Folate-metabolizing gene variants and pregnancy outcome of IVF

Reproductive BioMedicine Online (2011) 22, 603– 614

www.sciencedirect.com www.rbmonline.com

ARTICLE

Folate-metabolizing gene variants and pregnancy outcome of IVF ¨e M Laanpere a,*, S Altma a,c,g,h A Salumets

b,c

, T Kaart d, A Stavreus-Evers e, TK Nilsson f,

a

Department of Biotechnology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia; Department of Clinical Science, Intervention and Technology, Division of Obstetrics and Gynecology, Karolinska Institutet, Karolinska University Hospital Huddinge, 14186 Stockholm, Sweden; c Competence Centre on Reproductive Medicine and Biology, Tiigi 61b, 50410 Tartu, Estonia; d Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, Kreutzwaldi 62, 51014 Tartu, Estonia; e Department of Women’s and Children’s Health, Uppsala University, 75185 ¨ rebro University Hospital, 70185 O ¨ rebro, Sweden; g Department of Uppsala, Sweden; f Department of Clinical Chemistry, O h Obstetrics and Gynecology, University of Tartu, Puusepa 8, 51014 Tartu, Estonia; Nova Vita Clinic, Centre for Infertility Treatment and Medical Genetics, 74001 Tallinn, Estonia b

* Corresponding author. E-mail address: [email protected] (M Laanpere). Margit Laanpere obtained her MSc degree in biotechnology and biomedicine at the Faculty of Life Sciences, University of Tartu, Estonia in 2007. She then worked in scientific research at the Department of Biotechnology and later at the Department of Obstetrics and Gynaecology, both in University of Tartu, Estonia. Her research interests include nutrition, genetics and reproduction, specifically the effect of folate and other one-carbon metabolism nutrients and related gene variants on female and male fertility.

Abstract There is growing evidence that folate status and variation in folate-metabolizing genes are involved in female reproduc-

tive functions. This study evaluated the influence of maternal blood folate, vitamin B12, homocysteine and 10 folate pathway gene variants on IVF outcome. Also, the prevalence of these polymorphisms was compared in 439 female IVF patients and 225 fertile controls. MTHFR 677 CT heterozygotes had a higher proportion of good-quality embryos and an increased chance of pregnancy. MTHFR 1793 GA heterozygosity was associated with a lower percentage of previously failed IVF treatments. Heterozygosity for FOLR1 1816 C/delC and 1841 G/A was associated with a raised risk of pregnancy loss. The CTH 1208 GT genotype was associated with an increased chance of pregnancy and a smaller number of previously failed IVF cycles and the genotype frequency was lower in IVF patients with three or more previously failed IVF treatments compared with fertile controls. SLC19A1 80 GA heterozygotes had a decreased number of previously failed IVF treatments and were more prevalent among fertile controls. In conclusion, polymorphisms in folate-metabolizing genes may affect ovarian stimulation and pregnancy outcome of IVF, and heterozygous individuals, rather than the wild-type homozygotes, appeared to have more favourable outcomes. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: CTH, folate, FOLR1, MTHFR, SLC19A1, TCN2

1472-6483/$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rbmo.2011.03.002

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Introduction

Materials and methods

Assisted reproductive technologies have become widely accepted as a proven and routine treatment for infertility in industrialized countries (Andersen et al., 2007). Despite recent developments, only about one-third of IVF and intracytoplasmic sperm injection (ICSI) procedures result in live births (Andersen et al., 2007; Norwitz et al., 2001). It is crucial to understand the as-yet unidentified factors affecting IVF/ICSI pregnancy outcome in order to design safer and more effective infertility treatment. Growing evidence suggests that infertility treatment outcome may be modulated by maternal B vitamin status (reviewed in Laanpere et al. 2010). The water-soluble B vitamin folate is required for one-carbon biosynthetic and epigenetic processes that facilitate the synthesis and methylation of nucleic acids and proteins. Thus, folate is indispensable during periods of rapid cell growth and proliferation, which occur during follicular and embryonic development. Insufficient folate intake impairs fertility in animal models (Mohanty and Das, 1982; Mooij et al., 1992; Willmott et al., 1968) and causes early spontaneous abortions, birth defects and other adverse pregnancy outcomes in humans (George et al., 2002). Folate deficiency increases deoxyuridine monophosphate misincorporation into DNA (Blount et al., 1997; Courtemanche et al., 2004; Duthie and Hawdon, 1998), disrupts DNA integrity (Blount et al., 1997; Duthie and Hawdon, 1998), slows DNA replication and causes apoptosis and necrosis of the affected cells (Courtemanche et al., 2004; Huang et al., 1999; Kimura et al., 2004; Koury et al., 2000). Group B vitamins are critical epigenetic regulators (Menezo et al., 2010). Both dietary folate deficiency (Jacob et al., 1998; Rampersaud et al., 2000) and a common 677 C/T polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR) (Friso et al., 2002; Stern et al., 2000) cause defective DNA methylation, which may alter gene expression (Ingrosso et al., 2003) and lead to chromosome fragility (Lukusa and Fryns, 2008). Furthermore, dietary or genetically determined folate deficiency may result in an elevated serum homocysteine (Hcy) concentration (Jacques et al., 2001). High concentrations of Hcy, called hyperhomocysteinaemia, have been linked to several pathologies, including pregnancy complications (Berry et al., 1999; D’Uva et al., 2007; Del Bianco et al., 2004; Haggarty et al., 2006; James et al., 1999; Kumar et al., 2003; Lindblad et al., 2005; Nelen et al., 2000a,b; Owen et al., 1997; Wouters et al., 1993). Thus, Hcy has been suggested to be a mediator of the negative effect of insufficient folate status and the functional polymorphisms in genes of its metabolism. The aim of the present study was to examine the effect of 10 polymorphisms in folate pathway genes MTHFR, folate receptor 1 (FOLR1), transcobalamin II (TCN2), cystathionase (CTH) and solute carrier family 19, member 1 (SLC19A1) on blood serum concentrations of folate, vitamin B12 and Hcy in female IVF patients and to assess the associations between these genetic variants and ovarian stimulation and pregnancy outcomes of IVF in comparison with fertile controls.

Subjects All patients undergoing IVF or ICSI at the Nova Vita Clinic in Estonia from January 2004 to April 2007 were considered for participation in the current study. A total of 439 female IVF patients were recruited for the study. The primary diagnosis of infertility was male factor (abnormal sperm sample) in 111 of the couples (25.3%), female factor in 297 of the couples (67.7%) and unexplained infertility in 31 of the couples (7.1%). Among the cases of female-associated infertility, tubal occlusion (n = 169, 38.5% of total patients), polycystic ovary syndrome (PCOS) (n = 50, 11.4%), endometriosis (n = 35, 8.0%) and infertility due to other reasons (n = 43, 9.8%) were diagnosed. The mean age of the female IVF patients was 33.7 ± 4.6 years. In Estonia, all women trying to conceive are advised to take a folic acid supplement to ensure that 400 lg of folate is ingested daily. Female IVF patients had also been counselled to take vitamin and mineral supplements, but no follow up on supplement use was conducted. Ovarian stimulation was conducted according to either the gonadotrophin-releasing hormone (GnRH) antagonist or agonist protocol with recombinant FSH, as described previously (Altma ¨e et al., 2007). The ovarian stimulation outcome parameters included: (i) serum oestradiol concentration on the day of oocyte retrieval (pmol/l); (ii) amount of FSH administered per one oocyte obtained during oocyte retrieval (IU/oocyte); (iii) number of oocytes obtained during oocyte retrieval; (iv) number of goodquality embryos (four or more equally sized blastomeres with 20% cellular fragmentation as evaluated 44–46 h after insemination or ICSI); and (v) proportion (%) of good-quality embryos out of the total number of embryos. Patients with PCOS (n = 50) were excluded from the analysis of ovarian stimulation outcomes, because including women with markedly dysfunctional folliculogenesis and disturbed hormonal balances could confound the statistical analysis of the ovarian response to stimulation. IVF and ICSI were performed as described previously (Salumets et al., 2003). The IVF pregnancy outcomes analysed included: (i) pregnancy, indicated by a positive serum human chorionic gonadotrophin (HCG) test (10 IU/l) conducted 14 days after embryo transfer; (ii) clinical pregnancy, diagnosed by the presence of gestational sac(s) in the uterus on transvaginal sonography at 6–7 weeks of gestation; (iii) multiple pregnancy, diagnosed by the presence of more than one gestational sac on transvaginal sonography at 6–7 weeks of gestation; and (iv) pregnancy loss, defined as no clinical pregnancy after a positive HCG test. Women with ectopic pregnancy (n = 4) were excluded from the analyses of IVF pregnancy outcome. The treatment cycle studied was the first for 52.8% of the IVF patients (n = 232), the second for 28.7% of the IVF patients (n = 126), the third for 9.6% of the IVF patients (n = 42) and the fourth or more for 8.9% of the IVF patients (n = 39). Among the women for whom the current IVF treatment cycle was not the first, the previous IVF treatment outcomes were assessed using two parameters: (i) the number of previously failed IVF treatments (number of previous

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IVF treatments minus the number of previous IVF treatments resulting in live births); and (ii) the percentage of previously failed IVF treatments (proportion of previously failed IVF cycles out of the total number of IVF treatments). All of the IVF patients provided a blood sample for DNA extraction and a fasting serum sample for oestradiol, folate, vitamin B12 and Hcy analyses on the day of oocyte retrieval. For the control group of fertile women, genomic DNA samples were obtained from 225 women from the Estonian Genome Foundation. To include women with proven fertility, the inclusion criterion was four or more spontaneous pregnancies. The study chose not to match the controls to patients age-wise in order to be able to include subjects who were towards the end of their reproductive age and had had a chance to fulfil their reproductive potential. The mean age of the fertile controls was 43.1 ± 9.1 years. The mean number of pregnancies was 5.5 ± 1.6 and the mean number of live births was 3.3 ± 1.2. The majority of the pregnancies that did not result in live birth were terminated upon the women’s request (n = 484, 98.4%), the rest being spontaneous abortions (n = 3, 0.6%), ectopic pregnancies (n = 2, 0.4%) and stillbirths (n = 3, 0.6%). Serum samples were not available for the fertile control group. The Ethics Committee of the University of Tartu approved the study and informed consent was obtained from all participants.

species, free or protein-bound, found in serum) concentrations. Serum oestradiol concentrations were measured using a chemiluminescence immunoassay (Immulite 2000; Diagnostic Products Corporation, Los Angeles, CA, USA), which had a detection limit of 55 pmol/l, within-run coefficient of variation (CV) of 4.3–9.9% and total CV of 6.7–16.0%, respectively. A Chemiluminescent Microparticle Folate Binding Protein assay with the ARCHITECT Folate Reagent Kit (Abbott Laboratories, IL, USA) was used to determine serum folate concentrations. The kit had a detection limit of 2.70 nmol/l, within-run CV of 2.5–7.9% and total CV of 3.9–12.1%. Serum vitamin B12 concentrations were quantified using a Chemiluminescent Microparticle Intrinsic Factor assay with the ARCHITECT B12 Reagent Kit (Abbott Laboratories), which had a detection limit of 92 pmol/l, within-run CV of 2.7–5.6% and within-laboratory CV of 3.4–6.8%. Serum folate and vitamin B12 concentrations were quantified using the ARCHITECT i System (Abbott Laboratories). Serum Hcy concentrations were analysed by fluorescence polarization immunoassay using the IMx Homocysteine Reagent Pack and IMx Analyser (Abbott Laboratories). The kit had a detection limit of 0.5 lmol/l, within-run CV of 1.4–2.2% and total CV of 3.7–5.2%. Serum samples for biochemical marker analyses were available for 285 out of the 439 IVF patients.

Genotyping

Study design and statistical analyses

Genomic DNA was extracted from peripheral EDTA-blood using the salting-out method (Aljanabi and Martinez, 1997). PCR reactions were performed using the HotStarTaq DNA Polymerase Kit (Qiagen, Venlo, The Netherlands). Genotyping of the single-nucleotide polymorphisms (SNP) MTHFR 677 C/T (rs1801133), MTHFR 1298 A/C (rs1801131), MTHFR 1793 G/A (rs2274976), FOLR1 1314 G/A (rs2071010), FOLR1 1816 C/delC (rs3833748), FOLR1 1841 G/A (rs1540087), FOLR1 1928 C/T (rs9282688), TCN2 776 C/G (rs1801198), CTH 1208 G/T (rs1021737) and SLC19A1 80 G/A (rs1051266) was conducted by pyrosequencing using Pyro Gold Reagents and the PSQ 96 MA system (Biotage, Uppsala, Sweden) as described previously (Bo ¨ttiger et al., 2007a,b; Bo ¨ttiger and Nilsson, 2007; Nilsson et al., 2008). Pyrosequencing results were automatically analysed using the PSQ 96 MA 2.0.2 software (Biotage). DNA samples were available for genotyping for 400 female IVF patients and for all of the 225 fertile controls. All genotyped loci were in Hardy–Weinberg equilibrium in both patient and control groups. There was only one individual carrying the MTHFR 1793 AA genotype in the patient group and none in the fertile control group. As the AA patient did not belong to most of the IVF patient subgroups analysed, the AA genotype is not shown in the results of those analyses. The one MTHFR 1793 AA genotype was grouped together with the GA genotypes in other statistical analyses. Two FOLR1 SNP, 1816 C/delC and 1841 G/A, were always present simultaneously in the study groups, referring to complete linkage.

To investigate the relationship between folate metabolism and IVF outcome, a longitudinal cohort study was performed. A case–control study was used to compare folate metabolism related gene variants between fertile and infertile women. Statistical analyses were performed using the SAS system (SAS Institute). General linear and logistic models were used to determine: (i) the association of mean concentrations of the folate pathway biomarkers folate, vitamin B12 and Hcy with patient age; (ii) the difference in mean concentrations of the biomarkers between different genotypes in the IVF patients; (iii) the associations between polymorphisms and variation in ovarian stimulation and IVF pregnancy outcome; (iv) the effect of the biochemical markers on ovarian stimulation and IVF outcome; and (v) the associations between polymorphisms and the number of previously failed IVF attempts and the percentage of previously failed IVF procedures. All models were adjusted for patient age, number of ovaries and aetiology of infertility; ovarian stimulation outcome analyses were additionally adjusted for the stimulation protocol (either GnRH agonist or antagonist) and IVF outcome analyses for the number and quality of transferred embryos. Fisher’s exact test was used to estimate whether the genotype frequencies of the genetic variants differed significantly between fertile controls and all IVF patients as well as between fertile controls and IVF patient subgroups without clinical pregnancies and with three or more previously failed IVF attempts. Fisher’s exact test was also used to estimate whether prevalence of the combined genotypes of the two and three MTHFR polymorphisms (MTHFR 677–1298 and MTHFR 677–1298–1793, respectively) differed significantly between fertile controls and all IVF patients. Haploview (Barrett et al., 2005) was used to generate phased haplotypes from the genotype data of the

Biochemical analyses Fasting serum samples were analysed to determine oestradiol, folate, vitamin B12 and total Hcy (the sum of all Hcy

606 three MTHFR polymorphisms. Chi-squared test was employed to assess if the frequencies of the two- and three-locus haplotypes differed significantly between fertile controls and all IVF patients as well as between fertile controls and IVF patient subgroups without clinical pregnancies and patients with three or more previously failed IVF attempts. Data from statistical models adjusted for confounding factors are presented as least-square mean ± standard error of the mean (SE), unless otherwise indicated. A P-value <0.05 was considered statistically significant.

Results Serum folate, vitamin B12 and homocysteine concentrations in female IVF patients The mean ± standard deviation serum concentrations of the studied folate pathway biomarkers among 285 IVF patients were 12.4 ± 6.6 nmol/l for folate, 239.1 ± 94.0 pmol/l for vitamin B12 and 9.1 ± 2.3 lmol/l for Hcy. Folate and vitamin B12 concentrations fell below the reference range (6.8–38.5 nmol/l and 118–590 pmol/l, respectively) in 17.9% (n = 51) and 4.2% (n = 12) of the IVF patients, respectively. Hcy concentrations were higher than the reference interval (5–12 lmol/l) in 11.6% (n = 33) of the women. Folate concentrations were negatively correlated with Hcy concentrations (Pearson’s correlation coefficient r = 0.44, P < 0.0001), whereas vitamin B12 concentrations were positively correlated with folate concentrations (r = 0.23, P < 0.0001) and negatively with Hcy concentrations (r = 0.25, P < 0.0001). Folate and Hcy concentrations were not associated with IVF patient age, whereas mean vitamin B12 concentrations appeared to be somewhat higher in older women (regression coefficient b = 2.68, P = 0.036). The statistical significance of genotypic effects and the least-square means of serum folate, vitamin B12 and Hcy concentrations are presented in Table 1. When compared with subjects with the wild-type MTHFR 677 CC genotype (13.71 ± 0.61 nmol/l), mean folate concentrations were significantly lower in CT heterozygous (11.65 ± 0.65 nmol/l, P = 0.022) and TT homozygous (10.72 ± 1.35 nmol/l, P = 0.044) subjects. Individuals with the MTHFR 677 TT genotype (10.91 ± 0.44 lmol/l) had significantly higher Hcy concentrations than CC homozygotes (8.77 ± 0.20 lmol/l, P < 0.0001) and CT heterozygotes (9.23 ± 0.22 lmol/l, P = 0.001). Mean folate concentrations were elevated in patients with the MTHFR 1793 GA genotype (16.82 ± 1.93 nmol/l) compared with GG major homozygotes (12.33 ± 0.43 nmol/l, P = 0.024). Interestingly, FOLR1 1314 GA heterozygotes appeared to have higher serum folate concentrations (15.26 ± 1.25 nmol/l) than individuals with the major GG genotype (12.22 ± 0.45 nmol/l, P = 0.023). IVF patients with the TCN2 776 GG genotype had significantly higher serum folate concentrations (14.13 ± 1.00 nmol/l) than patients with the CC wild-type genotype (11.48 ± 0.76 nmol/l, P = 0.036). Furthermore, patients with the SLC19A1 80 GA genotype had significantly higher serum Hcy concentrations (9.57 ± 0.21 lmol/l) compared with wild-type GG homozygotes (8.68 ± 0.25 lmol/l, P = 0.007).

M Laanpere et al.

Ovarian stimulation outcome The mean oestradiol concentration was significantly higher in FOLR1 1928 CT heterozygotes (9588.24 ± 1297.40 pmol/l) compared with patients with the major CC genotype (3759.85 ± 164.09 pmol/l, P < 0.0001). Furthermore, oestradiol concentration was significantly higher in SLC19A1 80 AA subjects (4533.94 ± 386.17 pmol/l) compared with GA heterozygotes (3617.18 ± 239.88 pmol/l, P = 0.046). There was a non-significant trend for patients with the MTHFR 1298 CC genotype to require more FSH per oocyte (393.35 ± 59.57 IU) compared with major homozygous AA (271.29 ± 23.64 IU) and heterozygous AC (271.49 ± 25.24 IU) genotypes. Patients heterozygous for the FOLR1 1314 G/A polymorphism received significantly more FSH per oocyte (438.62 ± 49.40 IU) than patients with the GG genotype (262.43 ± 17.41 IU, P = 0.001) and had a higher oocyte fertilization rate (69.68 ± 3.15%) compared with patients with the GG genotype (62.63 ± 1.11%, P = 0.036). The proportion of good-quality embryos was significantly higher in subjects heterozygous for the MTHFR 677 C/T (48.36 ± 2.69%) compared with CC homozygotes (41.03 ± 2.27%, P = 0.038). The least-square means (± SE) of the ovarian stimulation outcome parameters in each genotype are shown in Supplementary Table 1 (available online only). Serum folate and Hcy did not affect ovarian stimulation outcome.

Pregnancy outcome of current IVF treatment Of the total 439 patients included in this study, 207 (47.2%) had a positive HCG test. Of these 30 (6.8%) experienced a pregnancy loss, four (0.9%) had an ectopic pregnancy and 173 (39.4%) had a normal clinical pregnancy, of which 48 (10.9%) were multiple pregnancies. Table 2 shows the chance of pregnancy, clinical pregnancy, multiple pregnancy and pregnancy loss among the IVF patients with different genotypes. The chance for pregnancy (adjusted odds ratio (aOR) 1.65, 95% confidence interval (CI) 1.04–2.62, P = 0.033) and clinical pregnancy (aOR 1.87, 95% CI 1.17–2.97, P = 0.009) were significantly higher in patients with the MTHFR 677 heterozygous CT genotype compared with patients with the major homozygous CC genotype. The MTHFR 677 minor homozygous TT genotype showed a non-significant tendency towards a decreased chance of pregnancy compared with the CT genotype (aOR 0.48, 95% CI 0.22–1.05). When only adjustments for age and transferred embryo quality were left in the statistical model, excluding aetiology of infertility, number of ovaries, stimulation protocol and number of embryos transferred, the lower probability for pregnancy in the case of a TT genotype was statistically significant (aOR 0.42, 95% CI 0.20–0.89, P = 0.025). Similarly, the decreased chance for clinical pregnancy in patients with the TT genotype compared with the CT genotype was statistically significant (aOR 0.43, 95% CI 0.20–0.93, P = 0.032). IVF patients with the CTH 1208 GT heterozygous genotype had an increased chance for clinical pregnancy compared with GG homozygotes, but the association did not reach statistical significance (aOR 1.50, 95% CI 0.94–2.41). When the statistical model was adjusted only for patient age and transferred embryo quality, both the chance for

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Table 1 Genotypic effects and serum folate, vitamin B12 and homocysteine (Hcy) concentrations in 285 IVF patients. Genotype

MTHFR 677 C/T Ala222Val CC CT TT MTHFR 1298 A/C Glu429Ala AA AC CC MTHFR 1793 G/A Arg594Gln GG GA FOLR1 1314 G/A GG GA AA FOLR1 1816 C/delC and 1841 G/A CC and GG CdelC and GA FOLR1 1928 C/T CC CT TCN2 776 C/G Arg259Pro CC CG GG CTH 1208 G/T Ser403Ile GG GT TT SLC19A1 80 G/A His27Arg GG GA AA

Biochemical markers of folate pathway Folate (nmol/l)

B12 (pmol/l)

Hcy (lmol/l)

P = 0.026 13.71 ± 0.61a 11.65 ± 0.65b 10.72 ± 1.35b NS 12.57 ± 0.59 12.69 ± 0.67 11.30 ± 1.68 P = 0.024 12.33 ± 0.43a 16.82 ± 1.93b P = 0.038 12.22 ± 0.45a 15.26 ± 1.25b 4.72 ± 6.62 NS 12.61 ± 0.44 10.81 ± 2.03 NS 12.52 ± 0.43 14.40 ± 4.72 NS 11.48 ± 0.76a 12.60 ± 0.60 14.13 ± 1.00b NS 11.66 ± 0.64 13.17 ± 0.65 13.54 ± 1.25 NS 13.57 ± 0.73 12.18 ± 0.63 11.42 ± 0.99

NS 241.8 ± 8.6 237.2 ± 9.1 239.0 ± 18.6 NS 236.6 ± 8.2 244.2 ± 9.2 234.0 ± 23.3 NS 238.6 ± 6.1 257.4 ± 26.1 NS 235.9 ± 6.3 265.0 ± 17.5 334.6 ± 92.7 NS 240.5 ± 6.1 218.6 ± 28.2 NS 239.7 ± 6.0 219.6 ± 65.6 NS 227.9 ± 10.5 240.3 ± 8.3 257.0 ± 14.0 NS 237.3 ± 8.8 242.1 ± 9.1 238.8 ± 17.6 NS 240.3 ± 10.1 239.5 ± 8.7 238.1 ± 13.8

P < 0.001 8.77 ± 0.20a 9.23 ± 0.22a 10.91 ± 0.44b NS 9.17 ± 0.20 9.16 ± 0.23 9.42 ± 0.58 NS 9.22 ± 0.15 8.49 ± 0.64 NS 9.24 ± 0.16 8.72 ± 0.43 8.95 ± 2.30 NS 9.17 ± 0.15 9.57 ± 0.70 NS 9.19 ± 0.15 8.32 ± 1.62 NS 9.26 ± 0.26 9.27 ± 0.21 8.81 ± 0.35 NS 9.20 ± 0.22 9.35 ± 0.22 8.52 ± 0.43 P = 0.026 8.68 ± 0.25a 9.57 ± 0.21b 9.17 ± 0.34

Values are least-square mean ± standard error of the mean. Statistical significance and values estimated using a general linear model considering confounding effects of female patient age, aetiology of infertility and number of ovaries. a,b Least-square means corresponding to the same gene and biochemical marker with different subscript letters are statistically significantly different (P < 0.05). CTH = cystathionase; FOLR1 = folate receptor a; MTHFR = methylenetetrahydrofolate reductase; NS = not statistically significant; SLC19A1 = solute carrier family 19 member 1; TCN2 = transcobalamin II.

pregnancy (aOR 1.59, 95% CI 1.02–2.49, P = 0.042) and for clinical pregnancy (aOR 1.62, 95% CI 1.03–2.55, P = 0.037) were significantly higher in GT heterozygotes compared with patients with the GG genotype. The heterozygous CdelC and GA genotypes of the FOLR1 polymorphisms 1816 C/delC and 1841 G/A were associated with a greater risk of pregnancy loss (aOR 6.02, 95% CI 1.23–29.33, P = 0.027). Chance for multiple pregnancy was not affected by any of the studied polymorphisms. Removing PCOS patients from the analyses of IVF pregnancy outcomes did not change the direction of presented genetic effects. None of the folate pathway biomarkers examined, including serum folate, vitamin B12 and Hcy, appeared to affect chance for

pregnancy, clinical or multiple pregnancy or risk for pregnancy loss.

Failure of previous IVF treatments The analysis of previously failed IVF treatments among different genotype groups (Supplementary Table 2, available online only) showed that the mean number of previously failed IVF cycles was significantly higher in patients with the CTH 1208 TT genotype (2.07 ± 0.23) compared with those with the GT genotype (1.48 ± 0.12, P = 0.025), as well as in patients with the SLC19A1 80 GG genotype (1.93 ± 0.13) compared with those with the GA genotype (1.45 ± 0.11, P = 0.006). The percentage of previously failed

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M Laanpere et al. Table 2 Probability of pregnancy, clinical pregnancy, multiple pregnancy and pregnancy loss among IVF patients with different genotypes. Genotype

MTHFR 677 C/T Ala222Val CT versus CC TT versus CC TT versus CT MTHFR 1298 A/C Glu429Ala AC versus AA CC versus AA CC versus AC MTHFR 1793 G/A Arg594Gln GA+AA versus GG FOLR1 1314 G/A GA versus GG AA versus GG AA versus GA FOLR1 1816 C/delC and 1841 G/A CdelC versus CC and GA versus GG FOLR1 1928 C/T CT versus CC TCN2 776 C/G Arg259Pro CG versus CC GG versus CC GG versus CG CTH 1208 G/T Ser403Ile GT versus GG TT versus GG TT–GT SLC19A1 80 G/A His27Arg GA versus GG AA versus GG AA versus GA

Probability of: Pregnancy

Clinical pregnancy

Multiple pregnancy

Pregnancy loss

NS 1.65 (1.04–2.62) 0.79 (0.37–1.71) 0.48 (0.22–1.05) NS 0.69 (0.44–1.09) 1.41 (0.61–3.25) 2.05 (0.87–4.82) NS 0.85 (0.34–2.05) NS 1.38 (0.69–2.74) 1.25 (0.19–8.37) 0.91 (0.12–6.65) NS 2.02 (0.70–5.85) NS 0.12 (0.01–1.08) NS 1.303 (0.79–2.14) 1.27 (0.67–2.40) 0.97 (0.54–1.75) NS 1.48 (0.93–2.35) 1.12 (0.57–2.21) 0.76 (0.38–1.51) NS 1.21 (0.74–1.97) 0.97 (0.51–1.82) 0.80 (0.44–1.45)

P = 0.023 1.87 (1.17–2.97) 0.96 (0.43–2.14) 0.52 (0.23–1.16) NS 0.71 (0.45–1.12) 0.91 (0.39–2.11) 1.29 (0.54–3.05) NS 0.74 (0.29–1.85) NS 1.24 (0.61–2.50) 0.68 (0.11–4.42) 0.55 (0.08–3.93) NS 0.91 (0.31–2.70) NS 0.18 (0.02–1.61) NS 1.28 (0.78–2.12) 0.99 (0.52–1.91) 0.78 (0.43–1.41) NS 1.50 (0.94–2.41) 1.03 (0.52–2.07) 0.69 (0.34–1.39) NS 1.15 (0.70–1.88) 1.02 (0.53–1.95) 0.88 (0.48–1.63)

NS 0.52 0.84 1.62 NS 0.55 0.47 0.85 NS 0.62 NS 2.59 ND ND NS 1.37 NS ND NS 0.68 0.46 0.68 NS 1.19 1.04 0.87 NS 1.23 0.52 0.42

NS 0.49 (0.19–1.22) 0.55 (0.09–3.20) 1.13 (0.19–6.89) NS 1.06 (0.41–2.79) 2.80 (0.76–10.32) 2.63 (0.68–10.18) NS 2.88 (0.46–18.17) NS 0.75 (0.18–3.16) 2.21 (0.14–33.87) 2.93 (0.16–55.03) P = 0.027 6.02 (1.23–29.33) NS ND NS 0.85 (0.30–2.42) 1.86 (0.55–6.30) 2.19 (0.74–6.49) NS 0.88 (0.34–2.28) 1.44 (0.37–5.65) 1.63 (0.43–6.25) NS 1.15 (0.43–3.07) 0.81 (0.20–3.23) 0.70 (0.19–2.55)

(0.23–1.16) (0.17–4.19) (0.32–8.21) (0.24–1.24) (0.09–2.48) (0.15–4.74) (0.10–3.64) (0.65–10.26)

(0.18–10.20)

(0.30–1.56) (0.14–1.51) (0.22–2.07) (0.52–2.72) (0.29–3.71) (0.25–3.04) (0.50–3.03) (0.13–2.02) (0.12–1.47)

Values are adjusted odds ratios (95% confidence intervals) for each genotype comparison. Associations between studied polymorphisms and IVF outcome estimated by logistic regression model adjusted for female patient age, number of ovaries, aetiology of infertility, stimulation protocol and number and quality of transferred embryos. Women with ectopic pregnancy (n = 4) were excluded from these analyses. CTH = cystathionase; FOLR1 = folate receptor a; MTHFR = methylenetetrahydrofolate reductase; ND = not determinable; NS = not statistically significant; SLC19A1 = solute carrier family 19 member 1; TCN2 = transcobalamin II.

IVF treatments was markedly higher in individuals with the MTHFR 1793 GG major homozygous genotype (94.12 ± 1.63%) compared with individuals with the GA heterozygous genotype (79.21 ± 6.20%, P = 0.021). Individuals with the FOLR1 1314 GA genotype showed a non-significant trend towards a lower percentage of previously failed IVF treatments (84.94 ± 4.84%) compared with individuals with the GG major homozygous genotype (94.00 ± 1.69%).

Genotypic frequencies in IVF patients and fertile controls No significant differences were observed in the genotype prevalence when all IVF patients were compared with the fertile controls. When only IVF patients that did not achieve clinical pregnancy in the current IVF treatment (n = 241) were considered, a non-significant trend for a difference from the fertile control group in the CTH 1208 G/T genotype

distribution was noted: the GG genotype was represented in 41.8% of the fertile controls and in 49.8% of the IVF patients with no clinical pregnancy; the GT genotype was represented in 47.6% of the fertile controls and in 36.9% of the IVF patients; and the TT genotype was represented in 10.7% of the fertile controls and in 13.3% of the IVF patients. In the same CTH 1208 G/T polymorphism, a significant difference (P = 0.036) in genotype distribution between the IVF patients who had experienced three or more previously failed IVF attempts (n = 36) and the fertile controls was detected: the GG genotype was represented in 41.8% of the controls and in 50.0% of the IVF patients; the GT genotype was represented in 47.6% of the fertile controls and in 27.8% of the IVF patients; and the TT genotype was represented in 10.7% of the controls and in 22.2% of the patients. Genotype distributions of the studied polymorphisms in all female IVF patients, patient subgroups and fertile controls are shown in Supplementary Table 3 (available online only).

Folate gene variants and IVF outcome

Figure 1 Distributions of the CTH 1208 G/T genotype among all female IVF patients, patient subgroups according to clinical pregnancy outcome and number of previous IVF failures and fertile controls. Fisher’s exact test was used to estimate whether the genotypic frequencies differed significantly among the studied groups. P-values of Fisher’s exact test are provided. *A generalized linear model revealed that individuals with the CTH 1208 GT genotype have a significantly reduced chance of belonging to the patient group with three or more previous IVF failures rather than the fertile control group, compared with individuals with the TT genotype (odds ratio 3.57, 95% confidence interval 1.27–10.04, P = 0.016). NS = not statistically significant.

Figure 1 shows the CTH 1208 G/T genotype distribution in the studied groups. The odds that an individual carrying the CTH 1208 GT genotype belonged to the group of fertile controls rather than to the IVF patient group with three or more failed IVF treatments were 3.6 times greater than for individuals carrying the TT genotype (OR 3.57, 95% CI 1.27–10.04, P = 0.016). The genotype distributions of the SLC19A1 80 G/A polymorphism were also slightly different between fertile controls and IVF patients with three or more previously failed IVF treatments; however, this difference did not reach statistical significance. When the odds of individuals with different SLC19A1 genotypes belonging to the IVF patient group with three or more failed IVF treatments rather than to the control group were calculated, an individual with the GA genotype was less likely to belong to the IVF patient group compared with an individual with the GG genotype (OR 0.39, 95% CI 0.17–0.87, P = 0.022). No significant difference in the frequency of genotype combinations of the two and three MTHFR polymorphisms (MTHFR 677–1298 and MTHFR 677–1298–1793) were found between IVF patients and fertile controls. Also, no statistically significant differences in the estimated haplotype distributions were found between all female IVF patients, patient subgroups (patients that did not achieve clinical pregnancy in the current IVF procedure and patients that had previously experienced three or more IVF failures) and fertile controls.

Discussion The present study demonstrates that polymorphisms in folate pathway genes are associated not only with significantly altered serum folate and homocysteine concentrations,

609 but also with ovarian stimulation and pregnancy outcome in IVF treatment. In accordance with previous findings (Bo ¨ttiger et al., 2007b; Frosst et al., 1995; McNulty et al., 2006), the MTHFR 677 C/T polymorphism was associated with significantly altered serum folate and Hcy concentrations. As enzyme activity and serum folate concentrations are the highest and Hcy concentrations are the lowest in individuals with the wild-type CC genotype, it has generally been considered the most beneficial genotype for health. However, the current findings show that in terms of IVF treatment outcome, the maternal MTHFR 677 heterozygous CT genotype is beneficial compared with the homozygous CC and TT genotypes with a greater proportion of good-quality embryos and an increased chance for both a positive HCG test and clinical pregnancy. In concordance with these results, it has been shown that women with the heterozygous MTHFR 677 CT genotype rather than the CC genotype have a slight but significantly increased chance of having a viable pregnancy with IVF treatment (Haggarty et al., 2006). Moreover, a previous study also demonstrated a higher occurrence of MTHFR 677 CT heterozygosity in a control group compared with females with unexplained infertility (Altma ¨e et al., 2010). In addition to the association between maternal MTHFR 677 CT heterozygosity and a greater chance for pregnancy achievement, fetuses with the CT genotype also appear to be more viable than fetuses with homozygous genotypes (Bae et al., 2007; Zetterberg et al., 2002). The reproductive advantage of the MTHFR 677 CT genotype could be explained by a favourable balance in the folate metabolism pathway (Figure 2) between methyl donor and nucleotide synthesis. The CC genotype is associated with efficient cellular methylation reactions (Friso et al., 2002; Stern et al., 2000), while the TT genotype is linked to folate cofactor balance that better supports DNA biosynthesis (Bagley and Selhub, 1998) and decreases deoxyuridine monophosphate misincorporation into DNA (Kapiszewska et al., 2005). The variant C allele of the second-most studied polymorphism, MTHFR 1298 A/C, has been linked to higher basal FSH concentrations and diminished response to ovarian stimulation (Rosen et al., 2007). In agreement, the current study found that the CC genotype had a tendency towards decreased ovarian responsiveness to FSH stimulation compared with the AA and AC genotypes. It has been postulated that the MTHFR 1298 A/C polymorphism may decrease the amount of folate cofactors available for nucleotide synthesis, thereby affecting DNA biosynthesis and increasing apoptosis in the granulosa cells, leading to an increased requirement for FSH for ovarian stimulation in the carriers of the variant C allele (Rosen et al., 2007). It has been previously shown that MTHFR 1793 GG wild-type adolescents have higher serum Hcy concentration than GA heterozygotes (Bo ¨ttiger et al., 2007b). The current study did not identify an association between this variant and serum Hcy concentrations. However, the MTHFR 1793 GG homozygous IVF patients did have significantly lower serum folate concentrations than the GA heterozygotes. Additionally, the MTHFR 1793 GA genotype appeared to be associated with a better IVF outcome when previous treatment cycles were considered: the percentage of previously failed IVF cycles was lower in patients with the MTHFR 1793

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Figure 2 Overview of folate-mediated one-carbon metabolism, consisting of two cycles that compete for folate cofactors: DNA biosynthesis (blue) and methylation (green). Homocysteine is catabolized in the trans-sulphuration pathway (pink). Genes tested in this study have been shown as blue ellipses with studied polymorphisms listed next to the gene. Tested biochemical markers (folate, homocysteine and vitamin B12) are shown in green. Dotted lines indicate compound transport into the cell. Dashed line represents biochemical reactions where all steps have not been shown. B12 = vitamin B12; BHMT = betaine-homocysteine methyltransferase; CTH = cystathionase; DHF = dihydrofolate; dTMP = deoxythymidine monophosphate; dUMP = deoxyuridine monophosphate; FOLR1 = folate receptor a; Hcy = homocysteine; MTHFR = methylenetetrahydrofolate reductase; MTR = methionine synthase; R = methyl acceptor, such as DNA or histones; SAH = S-adenosylhomocysteine; SAM = S-adenosylmethionine; SLC19A1 = solute carrier family 19 member 1; THF = tetrahydrofolate; TCN2 = transcobalamin II.

GA genotype compared with the GG genotype. This is in line with a recent study, which showed that the MTHFR 1793 GA genotype was underrepresented in infertile women compared with the general population (Altma ¨e et al., 2009), indicating a positive effect of the heterozygous genotype on fertility. The functional significance of the MTHFR 1793 G/A variation remains unknown. However, as the polymorphism appears to influence folate and Hcy concentrations, the effect on fertility may in part be mediated by these compounds. In the case of FOLR1 1314 G/A variation, the heterozygous genotype also appeared to be beneficial: compared with patients with the major GG genotype, the GA heterozygotes had significantly higher serum folate concentrations, higher oocyte fertilization rate and a tendency toward more previously successful IVF treatments. Heterozygosity for FOLR1 1816 C/delC and 1841 G/A polymorphisms appeared to increase the risk of pregnancy loss. Heterozygosity for the fourth FOLR1 SNP analysed, 1928 C/T, was associated with significantly increased oestradiol concentrations compared with the wild-type genotype. The SNP in the FOLR1 gene investigated in this study are located in the 50 untranslated region of the gene, encoded by exons 1–4, which could interfere with the regulatory regions of either of the two promoters situated upstream of exon 1 and intron 3 (Elwood et al., 1997) and lead to altered FOLR1 expression. FOLR1 encodes the folate receptor a, which is responsible for cellular uptake of folate and is expressed in granulosa

cells, epithelial cells of the Fallopian tube and the endometrium (Weitman et al., 1992) and placenta (Ross et al., 1994). Therefore, altered expression of the transporter may affect reproductive function through folate availability for cellular folate metabolism in these tissues. The TCN2 gene encodes a vitamin B12-binding protein that is responsible for delivering vitamin B12 to peripheral tissues. Previous studies have identified an association between the TCN2 776 GG genotype and lower concentrations of the circulating vitamin B12-transporter complex. However, similar to the current results, no differences in serum vitamin B12 concentrations have been identified (Afman et al., 2002; Miller et al., 2002; Namour et al., 2001; von Castel-Dunwoody et al., 2005). This suggests that the binding capacity of the recombinant transporter is reduced, which does not affect circulating concentrations of vitamin B12, but may decrease cellular transport and intracellular concentration of the vitamin. However, the finding of higher mean folate concentration in TCN2 776 GG homozygotes compared with CC homozygotes in this study’s IVF patient group is rather unexpected and the underlying mechanisms remain unclear. The CTH 1208 G/T polymorphism, which causes a Ser403Ile substitution in a conserved residue in the cystathionase protein, has been shown to increase serum Hcy concentrations in individuals with the TT genotype compared with individuals with the GG and GT genotypes (Altma ¨e et al., 2009; Wang et al., 2004). In the current study

Folate gene variants and IVF outcome population of female IVF patients, an association between this variant and Hcy was not observed. However, the CTH 1208 GT heterozygotes had an increased chance for a positive HCG test and clinical pregnancy. The positive effect of GT heterozygosity on fertility was also supported by the finding that GT heterozygotes had a lower number of previous IVF failures. Moreover, the frequency of the GT heterozygotes was higher in the fertile control group compared with both the IVF patients who did not achieve pregnancy in the current IVF treatment and the IVF patients that had experienced three or more IVF failures before the current IVF treatment cycle. As no functional tests have been carried out concerning this genetic variant, the underlying mechanism affecting fertility can only be postulated. CTH participates in the trans-sulphuration pathway, wherein Hcy is converted to cysteine. In addition, it catalyses a reaction converting cystine to thiocysteine, which decomposes to cysteine and hydrogen sulphide. The latter may act as a regulatory mediator (Lowicka and Beltowski, 2007), playing an important negative role in cell proliferation and survival (Yang et al., 2004). Hence, it is possible that highly active CTH, which is effective in cytotoxic Hcy removal, inhibits cell proliferation due to excess hydrogen sulphide production. If the CTH 1208 G/T polymorphism affects the concentrations or activity of CTH, a GT heterozygote might have a suitable concentration of active CTH enzyme. The SLC19A1 80 G/A polymorphism appeared to influence serum Hcy concentrations in the IVF patients, with increased Hcy concentrations in the GA heterozygous individuals compared with patients with the wild-type genotype. Higher Hcy concentrations are usually associated with adverse effects on fertility (Haggarty et al., 2006; Nelen et al., 2000a,b). However, in the current study, the GA genotype also showed a smaller number of previously failed IVF attempts than the GG genotype and was less prevalent among the IVF patients that had previously experienced three or more IVF failures compared with fertile controls. SLC19A1 encodes reduced folate carrier, a protein responsible for uptake of circulating folate in peripheral tissues (Matherly et al., 2007). The SLC19A1 80 G/A polymorphism results in the His27Arg amino acid change, which has been shown to have no effect on cellular folate intake in in-vitro studies (Whetstine et al., 2001). Therefore, it remains to be determined how the SLC19A1 80 G/A polymorphism modulates serum Hcy concentrations and influences IVF treatment outcome. This study did not detect significant differences in the combined genotypes of the MTHFR 677 C/T, 1298 A/C and 1793 G/A polymorphisms between IVF patients and controls; neither did the haplotype frequencies differ between the controls and IVF patients as well as IVF patient subgroups. In the study population the three MTHFR polymorphisms appear as 10 genotype combinations. It is possible that there were too few subjects in each genotype group to detect the cumulative effect of several polymorphisms on phenotype. Serum folate, vitamin B12 and Hcy concentrations did not appear to be associated with IVF pregnancy outcome in this study. Vitamin supplementation could mask or diminish the genotype-related effects within folate and homocysteine metabolism. Unfortunately, dietary folate and vitamin B12 intake could not be assessed, because data regarding

611 nutritional supplement use by the patients were not available, therefore folate and vitamin B12 intake could not have been taken into account in the data analyses. Although it is recognized that folate deficiency and hyperhomocysteinaemia may have pathophysiological effects that can be associated with impaired fertility and pregnancy complications (reviewed in Laanpere et al., 2010), it is postulated that the effect of the folate metabolism-related polymorphisms on pregnancy and miscarriage rates does not manifest exclusively nor primarily through its influence on blood folate or Hcy concentrations. Rather, it is proposed that the effect of these polymorphisms is mediated by the combination of and balance between several additional factors. The equilibrium that is created together with other metabolites in the whole folate-mediated one-carbon metabolism cycle and how it is reflected in cellular methylation status, thymidylate and purine synthesis, might be more important than the exact blood concentration of folate or Hcy. The hypothesis of folate pathway polymorphisms altering the flux of folates between DNA synthesis and methylation reactions is discussed in more detail in Laanpere et al. (2010). In addition to maternal folate pathway gene polymorphisms, respective fetal polymorphisms may also determine the viability of the pregnancy. For example, fetal human leukocyte antigen polymorphisms have been shown to affect assisted reproduction success (reviewed in Hviid, 2006), but as far as is known there are no studies examining fetal folate pathway genes in respect to IVF treatment outcome. Some insight has been gained studying MTHFR polymorphisms in spontaneously and therapeutically aborted fetuses. These studies are reviewed in Laanpere et al. (2010). However, the results are so far somewhat inconclusive. Notably, among several of the polymorphisms studied, particularly MTHFR 677 C/T and CTH 1208 G/T, heterozygous rather than wild-type homozygous individuals appeared to have favourable IVF outcomes. The positive effects of heterozygous genotypes of these variants on fertility detected in the current study are summarized in Figure 3. These findings are in line with the theory of heterozygote advantage, or overdominance, which suggests that genetic variation is maintained in natural populations due to greater viability and reproductive fitness among heterozygotes (Hansson and Westerberg, 2002). Studies have shown correlations between heterozygosity and fitness in numerous organisms, although meta-analysis suggests that the effects are weak (Chapman et al., 2009). In humans, the association between genome-wide SNP heterozygosity and physical fitness has been studied and an association has been reported with quantitative traits related to blood pressure, for example (Govindaraju et al., 2009). As far as is known, there are no data available on heterozygote advantage and fertility in humans. However, in female domesticated sheep, heterozygote advantage based on polymorphisms in genes that affect fecundity has been reported (Gemmell and Slate, 2006). It is possible that a similar reproductive advantage in humans is conferred by heterozygosity in the folate pathway genes. Clearly more research in this important field of infertility treatment is needed, especially in a bigger cohort. In the current study, if correction for multiple testing was used, several of the discussed findings would fail to reach

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References

Figure 3 Overview of the MTHFR 677 C/T and CTH 1208 G/T heterozygous genotypes’ association with positive IVF outcome and reproductive success.

statistical significance. Still, if further studies replicate the associations between folate pathway gene variants and IVF outcome, the next step would be to investigate how nutritional supplements affect infertility treatment outcome in different genotypes. It might be possible to ascertain genotypes that would most benefit from taking additional vitamins and thereby improve infertility treatment with respective genetic testing and nutritional supplementation. In conclusion, this study indicates that polymorphisms in folate pathway genes are associated with significantly altered ovarian stimulation and pregnancy outcome in IVF treatment and suggest a heterozygote advantage in reproductive fitness for several of the folate pathway gene variants.

Funding This study was supported by the Estonian Science Foundation (grant no. 6498), the Estonian Ministry of Education and Science (core grants nos. SF0180142Cs08 and SF0180044s09), Kristjan Jaak Stipendiumid, the European Union through the European Regional Development Fund through the Centre of Excellence in Genomics, the Estonian Biocentre and University of Tartu, the Enterprise Estonia (grant no. EU30200), the Swedish Research Council (205–7293), the Family Planning Foundation, Uppsala Uni¨ rebro. versity, Swedish Institute and Nyckelfonden, O

Acknowledgements We are grateful to all voluntary participants of the study, and to the staff at the Nova Vita Clinic and the Estonian Genome Foundation for collecting the samples.

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