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ARTICLE

MTHFR polymorphisms C677T andA1298C and associations with IVFoutcomes in Brazilian women

http://dx.doi.org/10.1016/j.rbmo.2014.02.0051472-6483/ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: D’Elia, PQ et al. MTHFR polymorphisms C677T and A1298C and associations with IVF outcomes in Bwomen. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.1016/j.rbmo.2014.02.005

Priscila Queiroz D’Elia a,*, Aline Amaro dos Santos b, Bianca Bianco b,c,Caio Parente Barbosa b,c, Denise Maria Christofolini b,c, Tsutomu Aoki a

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: priqueirozbiomedica@hotmail.com (PQ D’Elia).

Abstract The aim of this

Priscila Queiroz D’Elia 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.

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

number and maturity of oocytes retrieved may be related to the MTHFR polymorphisms C677T and A1298C. RBMOnline

ª 2014, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: assisted reproduction, embryos, genetic, ICSI, MTHFR, polymorphism

Introduction

Currently, more than 70 million couples are affected byinfertility problems worldwide. To help these couples,

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,

razilian

2 PQ D’Elia et al.

significantly improving the success rates of the procedures(Allen and Reardon, 2005).

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).

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).

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

Please cite this article in press as: D’Elia, PQ et al. MTHFR polymorphismswomen. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.10

to become pregnant than those with the wild-type 1298AAgenotype (Azem et al., 2004; Haggarty et al., 2006).

The aim of this study was to investigate the associationbetween the MTHFR polymorphisms C677T and A1298C andIVF outcomes in women undergoing assisted reproductiontreatment.

Materials and methods

Patient population

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 >15.0 · 106/ml and <50% motility. Thisselection produced a group of 402 couples presenting malefactor infertility. Further selection criteria for this studywere: female age �37 years, normal serum FSH and prolac-tin concentrations, no minimal or mild endometriosis, notubal factor, no unexplained infertility or recurrent preg-nancy loss. This rigorous selection produced a group of 82patients who met all criteria and had all informationcompleted.

Sperm count

Evaluation of sperm samples was made according to theWorld Health Organization to analyse concentration, motil-ity and morphology. Based on this analysis, oligoasthenote-ratospermia (OAT) were classified: mild OAT (motilespermatozoa >5 million); moderate OAT (motile spermato-zoa 3–5 million); and severe OAT (motile spermatozoa <3million). Sperm morphology were evaluated considering�4% normal sperm morphology as normozoospermic and<4% as teratozoospermic (WHO, 2010).

Ovarian stimulation

The protocol was performed by using exogenousrecombinant gonadotrophins (100–200 IU/day; Puregon;Schering-Plough, New Jersey, USA), starting on cycle day2. When the leading follicle reached 14 mm in diameter,endogenous LH release was suppressed by a gonadotrophin-releasing hormone antagonist analogue (ganirelix acetate;Orgalutran; Schering-Plough) until the day of human cho-rionic gonadotrophin (HCG) administration. When theleading follicle reached 18 mm in diameter, a total doseof 250 lg HCG was administered (Ovidrel; Merck-Serono,Darmstadt, Germany), and 36 h thereafter, ultra-

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MTHFR polymorphisms C677T/A1298C and IVF outcomes in Brazilian women 3

sound-guided transvaginal oocyte retrieval under sedationwas performed.

Oocytes, embryo selection and embryo transfer

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 16–18 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 <20% fragmentation and an adequate number of cellsfor transfer were considered good quality (Veeck, 1999).

Up to two embryos were transferred in patients aged�35 years and a maximum of three embryos in patients aged>35 years on day 3 or 5 after fertilization. Luteal-phase sup-port was done with vaginal progesterone (Utrogestan; Farm-oquımica, 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.

Genotype determination

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 95�C for15 s and annealing/extension at 60�C for 1 min, as recom-mended by the manufacturer.

Statistical analysis

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 Shapiro–Wilk test (P < 0.05)was used and data were presented as median and interquar-tile range. To compare quantitative variables according togenotypes, the Mann–Whitney test for two variables andthe Kruskal–Wallis test for three variables were used. A dif-ference was considered statistically significant if P < 0.05.To test for associations between genotypes and qualitativevariables, the chi-squared test was used.

Results

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

Please cite this article in press as: D’Elia, PQ et al. MTHFR polymorphismswomen. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.10

of the mutated allele. Regarding the age groups, there wasno statistically significant difference (31.0 versus32.0 years, respectively).

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).

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).

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).

Discussion

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

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Table 1 Laboratory variables and clinical pregnancy rate according to presence of mutated alleles for MTHFR polymorphismsC677T and A1298C.

Variable C677T polymorphism A1298C polymorphism

Normal (n = 35) Mutated (n = 47) Normal (n = 44) Mutated (n = 38)

Age (years)a 31.0 (29.0–34.0) 32.0 (29.0–35.0) 32.0 (29.0–34.0) 31.0 (29.0–34.0)Oocytes recovered (n) 8.0 (5.0–13.0) 9.0 (6.0–15.0) 10.5 (7.5–16.0) 8.0 (5.0–12.0)a

Immature oocytes (%) 7.7 (0–15.4) 16.7 (7.7–28.6)b 11.8 (0–26.6) 13.8 (0–23.8)Mature oocytes (%) 86.7 (76.9–100) 75.0 (62.5–84.6)c 81.4 (66.7–81.4) 80.0 (66.7–87.5)Normal fertilization (%) 60.0 (33.3–75.0) 57.1 (40.0–75.0) 57.7 (50.0–75.0) 63.3 (33.3–75.0)Not fertilized (%) 25.0 (10.0–40.0) 25.0 (11.1–37.5) 25.0 (10.0–36.9) 25.0 (11.1–40.0)Good-quality embryos (%) 66.7 (0–85.7) 60.0 (25.0–83.3) 52.3 (8.3–75.0) 66.7 (25.0–100)Clinical pregnancy rate (%) 33.3 36.6 39.5 30.6

Values are median (interquartile range).aP = 0.044, Mann–Whitney test.bP = 0.003, Mann–Whitney test.cP = 0.006, Mann–Whitney test.

Table 2 Laboratory variables and clinical pregnancy rate according to presence or absence of MTHFR polymorphisms C677Tand A1298C.

Variables No polymorphism (n = 19) At least one polymorphism (n = 41) Both polymorphisms (n = 22)

Age (years) 31.0 (29.0–34.0) 32.0 (29.0–34.0) 31.5 (30.0–35.0)Oocytes recovered (n) 11.0 (8.0–13.0) 8.0 (6.0–16.0) 8.0 (6.0–12.0)Mature oocytes (%) 88.9 (76.9–100) 81.0 (62.5–90.0) 76.8 (66.7–83.3)Normal fertilization (%) 60.0 (36.3–83.3) 57.1 (50.0–75.0) 61.9 (40.0–75.0)Not fertilized (%) 25.0 (8.3–43.8) 25.0 (12.5–36.4) 25.0 (8.3–40.0)Good-quality embryos (%) 66.7 (0–70.0) 50.0 (25.0–80.0) 70.8 (25.0–100)Clinical pregnancy rate (%) 33.3 40.0 28.6

Values are median (interquartile range). There were no statistically significant differences between the groups.

4 PQ D’Elia et al.

pregnancy loss (Isolato et al., 2000; Nelen et al., 1998;Zetterberg et al., 2002). The present study investigated asample of 82 female patients aged �37 years and associa-tions between MTHFR polymorphisms and IVF outcome.

A statistically significant decrease in oocyte maturitywhen the MTHFR polymorphism 677 was observed. Previousstudies suggest that the presence of the mutant allele of the677 polymorphism can influence the concentration of intra-follicular homocysteine, resulting in a hostile environmentfor oocyte maturation (Boxmeer et al., 2008; Szymanskiand Kazdepka-Zieminska, 2003).

This study also observed a statistically significant differ-ence in the number of oocytes retrieved in patients withmutated alleles for the MTHFR 1298 polymorphism. It is pos-tulated that the MTHFR mutation A1298C, both in its homo-zygous mutant (CC) or heterozygous (AC) state, does notseem to cause elevations in plasma homocysteine concen-tration when it occurs individually (van der Put et al., 1998;Weisberg et al., 1998, 2001).

It has been considered that the A1298C polymorphismmay bring harm when appearing concurrently with the het-erozygous mutation for the C677T polymorphism (doubleheterozygous), producing a 677CT/1298AC genotype (vander Put et al., 1998; Weisberg et al., 1998, 2001). There-fore, the current study divided the patients into three

Please cite this article in press as: D’Elia, PQ et al. MTHFR polymorphismswomen. Reproductive BioMedicine Online (2014), http://dx.doi.org/10.10

groups to compare the effect of the presence of the poly-morphism at position 1298 concomitantly with the polymor-phism at position 677 versus the presence of at least onepolymorphism versus absence of both polymorphisms. Thestatistical analyses of the results showed a nonsignificanttendency towards a higher proportion of mature oocytesin patients without any polymorphism. The double heterozy-gous haplotype seemd to have no impact on outcome.

A previous study published by Dobson et al. in 2007 eval-uated 197 couples undergoing assisted reproduction cyclesand investigated the association between MTHFR polymor-phisms C677T and A1298C in men and women in relationto laboratory results and pregnancy outcomes. The studyfound no association in any of the evaluations betweenthe presence of polymorphism and laboratory results suchas number and quality of embryos transferred and preg-nancy rate in the assisted fertilization cycles performed(Dobson et al., 2007).

Another study also associated the presence of the MTHFRpolymorphism C677T with embryo quality by evaluating thegenetic variants of folate metabolism and IVF outcomes in439 women undergoing assisted fertilization cycles. Thestudy concluded that patients with a heterozygous CT geno-type have higher proportions of better-quality embryos andincreased chances of pregnancy when compared with

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MTHFR polymorphisms C677T/A1298C and IVF outcomes in Brazilian women 5

patients with homozygous TT genotypes or CC genotypes(Laanpere et al., 2011).

Some studies have shown that a high homocysteine con-centration in the follicular fluid may cause a decrease in celldivision and high embryonic fragmentation, which means adecrease in oocyte as well as embryonic quality (Aitkenet al., 1991; Berker et al., 2009). Since folate and methio-nine metabolism is required for transmitting methyl groupsfor DNA methylation, and methylation is essential forembryo regulation processes such as imprinting, it is possi-ble that the apparently good-quality embryos are in factepigenetically unstable due to a disruption in the folatepathway caused by a MTHFR polymorphism (Market Velkeret al., 2012).

The present work has limitations which did not allowaccurate assessment of the impact of different types ofmale infertility on normal fertilization rates and embryoresults. Also, a possible bias may have been introduced bythe fact that, although all couples in the study group pre-sented infertility due to a male factor, they had had no pre-vious pregnancy: thus, this work cannot rule out thepossibility that at least some of the female patients mightpresent some other cause of infertility. Another limitationis the small number of patients, which may have reducedstatistical power and, consequently, produced results lesssignificant than expected. A strength is that the patientgroup was highly selected so as to avoid bias with otherdiseases.

Although MTHFR mutations have lately been linked todiseases such as Down’s syndrome (James et al., 1999), neu-ral tube defects and other malformations (Finnell et al.,1998; van der Put et al., 1995), research focusing on preg-nancy complications (Ray and Laskin, 1999) and recurrentpregnancy loss (Isolato et al., 2000; Nelen et al., 1998;Zetterberg et al., 2002) and the role of polymorphismsC677T and A1298C in human fertility is at an early stage,with only a few studies exploring the real influence of thesepolymorphisms on IVF cycles. The current study is, there-fore, a contribution to the literature, providing more labo-ratory data on IVF related to these polymorphisms.

In conclusion, this study demonstrated a significanteffect of the MTHFR polymorphisms C677T and A1298C onoocyte maturity and recovery. Moreover, interesting associ-ations were found regarding good-quality embryos ofpatients presenting a mutant allele for the MTHFR polymor-phism A1298C. Collaborative studies on larger patient sam-ples may contribute significantly to the elucidation of theeffect of MTHFR polymorphisms on IVF outcomes.

Acknowledgements

The authors thank all patients who participated in thisstudy. They are also grateful for the support from the IdeiaFertility Institute, Fundacao de Amparo a Pesquisa do Esta-do de Sao Paulo (2010/16274-4), Capes and CNPq (476116/2010-4).

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Declaration: The authors report no financial or commercialconflicts of interest.

Received 6 June 2013; refereed 4 February 2014; accepted 5February 2014.

C677T and A1298C and associations with IVF outcomes in Brazilian16/j.rbmo.2014.02.005