MTHFR polymorphisms C677T and A1298C and associations with IVF outcomes in Brazilian women

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<ul><li><p>Reproductive BioMedicine Online (2014) xxx, xxxxxx</p><p> iencedi rec t . comwww.rbmonl ine .com</p><p>ARTICLE</p><p>MTHFR polymorphisms C677T andA1298C and associations with IVFoutcomes in Brazilian women</p><p> 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.</p><p>Please cite this article in press as: DElia, PQ et al. MTHFR polymorphisms C677T and A1298C and associations with IVF outcomes in Bwomen. Reproductive BioMedicine Online (2014),</p><p>Priscila Queiroz DElia a,*, Aline Amaro dos Santos b, Bianca Bianco b,c,Caio Parente Barbosa b,c, Denise Maria Christofolini b,c, Tsutomu Aoki a</p><p>a Faculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo 01221-020, Brazil; b Faculdade de Medicina do ABC,Santo Andre 09060-650, Brazil; c Instituto Ideia Fertil de Saude Reprodutiva, Santo Andre 09060-650, Brazil* Corresponding author. E-mail address: (PQ DElia).</p><p>Abstract The aim of this</p><p>Priscila Queiroz DElia is an embryologist. She obtained a degree in biomedical sciences at the UniversidadeMetodista de Sao Paulo in 2004, speciality in human assisted reproduction at the Associacao Instituto Sapientiaein 2005 and here PhD at the Faculdade de Ciencias Medicas da Santa Casa de Sao Paulo in 2012. Her majorinterests are embryology and gamete manipulation and has experience in IVF laboratory.</p><p>study was to investigate the association between MTHFR gene polymorphisms and IVF outcomes in Brazilianwomen undergoing assisted reproduction treatment. A prospective study was conducted in the Human Reproduction Department atthe ABC University School of Medicine and the Ideia Fertility Institute between December 2010 and April 2012. The patient populationwas 82womenundergoingassisted reproductioncycles. TheMTHFRpolymorphismsC677TandA1298Cwereevaluated and comparedwithlaboratory results andpregnancy rates.TheC677Tvariantwas associatedwithproportions ofmature (P = 0.006) and immature (P = 0.003)oocyteswhereas theA1298Cvariantwasassociatedwithnumberof oocytes retrieved (P = 0.044). Thepolymorphisms,whether aloneor incombination, were not associatedwith normal fertilization, good-quality embryo or clinical pregnancy rates. This study suggests that the</p><p>number and maturity of oocytes retrieved may be related to the MTHFR polymorphisms C677T and A1298C. RBMOnline</p><p> 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.</p><p>KEYWORDS: assisted reproduction, embryos, genetic, ICSI, MTHFR, polymorphism</p><p>Introduction</p><p>Currently, more than 70 million couples are affected byinfertility problems worldwide. To help these couples,</p><p>assisted reproduction treatment has been an essential toolever since 1978, when the first assisted reproduction baby,Louise Brown, was born in the UK. Over the last 35 years,great strides have been made in assisted reproduction,</p><p>razilian</p>mailto:priqueirozbiomedica@hotmail.com</li><li><p>2 PQ DElia et al.</p><p>significantly improving the success rates of the procedures(Allen and Reardon, 2005).</p><p>It is believed that folate, an important B vitamin, is ofcrucial importance in the human reproduction process (Tam-ura and Picciano, 2006). Folate deficiency can be caused bydietary or genetic factors and can compromise the functionof metabolic pathways such as amino acid metabolism,purine and pyrimidine synthesis and methylation of nucleicacids, proteins and lipids, leading to an increase in homocys-teine concentration (Jacques et al., 2001). Homocysteine,an amino acid that originates from the degradation of methi-onine, is involved in the pro-oxidant/antioxidant balance inhuman tissues (Lucock, 2000). Some of the adverse effectsof folate deficiency and accumulation of homocysteine onthe female reproductive functions include deficient cell divi-sion and production of inflammatory cytokines (Gmyreket al., 2005), and folate deficiency and hyperhomocysteina-emia can also compromise fertility and lead to pregnancycomplications because these processes are involved in thedevelopment of oocytes, preparation of endometrial recep-tivity, implantation of the embryo and pregnancy (Tamuraand Picciano, 2006). In folliculogenesis, hyperhomocysteina-emia can activate apoptosis, leading to follicular atresia(Forges et al., 2007) and affecting the maturity of oocytes(Szymanski and Kazdepka-Zieminska, 2003) and the qualityof embryos cultured in vitro (Ebisch et al., 2006). 5,10-Methylenetetrahydrofolate reductase (MTHFR) catalysesthe irreversible conversion of 5,10-methylenotetrahydrofo-late to 5-methylenotetrahydrofolate, which serves as amethyl donor in the remethylation of homocysteine tomethionine (Ueland et al., 2001).</p><p>A missense mutation in exon 4 of the MTHFR gene, acytosine-to-thymine substitution at position 677 (C677T)that converts an alanine codon to a valine codon, causesthermolability of MTHFR (Frosst et al., 1995). A secondgenetic polymorphism in exon 7 of MTHFR results from anadenine-to-cytosine substitution at position 1298 (A1298C;van der Put et al., 1998; Viel et al., 1997; Weisberg et al.,1998). The specific activity of the MTHFR enzyme is reducedby 35% in the presence of the heterozygous genotype 677CTand by 70% in the presence of the homozygous genotype677TT compared with the normal 677CC genotype (Balaghiand Wagner, 1993; De Cabo et al., 1994; Wainfan and Poiri-er, 1992). The A1298C mutation, both in the homozygousmutant (CC) and in the heterozygous (AC) state, does notseem to cause elevations in plasma homocysteine concen-tration. However, the combination of both heterozygousmutation (double heterozygote: 677CT/1298AC produces agenotype that results in a significant increase in plasmahomocysteine concentration (van der Put et al., 1998; Weis-berg et al., 1998, 2001).</p><p>Mutations in MTHFR have been associated with manydevelopmental abnormalities and pregnancy loss (Isolatoet al., 2000; Nelen et al., 1998; Zetterberg et al., 2002),but their role in fertility has not been extensively studiedso far. Previous studies have demonstrated that MTHFR isexpressed in human oocytes and preimplantation embryos(Dobson et al., 2004). Moreover, an increase in the incidenceof theMTHFR 677TT mutant genotype, albeit not significant,was demonstrated in women failing at least four cycles ofIVF, and another study found that women with the MTHFR1298CC mutant genotype who underwent IVF were less likely</p><p>Please cite this article in press as: DElia, PQ et al. MTHFR polymorphismswomen. Reproductive BioMedicine Online (2014),</p><p>to become pregnant than those with the wild-type 1298AAgenotype (Azem et al., 2004; Haggarty et al., 2006).</p><p>The aim of this study was to investigate the associationbetween the MTHFR polymorphisms C677T and A1298C andIVF outcomes in women undergoing assisted reproductiontreatment.</p><p>Materials and methods</p><p>Patient population</p><p>This study was conducted at the Human ReproductionDepartment of the ABC School of Medicine, Ideia FertilityInstitute, Santo Andre, Brazil, from December 2010 to April2012. The protocol was approved by the Ethics Committeeof the ABC School of Medicine (reference no. 097/2010,approved 13 May 2010). Written informed consent wasobtained from all participants. During this period of time,1432 IVF cycles were performed. In order to understandthe interference of the MTHFR polymorphisms C677T andA1298C, this study selected women undergoing oocyteretrieval after ovarian stimulation for assisted reproductiontreatment that presented infertility due to a male factor.The characterization of male factor infertility was madeby sperm analysis, including individuals who presentedsperm count values &gt;15.0 106/ml and 5 million); moderate OAT (motile spermato-zoa 35 million); and severe OAT (motile spermatozoa </p></li><li><p>MTHFR polymorphisms C677T/A1298C and IVF outcomes in Brazilian women 3</p><p>sound-guided transvaginal oocyte retrieval under sedationwas performed.</p><p>Oocytes, embryo selection and embryo transfer</p><p>Oocytes were classified according to the degree of nuclearmaturation: MII, mature metaphase II; MI, immature meta-phase; or PI, prophase. To perform intracytoplasmic sperminjection, only MII oocytes were used. About 1618 h afterthe procedure, normal fertilization was confirmed by iden-tifying the presence of two pronuclei using an invertedmicroscope (40; TS100-F; Nikon). Fertilization status wasclassified as normal, abnormal or not fertilized. Embryoswith 35 years on day 3 or 5 after fertilization. Luteal-phase sup-port was done with vaginal progesterone (Utrogestan; Farm-oqumica, Brazil) three times a day, starting the day afteroocyte retrieval. Pregnancies were confirmed by measuringserum bHCG concentration on day 12 after embryo transfer.Cases with positive bHCG but no identified gestational sac onultrasound examination were considered biochemical preg-nancies. To be considered as a clinical pregnancy, an intra-uterine gestational sac had to be identified by transvaginalultrasound examination after 5 or 6 weeks of pregnancy.</p><p>Genotype determination</p><p>Peripheral blood (15 ml) was collected for genomic DNAanalysis. TaqMan SNP genotyping assays for the two com-mon MTHFR polymorphisms, designed by Applied Biosystems(Foster City, CA, USA), have previously been used success-fully (Ulvik and Ueland, 2001). The assays were performedusing TaqMan Genotyping Master Mix with 50 ng DNA. ThePCR conditions were 40 cycles of denaturation at 95C for15 s and annealing/extension at 60C for 1 min, as recom-mended by the manufacturer.</p><p>Statistical analysis</p><p>All statistical analyses were performed using Stata Data ver-sion 11.0 (Stata, College Station, Texas, USA). In view of thenonnormality of the data, the ShapiroWilk test (P &lt; 0.05)was used and data were presented as median and interquar-tile range. To compare quantitative variables according togenotypes, the MannWhitney test for two variables andthe KruskalWallis test for three variables were used. A dif-ference was considered statistically significant if P &lt; 0.05.To test for associations between genotypes and qualitativevariables, the chi-squared test was used.</p><p>Results</p><p>MTHFR genotyping analysis for the loci 677 and 1298 wasperformed in 82 patients receiving treatment due to malefactor infertility. The patients were divided into twogroups: normal (with no mutated allele) and mutated (withat least one mutated allele), considering a dominant effect</p><p>Please cite this article in press as: DElia, PQ et al. MTHFR polymorphismswomen. Reproductive BioMedicine Online (2014),</p><p>of the mutated allele. Regarding the age groups, there wasno statistically significant difference (31.0 versus32.0 years, respectively).</p><p>Based on the 677 genotype evaluation, 35 patients wereclassified as normal homozygote (normal) and 47 patients asmutated homozygote or heterozygote. As for the laboratoryvariables, comparing the normal group with the mutatedgroup, no differences were found regarding the number ofoocytes retrieved (8.0 versus 9.0), normal fertilization rate(60.0% versus 57.1%), oocytes not fertilized (25.0% versus25.0%), good-quality embryos (66.7% versus 60.0%) and clin-ical pregnancy rate (33.3% versus 36.6%, respectively). How-ever, there were statistically significant differencesbetween the groups regarding mature oocytes retrieved(86.7% versus 75.0%, respectively; P = 0.006) and immatureoocytes retrieved (7.7% versus 16.7%, respectively;P = 0.003) (Table 1).</p><p>Regarding the MTHFR polymorphism 1298, 44 patientspresented normal alleles and 38 patients presented mutatedalleles. There were no statistically significant differencesbetween the normal and mutated groups with regard toage (32.0 versus 31.0 years, respectively). As for laboratoryvariables, comparing the normal group with the mutatedgroup, the number of oocytes retrieved was higher in thenormal group (10.5 versus 8.0, respectively; P = 0.044).There were no significant differences for the other labora-tory evaluations: mature oocytes retrieved (81.4% versus80.0%; P = 0.590), normal fertilization rate (57.7% versus63.3%), oocytes not fertilized (25.0 versus 25.0), good-quality embryos (52.3% versus 66.7%) and clinical pregnancyrate (39.5% versus 30.6%, respectively) (Table 1).</p><p>To determine if the concomitant occurrence of polymor-phisms in both loci (677 and 1298) could influence out-comes, the patients were divided into another threegroups: patients without either polymorphism (n = 19),patients with at least one polymorphism (n = 41) andpatients with both polymorphisms (n = 22). There were nostatistically significant differences with regard to age (31.0versus 32.0 versus 31.5 years), number of oocytes collected(11.0 versus 8.0 versus 8.0), normal fertilization rate (60.0%versus 57.1% versus 61.9%, respectively), oocytes not fertil-ized (25.0% versus 25.0% versus 25.0%, respectively),good-quality embryos (66.7% versus 50.0% versus 70.8%,respectively) and clinical pregnancy rate (33.3% versus40.0% versus 28.6%, respectively). However, there was anonsignificant tendency towards a higher proportion ofmature oocytes in the group of patients without either poly-morphism compared with those with one or two polymor-phisms (88.9% versus 81.0% versus 76.8%, respectively;Table 2).</p><p>Discussion</p><p>The MTHFR gene encodes the 5-methylenetetrahydrofolatereductase enzyme (MTHFR), which is related to folatemetabolism (Goyette et al., 1998). Consequently, polymor-phisms in MTHFR modify the circulating concentrations offolate and homocysteine. It is known that folate deficiencyand hyperhomocysteinaemia are risk factors for infertility.Furthermore, MTHFR polymorphisms have also been associ-ated with fetal malformations and spontaneous recurrent</p><p>C677T and A1298C and associations with IVF outcomes in Brazilian16/j.rbmo.2014.02.005</p></li><li><p>Table 1 Laboratory variables and clinical pregnancy rate according to presence of mutated alleles for MTHFR polymorphismsC677T and A1298C.</p><p>Variable C677T polymorphism A1298C polymorphism</p><p>Normal (n = 35) Mutated (n = 47) Normal (n = 44) Mutated (n = 38)</p><p>Age (years)a 31.0 (29.034.0) 32.0 (29.035.0) 32.0 (29.034.0) 31.0 (29.034.0)Oocytes recovered (n) 8.0 (5.013.0) 9.0 (6.015.0) 10.5 (7.516.0) 8.0 (5.012.0)a</p><p>Immature oocytes (%) 7.7 (015.4) 16.7 (7.728.6)b 11.8 (026.6) 13.8 (023.8)Mature oocytes (%) 86.7 (76.9100) 75.0 (62.584.6)c 81.4 (66.781.4) 80.0 (66.787.5)Normal fertilization (%) 60.0 (33.375.0) 57.1 (40.075.0) 57.7 (50.075.0) 63.3 (33.375.0)Not fertilized (%) 25.0 (10.040.0) 25.0 (11.137.5) 25.0 (10.036.9) 25.0 (11.140.0)Good-quality embryos (%) 66.7 (085.7) 60.0 (25.083.3) 52.3 (8.375.0) 66.7 (25.0100)Clinical pregnancy rate (%) 33.3 36.6 39.5 30.6</p><p>Values are median (interquartile range).aP = 0.044, MannWhitney test.bP...</p></li></ul>