 428 bp), shown in lanes 4, 5 and 7 and the CC genotype (875 bp and 428bp), shown in lane 1. 55.9% had the TT genotype, 39.1% the TC genotype, and 5.0% the CC genotype. In a co-dominant model with univariate logistic regression analysis, the d CC genotype had a significant association with RPL 4 M. M. HEIDARI ET AL. statistically significant difference in the AGT rs4762 C allele and ACE rs4340 I allele prevalence between patients and controls (p ¼ 0.105 and p ¼ 0.65, respectively), whereas the AGT rs699 T allele prevalence was significantly higher in the patients (p ¼ 0.020). For AGTR1 rs5186 polymorphism, results of allelic frequencies analysis showed a significant association between RPL and AGTR1 rs5186 A allele (p ¼ 0.003). Downloaded by [UAE University] at 14:18 25 October 2017 Discussion Figure 2. Agarose gel electrophoresis of (A) the tetra-primer PCR products of AGTR1. The AA genotype (rs5185) (497 bp and 354), shown in lanes 1, 2, 4 and 5 and the AC genotype (497 bp, 354 bp and 188bp), shown in lanes 3 and 6. (B) the PCR products of ACE. The II genotype (rs4340) (491 bp), the I/D genotype (491 bp and 203 bp), the D/D genotype (203 bp). (OR ¼ 5.97, 95% CI: 1.28–27.82, p ¼ 0.015). According to the recessive model, an association was found in women with RPL (OR: 0.18, 95% CI: 0.04–0.85; p ¼ 0.031) (Table 3). The results of AGTR1 rs5186 (A1166C) polymorphism analysis in RPL women were as follows: 68.3% had the AA genotype, 30.2% the AC genotype, and 1.5% the CC genotype. According to the co-dominant model with univariate logistic regression analysis, the AC genotype had an association with RPL (OR ¼ 1.99, 95% CI: 1.22–3.07, p ¼ 0.004). Dominant models also showed, an association between AC genotype and RPL (dominant model OR: 1.98, 95% CI: 1.25–3.12; p ¼ 0.003) (Table 4). The results of ACE rs4340 (insertion/deletion) polymorphism analysis in RPL women showed that 25.2% had the II genotype, 50.5% the ID genotype, and 24.3% the DD genotype. There was no significant difference in rs4340 polymorphism genotype distribution between patients and controls (Table 4). Finally, we investigated the allelic frequencies of AGT, AGTR1 and ACE polymorphisms in all of the patients and healthy subjects. There was no Researchers in different countries have examined many genes in patients with idiopathic RPL, including those involved in immune tolerance, inflammation, thrombosis and the cardiovascular system (Buchholz et al., 2004). The molecular impairments in the RAS system in women developing complications during pregnancy has been widely demonstrated (Jeon et al., 2013) and show that the plasma levels of renin activity, renin concentration, angiotensinogen and angiotensin II are all lower in women with pregnancy complications as compared with pregnant women who have not experienced such problems (Mignini, Villar, & Khan, 2006). AGT, AGTR1 and ACE genes are members of the RAS complex and human linkage studies have shown that polymorphisms in these genes leads to improper functioning of the RAS system and may be risk factors for RPL (Afshariani et al., 2014). At the cellular level, these genes can impact on growth, differentiation and apoptosis in prenatal development (Johren, Dendorfer, & Dominiak, 2004; Vaiman, 2015; Vinson, Barker, & Puddefoot, 2012). In the present study, we investigated the association between genetic variation in AGT (rs4762 and rs699), AGTR1 (rs5186) and ACE (rs4340) in recurrent pregnancy loss. Our results show statistically significant associations between genotype frequencies of AGT and AGTR1 polymorphisms with Iranian idiopathic RPL. Of the few studies which have focused on the role of AGT and AGTR1 gene polymorphisms in the pathogenesis idiopathic RPL. Hefler et al. investigated the single base rs699 polymorphism of AGT, but did not observe any significant differences between genotypes and allele frequencies with idiopathic recurrent miscarriage (Hefler et al., 2002). Choi et al. compared the AGTR1 rs5186 and AGT rs699 polymorphisms in 251 Korean patients with idiopathic recurrent spontaneous abortions and 126 healthy controls. Their results indicated no significant differences between patients and controls with respect to the AGT rs699 and AGTR1 rs5186 polymorphisms (Choi et al., 2011). Fatini et al. evaluated the AGTR1 gene polymorphisms in RAS patients as possible risk factors for fetal loss. Their HUMAN FERTILITY 5 Table 3. Genotype and allele frequencies and dominant and the recessive models of inheritance analysis of AGT rs4762 and rs699 polymorphisms in idiopathic RPL women and controls. Downloaded by [UAE University] at 14:18 25 October 2017 AGT rs4762 (C521T) Co-dominant model CC CT TT Dominant model CC CT þ TT Recessive model TT CC þ CT Allele frequency C T AGT rs699 (T702C) Co-dominant model TT TC CC Dominant model TT TC þ CC Recessive model CC TT þ TC Allele Frequency T C Patients (n ¼ 202) Controls (n ¼ 210) OR (95% CI) p Value 129 (63.9%) 73 (36.1%) 0 (0.0%) 156 (74.3%) 54 (25.7%) 0 (0.0%) 1 (ref.) 1.63 (1.07–2.49) – 0.022 – 129 (63.9%) 73 (36.1%) 156 (74.3%) 54 (25.7%) 1 (ref.) 1.63 (1.07–2.49) 0.022 0 (0.0%) 202 (100%) 0 (0.0%) 210 (100%) – – – 331 (81.9%) 73 (18.1%) 336 (83.2%) 54 (16.8%) 1.37 (0.93–2.01) 0.105 113 (55.9%) 79 (39.1%) 10 (5.0%) 135 (64.3%) 73 (34.8%) 2 (0.9%) 1 (ref.) 1.30 (0.87–1.94) 5.97 (1.28–27.82) 0.210 0.015 113 (55.9%) 89 (44.1%) 135 (64.3%) 75 (35.7%) 1 (ref.) 1.42 (0.95–2.11) 0.084 10 (5.0%) 192(95.0%) 2 (0.9%) 208 (99.1%) 1 (ref.) 0.18 (0.04–0.85) 0.031 303 (73.7%) 101 (26.3%) 343 (81.7%) 77 (18.3%) 1.48 (1.06–2.07) 0.020 OR: odds ratio; CI: confidence interval. Table 4. Genotype and allele frequencies and dominant and the recessive models of inheritance analysis of AGTR1 rs5186 and ACE rs4340 polymorphisms in idiopathic RPL women and controls. AGTR1 rs5186 (A1166C) Co-dominant model AA AC CC Dominant model AA AC þ CC Recessive model CC CC þ AC Allele frequency A C ACE rs4340 (I/D) Co-dominant model DD ID II Dominant model DD ID þ II Recessive model II DD þ ID Allele frequency D I Patients (n ¼ 202) Controls (n ¼ 210) OR (95% CI) p Value 138 (68.3%) 61 (30.2%) 3 (1.5%) 171 (81.4%) 38 (18.1%) 1 (0.5%) 1 (ref.) 1.99 (1.22–3.07) 3.72 (0.38–36.14) 0.004 0.258 138 (68.3%) 64 (31.7%) 171 (81.4%) 40 (18.6%) 1 (ref.) 1.98 (1.25–3.12) 0.003 3 (1.5%) 199 (98.5%) 1 (0.5%) 209 (99.5%) 1 (ref.) 0.32 (0.03–3.01) 0.322 337 (83.4%) 67 (16.6%) 380 (90.5%) 40 (9.5%) 1.98 (1.24– 2.87) 0.003 51 (25.2%) 102 (50.5%) 49 (24.3%) 70 (33.4%) 99 (47.1%) 41 (19.5%) 1 (ref.) 1.41 (0.89–2.22) 1.64 (0.95–2.84) 0.135 0.087 51 (25.2%) 151 (74.8%) 70 (33.4%) 140 (66.6%) 1 (ref.) 1.48 (0.96–2.27) 0.072 49 (24.3%) 153 (75.7%) 41 (19.5%) 169 (80.5%) 1 (ref.) 0.76 (0.48–1.21) 0.246 204 (50.5%) 200 (49.5%) 239 (59.2%) 181 (40.8%) 1.29 (0.98–1.70) 0.065 OR: odds ratio; CI: confidence interval. results showed a significant association of the AGTR1 rs5186 CC genotype in patients with fetal loss (Fatini et al., 2000) and Buchholz et al. (2004) reported that the prevalence of genotype AGTR1 CC is similar in RPL patients and controls. The association between RPL and ACE I/D polymorphism has been examined but with incompatible results. Yang et al. (2012) described the association between RPL and ACE I/D polymorphism by metaanalysis. This research was based on 1264 patients and Downloaded by [UAE University] at 14:18 25 October 2017 6 M. M. HEIDARI ET AL. 845 healthy controls in 9 studies. Their most important finding was that the D allele of the ACE I/D polymorphism emerged as a potentially major risk factor for predisposition to RPL in some populations but not others. In the present study population a significant association between ACE polymorphism with unexplained RPL was not observed. Similar results were presented by Goodman et al. and Vettriselvi et al., but not in other studies (Aarabi et al., 2011; Bagheri et al., 2010; Goodman, Hur, Goodman, Jeyendran, & Coulam, 2009; Vettriselvi, Vijayalakshmi, Paul, and Venkatachalam, 2008). These findings indicate a possible role for ethnic differences as a result of genetic background and effects of the environment; an area for future study as is the effect of age. Since recurrent pregnancy loss is a complex disorder in which genetic backgrounds and environmental factors interact in determining its pathogenesis, further studies are therefore needed to search for potential influences of the AGTR1 and AGT gene polymorphisms in pathogenesis of RPL which may be affected via gene–gene and gene–environment interactions. Acknowledgements This research was supported by Yazd University. The authors thank all of the patients to provide blood samples for scientific research and their cooperation was essential for our work. Disclosure statement The authors declare that they have no conflict of interests. Funding This research was supported by Yazd University. References Aarabi, M., Memariani, T., Arefi, S., Aarabi, M., Hantoosh Zadeh, S., Akhondi, M.A., & Modarressi, M.H. (2011). Polymorphisms of plasminogen activator inhibitor-1, angiotensin converting enzyme and coagulation factor XIII genes in patients with recurrent spontaneous abortion. Journal of Maternal, Fetal and Neonatal Medicine, 24, 545–548. doi: 10.3109/14767058.2010.511331. Abdollahi, M., Gaunt, T., Syddall, H., Cooper, C., Phillips, D., Ye, S., & Day, I. (2005). Angiotensin II type I receptor gene polymorphism: anthropometric and metabolic syndrome traits. Journal of Medical Genetics, 42, 396–401. doi: 10. 1136/jmg.2004.026716. Afshariani, R., Roozbeh, J., Sharifian, M., Ghaedi, M., Samsami Dehaghani, A., & Ghaderi, A. (2014). Association between angiotensinogen M235T polymorphism and preeclampsia in Iranian pregnant women. Journal of Family and Reproductive Health, 8, 169–173. Retrieved from: http:// jfrh.tums.ac.ir/index.php/jfrh/article/view/190 Akolekar, R., Bower, S., Flack, N., Bilardo, C., & Nicolaides, K. (2011). Prediction of miscarriage and stillbirth at 11–13 weeks and the contribution of chorionic villus sampling. Prenatal Diagnosis, 31, 38–45. doi: 10.1002/pd.2644. Alkanli, N., Sipahi, T., Kilic, T.O., & Sener, S. (2014). Lack of association between ACE I/D and AGTR1 A1166C gene polymorphisms and preeclampsia in Turkish pregnant women of Trakya region. Journal of Gynecology and Obstetrics, 2, 49–53. doi: 10.11648/j.jgo.20140204.11. Andersen, A., Wohlfahrt, J., Christens, P., Olsen, J., & Melbye, M. (2000). Maternal age and fetal loss: Population based register linkage study. Bmj (Clinical Research Ed.), 320, 1708–1712. doi: 10.1136/bmj.320.7251.1708. Bagheri, M., Abdi Rad, I., Omrani, M.D., & Nanbaksh, F. (2010). Polymorphisms of the angiotensin converting enzyme gene in Iranian Azeri Turkish women with unexplained recurrent pregnancy loss. Human Fertility, 13, 79–82. doi: 10.3109/14647273.2010.484844. Berry, C.W., Brambati, B., Eskes, T.K.A.B., Exalto, N., Fox, H., Geraedts, J.P.M., … Hustin, J. (1995). The EuroTeam Early Pregnancy (ETEP) protocol for recurrent miscarriage. Human Reproduction, 10, 1516–1520. doi: 10.1093/HUMREP/10.6.1516. Branch, D., Gibson, M., & Silver, R. (2010). Clinical practice. Recurrent miscarriage. The New England Journal of Medicine, 363, 1740–1747. doi: 10.1056/NEJMcp1005330. Buchholz, T., Lohse, P., Kosian, E., & Thaler, C. (2004). Vasoconstrictively acting AT1R A1166C and NOS3 4/5 polymorphisms in recurrent spontaneous abortions (RSA). American Journal of Reproductive Immunology, 51, 323–332. doi: 10.1111/j.1600-0897.2004.00163.x. Choi, Y.S., Kwon, H., Kim, J.H., Shin, J.E., Choi, Y., Yoon, T.K., Choi, D.H., & Kim, N.K. (2011). Haplotype- based association of ACE I/D, AT1R 1166A > C,and AGT M235T polymorphisms in renin-angiotensin-aldosterone system genes in Korean women with idiopathic recurrent spontaneous abortions. European Journal of Obstetrics, Gynecology and Reproductive Biology, 158, 225–228. doi: 10.1016/j.ejogrb.2011.04.028. Christiansen, O.B., Steffensen, R., Nielsen, H., & Varming, K. (2008). Multifactorial etiology of recurrent miscarriage and its scientific and clinical implications. Gynecologic and Obstetric Investigation, 66, 257–267. doi: 10.1159/000149575. Ding, X., Zhang, N., Cai, Y., Li, S., Zheng, C., Jin, Y., Yu, T., Wang, A., & Zhou, X. (2012). Down-regulation of tumor suppressor MTUS1/ATIP is associated with enhanced proliferation, poor differentiation and poor prognosis in oral tongue squamous cell carcinoma. Molecular Oncology, 6, 73–80. doi: 10.1016/j.molonc.2011.11.002. Fatini, C., Gensini, F., Battaglini, B., Prisco, D., Cellai, A.P., Fedi, S., Marcucci, R., Brunelli, T., Mello, G., Parretti, E., Pepe, G., & Abbate, R. (2000). Angiotensin-converting enzyme DD genotype, angiotensin type 1 receptor CC genotype, and hyperhomocysteinemia increase first-trimester fetal-loss susceptibility. Blood Coagulation and Fibrinolysis, 11, 657–656. Retrieved from: https://insights.ovid.com/ pubmed?pmid¼1108528610. 1097/00001721-200010000-00010 Fatini, C., Gensini, F., Sticchi, E., Battaglini, B., Prisco, D., Fedi, S., Brunelli, T., Marcucci, R., Conti, A.A., Gensini, G.F., & Abbate, R. (2003). ACE DD genotype: An independent predisposition factor to venous thromboembolism. European Journal of Clinical Investigation, 33, 642–647. doi: 10.1046/ j.1365-2362.2003.01185.x. Downloaded by [UAE University] at 14:18 25 October 2017 HUMAN FERTILITY Goodman, C., Hur, J., Goodman, C.S., Jeyendran, R.S., & Coulam, C. (2009). Are polymorphisms in the ACE and PAI1 genes associated with recurrent spontaneous miscarriages? American Journal of Reproductive Immunology, 62, 365–370. doi: 10.1111/j.1600-0897.2009.00744.x. Guo, D.F., Furuta, H., Mizukoshi, M., & Inagami, T. (1994). The genomic organization of human angiotensin II type 1 receptor. Biochemical and Biophysical Research Communications, 200, 313–319. doi: 10.1006/bbrc.1994.1450. Hall, J. (2003). Historical perspective of the renin-angiotensin system. Molecular Biotechnology, 24, 27–39. doi: 10.1385/ MB:24:1:27. Hefler, L., Tempfer, C., Bashford, M., Unfried, G., Zeillinger, R., Schneeberger, C., … Huber, J. (2002). Polymorphisms of the angiotensinogen gene, the endoyhelial nitric oxide synthase gene, and the interleukin-1b gene promotor in women with idiopathic recurrent miscarriage. Molecular Human Reproduction, 8, 95–100. doi: 10.1093/molehr/8.1.95. Hyde, K.J., & Schust, D.J. (2015). Genetic considerations in recurrent pregnancy loss. Cold Spring Harbour Perspectives in Medicine, 5, a023119. doi: 10.1101/cshperspect.a023119. Jauniaux, E., Farquharson, R., Christiansen, O., & Exalto, N. (2006). Evidence-based guidelines for the investigation and medical treatment of recurrent miscarriage. Human Reproduction, 22, 2216–2222. doi: 10.1093/humrep/del150. Jeon, Y.J., Kim, J.H., Lee, B.E., Rah, H.C., Shin, J.E., Kang, H., … Kim, N.K. (2013). Association between polymorphisms in the renin-angiotensin system genes and prevalence of spontaneously aborted fetuses. American Journal of Reproductive Immunology, 70, 238–245. doi: 10.1111/aji.12110. Johren, O., Dendorfer, A., & Dominiak, P. (2004). Cardiovascular and renal function of angiotensin II type-2 receptors. Cardiovascular Research, 62, 460–467. doi: 10. 1016/j.cardiores.2004.01.011. Khatami, M., Heidari, M., Hadadzadeh, M., Schiber-Mojdehkar, B., Bitaraf Sani, M., & Houshmand, M. (2017). Simultaneous genotyping of the Rs4762 and Rs699 Polymor-phisms in angiotensinogen gene and correlation with Iranian CAD Patients with Novel Hexa-primer ARMS-PCR. Iranian Journal of Public Health, 46, 811–819. Retrieved from: http://ijph.tums.ac.ir/index.php/ijph/article/view/10085 Kolte, A.M., Nielsen, H.S., Moltke, I., Degn, B., Pedersen, B., Sunde, L., Nielsen, F.C., & Christiansen, O.B. (2011). A genome-wide scan in affected sibling pairs with idiopathic recurrent miscarriage suggests genetic linkage. MHR: Basic Science of Reproductive Medicine, 17, 379–385. doi: 10. 1093/molehr/gar003. Kusmierska-Urban, K., Rytlewski, K., & Reron, A. (2013). w układu renina-angiotensyna Wybrane polimorfizmy geno w patogenezie nadcisnienia w przebiegu cia˛_zy [Selected polymorphisms of Renin-Angiotensin System in the pathology of hypertensive disorders of pregnancy]. Ginekologia Polska, 84, 214–218. Retrieved from: http://68.indexcopernicus.com/abstracted.php?level ¼ 5&ICID ¼ 1043523 Lv, L.L., & Liu, B.C. (2015). Role of non-classical renin-angiotensin system axis in renal fibrosis. Frontiers in Physiology, 6, 117. doi: 10.3389/fphys.2015.00117. Mignini, L.E., Villar, J., & Khan, K.S. (2006). Mapping the theories of preeclampsia: The need for systematic reviews of mechanisms of the disease. American Journal of Obstetrics and Gynecology, 194, 317–321. doi: 10.1016/j.ajog.2005.08.065. 7 Paul, M., Mehr, A.P., & Kreutz, R. (2006). Physiology of local renin-angiotensin systems. Physiological Reviews, 86, 747–803. doi: 10.1152/physrev.00036.2005. Practice Committee of American Society for Reproductive Medicine. (2012). Multiple gestation associated with infertility therapy: An American Society for Reproductive Medicine Practice Committee opinion. Fertility and Sterility, 97, 825–834. doi: 10.1016/j.fertnstert.2011.11.048. Rai, R., & Regan, L. (2006). Recurrent miscarriage. Lancet (London, England), 368, 601–611. doi: 10.1016/S0140-6736(06)69204-0. Rigat, B., Hubert, C., Alhenc-Gelas, F., Cambien, F., Corvol, P., & Soubrier, F. (1990). An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. Journal of Clinical Investigation, 86, 1343–1346. doi: 10.1172/JCI114844. Rull, K., Nagirnaja, L., & Laan, M. (2012). Genetics of recurrent miscarriage: Challenges, current knowledge, future directions. Frontiers in Genetics, 3, 34. doi: 10.3389/fgene.2012.00034. Su, M.-T., Lin, S.-H., & Chen, Y.-C. (2011). Genetic association studies of angiogenesis- and vasoconstriction- related genes in women with recurrentpregnancy loss: A systematic review and meta-analysis. Human Reproduction Update, 17, 803–812. doi: 10.1093/humupd/dmr027. Vaiman, D. (2015). Genetic regulation of recurrent spontaneous abortion in humans. Biomedical Journal, 38, 11–24. doi: 10.4103/2319-4170.133777. Vettriselvi, V., Vijayalakshmi, K., Paul, S.F., & Venkatachalam, P. (2008). ACE and MTHFR gene polymorphisms in unexplained recurrent pregnancy loss. Journal of Obstetrics and Gynaecology Research, 34, 301–306. doi: 10.1111/j.14470756.2008.00792.x. Vinson, G., Barker, S., & Puddefoot, J. (2012). The reninangiotensin system in the breast and breast cancer. Endocrine Related Cancer, 19, R1–19. doi: 10.1530/ERC-110335. Wang, Z., Wang, P., Wang, X., He, X., Wang, Z., Xu, D., Hu, J., & Wang, B. (2013). Significant association between angiotensin-converting enzyme gene insertion/deletion polymorphism and risk of recurrent miscarriage: A systematic review and meta-analysis. Metabolism, 62, 1227–1238. doi: 10.1016/j.metabol.2013.03.003. Wegman-Ostrosky, T., Soto-Reyes, E., Vidal-Millan, S., & Sanchez-Corona, J. (2015). The renin-angiotensin system meets the hallmarks of cancer. Journal of the ReninAngiotensin-Aldosterone System, 16, 227–233. doi: 10.1177/ 1470320313496858. Xi, B., Zeng, T., Liu, L., Liang, Y., Liu, W., Hu, Y., & Li, J. (2011). Association between polymorphisms of the reninangiotensin system genes and breast cancer risk: A meta-analysis. Breast Cancer Research and Treatment, 130, 561–568. doi: 10.1007/s10549-011-1602-3. Yang, C., Fangfang, W., Jie, L., Yanlong, Y., Jie, W., Xuefei, L., Xuerong, Z., & Yanling, H. (2012). Angiotensin-converting enzyme insertion/deletion (I/D) polymorphisms and recurrent pregnancy loss: a meta-analysis. Journal of Assisted Reproduction and Genetics, 29, 1167–1173. doi: 10.1007/ s10815-012-9870-3. Zhang, K., Zhou, B., & Zhang, L. (2013). Association study of angiotensin II type 1 receptor: A1166C (rs5186) polymorphism with coronary heart disease using systematic meta-analysis. Journal of the Renin-Angiotensin-Aldosterone System, 14, 181–188. doi: 10.1177/1470320312447652.