Common 677C→T mutation of the 5,10-methylenetetrahydrofolate reductase gene affects follicular estradiol synthesis

GENETICS Common 677C/T mutation of the 5, 10-methylenetetrahydrofolate reductase gene affects follicular estradiol synthesis Stephanie Hecht,a Roman Pavlik,a Peter Lohse, M.D.,b Ulrich Noss, M.D.,c Klaus Friese, M.D.,a and Christian J. Thaler, M.D.a a

Department of Obstetrics and Gynecology, Division of Endocrinology and Reproductive Medicine, and b Department of Clinical Chemistry, Grosshadern, Ludwig Maximilians-University; and c Centre for Reproductive Medicine, Munich, Germany

Objective: To investigate the influence of the 5,10-methylenetetrahydrofolate reductase 677C/T mutation on the E2 synthesis in human granulosa cells (GCs). Design: In vitro cell culture study. Setting: Research laboratory of a university hospital. Patient(s): Follicular fluids (n ¼ 139) and GCs (n ¼ 66) were obtained from patients undergoing controlled ovarian hyperstimulation for IVF with or without ICSI. Intervention(s): Granulosa cells were cultured for a total of 5 days. On day 3, the cells either were stimulated with recombinant (r-) FSH or r-LH (80 IU/L for 48 h) or were sham stimulated. Main Outcome Measure(s): Estradiol and protein content were measured in the pooled follicular fluids of each individual. At the end of each GC-culturing period, the concentrations of E2 were measured in the supernatants of triplicate cultures by immunoassays. The 5,10-methylenetetrahydrofolate reductase 677C/T genotype was determined by RFLP analysis. Result(s): The E2-protein ratio of homozygous T/T carriers was significantly lower compared with that of homozygous C/C individuals. Furthermore, basal and r-FSH– as well as r-LH–stimulated E2 synthesis of GC obtained from homozygous T/T patients was significantly reduced, compared with GC from heterozygous C/T and homozygous C/C subjects. Conclusion(s): Decreased E2 in follicular fluid and decreased E2 synthesis of GC from homozygous T/T individuals suggest that reduced follicular E2 is a result of impaired E2 production of human GC. (Fertil Steril 2009; 91:56–61. 2009 by American Society for Reproductive Medicine.) Key Words: MTHFR 677C/T mutation, estradiol synthesis, granulosa cells, follicular fluid

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the irreversible conversion of 5,10methylenetetrahydrofolate into 5-methyltetrahydrofolate, which serves as a methyl donor in the remethylation of homocysteine to methionine (1). A 677C/T/p.Ala222Val missense mutation produces a thermolabile variant of the enzyme that has a 70% reduction in enzymatic activity and leads to increased plasma homocysteine concentrations Received March 14, 2007; revised and accepted November 5, 2007. Authors S.H. and R.P. contributed equally to the work and both should be considered to be the first author. Supported by the Department of Obstetrics and Gynecology, Grosshadern, Ludwig-Maximilians-University, Munich, Germany. Presented at the 56th National Congress of Gynecology and Obstetrics, Berlin, Germany, September 19–22, 2006. Reprint requests: Christian J. Thaler, M.D., Department of Obstetrics and Gynecology, Division of Endocrinology and Reproductive Medicine, Grosshadern, Ludwig-Maximilians-University, Marchioninistrasse 15, D-81377 Munich, Germany (FAX: 49-89-7095-7588; E-mail: thaler@ med.uni-muenchen.de).

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under conditions of low folate intake (2) . The 677T allele has been related to the occurrence of neural-tube and other birth defects (3, 4), Down syndrome (5), and pregnancy complications (6). We have reported elsewhere that the MTHFR 677C/T mutation is significantly less prevalent in mothers of spontaneously conceived dichorionic twins compared with in those with singleton pregnancies (7). Moreover, we have shown that assisted reproductive technology (ART) patients carrying one or two T alleles exhibited reduced ovarian responsiveness with regard to recombinant (r-) FSH stimulation. Although they required higher amounts of r-FSH for controlled ovarian hyperstimulation, these patients had significantly lower concentrations of serum E2 and produced significantly fewer oocytes for retrieval (8). Up to now, it has not been known whether reduced E2 serum concentrations in T-allele carriers are solely a result of fewer follicles or whether E2 production of granulosa cells

Fertility and Sterility Vol. 91, No. 1, January 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

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(GCs) is in addition impaired in follicles of affected individuals. Therefore, we have studied whether E2 concentrations in follicular fluids of ART patients differ with respect to the three MTHFR genotypes. Furthermore, we have examined whether the MTHFR 677C/T mutation has a direct influence on the E2 synthesis of cultured human GCs.

MATERIALS AND METHODS Patient Population This prospective study consisted of 139 women undergoing oocyte retrieval after controlled ovarian hyperstimulation for ART procedures at our infertility clinic in Munich, Germany. The subjects were recruited between September 2003 and March 2004. All patients who were 20–45 years of age and had tested negative for HIV-1 and -2, hepatitis-B and -C, and were immune to rubella were included. In vitro fertilization or intracytoplasmic sperm injection (ICSI) were performed for tubal-factor infertility (n ¼ 23), male-factor infertility (n ¼ 49), endocrine sterility (n ¼ 49), or idiopathic sterility (n ¼ 18). Tubal factor was defined as bilateral blockage of tubes, as diagnosed by laparoscopic chromopertubation. Male-factor infertility was classified according to the 1992 World Health Organization criteria. Endocrine sterility was defined as World Health Organization type II anovulation: women who were resistant to clomiphene citrate therapy and/or low-dose FSH stimulation and who did not become pregnant after at least four cycles of treatment with stimulation and ovulation induction. Couples were defined as being idiopathically sterile if complete infertility workup did not provide any explanation for their infertility and if no pregnancy was achieved after at least four cycles of follicular monitoring, with or without stimulation and timed intrauterine insemination. Of all participating women, 43.9% underwent their first; 29.4%, their second; 25.2%, their third; and 1.4%, their fourth cycle of IVF with or without ICSI. All women were supplemented with folic acid (0.4 mg by mouth, once per d) during the entire time period of the ART treatment to reduce the risk of neural-tube defects in the newborn. Age, body mass index, smoking status, number of menstrual cycles per year, total amount of required r-FSH, and number of retrieved oocytes were recorded prospectively. Data are expressed as mean  SD. The MTHFR 677C/T genotype was determined after oocyte retrieval.

Ethical Considerations The study was approved by the Ethics Committee of the Medical Faculty of the University of Munich. Written informed consent was obtained from all participants, and the study was performed according to the guidelines of the Declaration of Helsinki. All samples and clinical information were anonymized. Fertility and Sterility

Follicular-Fluid Collection Follicular fluid was obtained from 139 women undergoing oocyte retrieval for IVF with or without ICSI. Development of multiple follicles was induced after GnRH down-regulation by intranasal application of Nafarelin (Synarela; Pharmacia GmbH, Karlsruhe, Germany) starting at day 23 of the pretreatment cycle, followed by stimulation with human r-FSH (Gonal F; Serono, Unterschleissheim, Germany). Human chorionic gonadotropin (10,000 IU, Pregnesin; Serono) was administered after follicles had reached a diameter of >18 mm, as determined by vaginal sonography. Thirty-six hours later, ultrasound-guided transvaginal oocyte retrieval was performed under general anesthesia. Follicular fluids of individual patients were collected and pooled after obtaining the oocytes. Samples with massive blood or flushing fluid contamination were excluded. Follicular fluid samples were immediately processed by centrifuge for 10 minutes at 1,000  g, and supernatants were aliquoted and stored at 70 C until further assayed.

Luteinized GC Culture Subjects for this prospective observational study were recruited during a time period of 6 months. To avoid systematic mistakes caused by the preparation of several successive GC cultures during the same day, we did not establish cell cultures from all subjects. Instead, patients were randomly selected by a computer program (Excel; Microsoft, Redmond, WA). After centrifugation of pooled follicular aspirates at 1,000  g for 10 minutes, the cell pellet was washed with Dulbecco’s phosphate-buffered saline (Invitrogen, Karlsruhe, Germany), layered on a 40% Percoll gradient (Amersham Biosciences, Freiburg, Germany), and processed by centrifuge at 2,000  g for 10 minutes. Granulosa cells were collected at the Percoll–phosphate-buffered saline interface, washed, and trypsinated at 37 C for 20 minutes with a trypsin–ethylenediamine tetraacetate solution (Invitrogen) at final trypsin and ethylenediamine tetraacetate concentrations of 0.5 g/L and 0.20 g/L, respectively. The resulting cell suspension was subjected to gentle repeated pipetting through a narrow-bore pipette tip to facilitate cell dispersal and then was filtered through a 40-mm filter (Falcon; BD Biosciences Discovery Labware, Bedford, MA). The suspension was washed free of enzyme by using Dulbecco’s phosphate-buffered saline containing 1% fetal calf serum (Invitrogen). The recovered cells were resuspended in 1 mL of cooled (4 C) culture medium (M199 þ Earles’ þ L-glutamine; Invitrogen), supplemented with 10% fetal calf serum and gentamicin (0.1 mg/mL; Invitrogen). To remove CD14- and CD45-positive blood cells (such as leukocytes, monocytes, and macrophages), GCs were further purified by incubation for 30 minutes at 4 C with Dynabeads M-450 CD14 and CD45 (Dynal Biotech, Hamburg, Germany) under gentle rotation, as described elsewhere (9). Leukocytes, monocytes, and macrophages rosetted with immunomagnetic beads were removed by using a Dynal magnetic particle concentrator. 57

Cell number and viability of purified GCs were assessed in a Neubauer chamber by trypan blue (0.5%; Biochrom AG, Berlin, Germany) exclusion (vitality of >91.3%). The cells were distributed in 200 mL of supplemented M199 at 20,000 cells per well into fibronectin-precoated (0.5 mg per well; fibronectin from bovine plasma; Sigma-Aldrich Chemie GmbH, Munich, Germany), 96-well dishes (Costar; Corning Incorporated, NY) and were incubated at 37 C in a humidified atmosphere containing 5% CO2. The GCs were left to attach for 72 hours, before nonattached cells and medium were removed.

Treatment Protocol for the Human GCs The human GCs were cultured for a total of 5 days in medium (M199 þ Earles’ þ L-glutamine; Invitrogen) supplemented with 10% fetal calf serum and gentamicin (0.1 mg/mL; Invitrogen). Experiments were performed after 72 hours, because cells were found to be maximally responsive to gonadotropin stimulation after 3 days in culture (Hecht S, Pavlik R, unpublished data). Cells were treated with either 80 IU/L of r-FSH (Gonal F, 75 IU; Serono) or 80 IU/L of r-LH (Luveris; Serono) for 48 hours or were sham stimulated. Androstenedione (106 mol/L; Sigma-Aldrich Chemie GmbH) was added to all cultures as a substrate. Supernatants of triplicate cultures were collected after 48 hours and pooled. Estradiol concentrations of the pooled supernatants were measured by commercial EIA kits, as described in the next subsection.

Protein Assay and E2 Enzyme Immunoassay Protein concentrations were determined by using the Bradford method (10) with Coomassie brilliant blue G-250 dye (Bio-Rad Laboratories, Munich, Germany), using bovine serum albumin (Bio-Rad Laboratories) as protein standard. Samples were diluted 1:500 with distilled water and analyzed in duplicate. Estradiol concentrations were measured by a specific enzyme immunoassay kit (DSL-10-4300 ACTIVE Estradiol EIA Kit; Diagnostic Systems Laboratories Deutschland GmbH, Sinsheim, Germany), according to the manufacturer’s instructions. The detection limit for the assay is reported to be 7 pg/mL for E2. Both intra-assay and interassay coefficients of variation were <10%. Follicular fluid was diluted 1:500 with distilled water, and supernatants of GCs were diluted 1:8 with Standard A diluent (Diagnostic Systems Laboratories Deutschland GmbH). All samples were assayed in duplicate. To exclude a possible effect of the flushing media, we normalized follicular E2 concentrations to follicular protein concentrations. Estradiol concentrations of the GC culture supernatants, calculated E2–protein ratios of follicular fluids, and calculated stimulatory effects of r-FSH and r-LH in human GC (r-FSH [E2]/basal [E2] and r-LH [E2]/basal [E2]) were compared with respect to the three MTHFR 677C/T genotypes C/C, C/T, and T/T. 58

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Isolation of DNA and Determination of the MTHFR 677C/ T Genotype Ethylenediamine tetraacetate blood was drawn from all patients before follicle aspiration. Genomic DNA was extracted from leukocytes by using the QIAmp DNA blood mini kit (QIAGEN GmbH, Hilden, Germany). Amplification by polymerase chain reaction was performed with the MTHFR-specific primer pair MT-1 (50 -TTGAGGCT GACCTGAAGCACTTG-30 ) and MT-2 (50 -AGGACGGT GCGGTGAGAGTG-30 ) for 40 cycles (95 C for 20 seconds, 65 C for 20 seconds, and 72 C for 20 seconds). The resulting PCR products were digested by using the restriction endonuclease HinfI (50 -G/ANTC-30 ; New England BioLabs, Inc., Beverly, MA) and analyzed on 2% low–melting point agarose (Invitrogen) gels. The mutant T allele was characterized by two fragments, 176 and 43 base pairs in length, whereas the normal C allele was not cleaved by the enzyme. Statistical Analysis Statistical significance was assessed by using the nonparametric Mann-Whitney U test. The level of significance was set at P<.05. We used SPSS for Windows 12.0.1. (SPSS Inc., Chicago, IL) to generate box plots for the data.

RESULTS The study population consisted of 139 ART patients. Follicular fluid was collected from all 139 women, whereas GC cultures were established for a randomly selected subset of 66 subjects. Characteristics of the patient population and frequencies of the C/C, C/T, and T/T genotypes among our patients are given in Table 1. Overall, homozygosity for the C allele was observed in 41.1% (n ¼ 57); compound heterozygosity, in 46% (n ¼ 64); and homozygosity for the T allele, in 12.9% (n ¼ 18) of the subjects. Age, body mass index, smoking status, number of menstrual cycles per year, total amount of required r-FSH, and number of retrieved oocytes did not differ significantly between the three MTHFR 677C/T genotype groups. The protein concentrations of the follicular fluids also were not statistically significantly different between the three MTHFR 677C/T genotypes. In contrast, E2 synthesized and released from GCs of homozygous T/T patients under basal conditions (4,263  1,677 pg/mL) was significantly reduced in comparison to the cases of heterozygous C/T (9,889  7,080 pg/mL; P¼.006) and homozygous C/C (12,205  9,466 pg/mL; P¼.006) subjects (Fig. 1A). Accordingly, the calculated E2–protein ratio of follicular fluids of homozygous T/T carriers was significantly lower than that of homozygous C/C individuals (P¼.035; Fig. 2), whereas the E2–protein ratio of heterozygous C/T individuals (5,177  3,418 pg E2 per milligram of protein) was not statistically different when compared with either homozygous C/C (5,287  2,474 pg E2 per milligram of protein; P¼.273) or homozygous T/T

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TABLE 1 Characteristics of the 139 patients studied with regard to their MTHFR 677C/T genotype. MTHFR C677/T genotype

n

C/C C/T T/T

57 41.1 33.4  5.3 64 46.0 34.6  4.2 18 12.9 35.7  4.1

%

Age (y)

Menstrual Body mass cycles per ya Total amount (n [ 125) of r-FSH (IU) index Smokers (%) 22.1  6.0 22.2  5.4 23.2  4.1

21.27 25 20

14.03  3.91 14.22  4.60 15.40  5.79

1,940  768 1,815  635 2,046  808

No. of retrieved oocytes 16.3  9.2 15.8  7.4 15.1  7.4

Note: Data are mean  SD unless otherwise indicated. Age, body mass index, smoking status, number of menstrual cycles per year, and total amount of required r-FSH did not differ significantly between the three MTHFR 677C/T genotypes C/C, C/T, and T/T. a Only participants with spontaneous menstrual cycles (n ¼ 125) were included. Hecht. Lower estradiol as a result of MTHFR 677T. Fertil Steril 2009.

(3,954  1,858 pg E2 per milligram of protein; P¼.222) subjects. Furthermore, r-FSH– and r-LH–stimulated E2 synthesis of GCs obtained from homozygous T/T individuals (r-FSH– stimulated: 7,173  2,853 pg/mL; r-LH–stimulated: 8,600  3,340 pg/mL) also was significantly reduced when compared with heterozygous C/T (r-FSH–stimulated: 18,570  13,343 pg/mL; P¼.027; r-LH–stimulated: 22,481  12,830 pg/mL; P¼.014) or homozygous C/C (r-FSH–stimulated: 18,339  12,933 pg/mL; P¼.016; r-LH–stimulated: 25,213  16,801 pg/mL; P¼.004) subjects (Fig. 1B and C). The E2 production of GCs from heterozygous C/T and homozygous C/C patients, in contrast, was not statistically significantly different (basal: P¼.402; r-FSH–stimulated: P¼.971; r-LH–stimulated: P¼.889). The calculated stimulatory effect (E2 of control group/E2 of stimulated group) of r-FSH and r-LH on human GCs also was not statistically different between the three MTHFR genotypes.

DISCUSSION In the present study, we observed not only significantly reduced E2 concentrations in the follicular fluid of homozygous 677T allele carriers but also a significant decrease of the E2 synthesis in GCs from these individuals. The results obtained were in very good agreement with our clinical data from our previous study, which had demonstrated lower E2 concentrations in the serum of heterozygous and homozygous ART patients undergoing controlled ovarian hyperstimulation (8). Because our previous study also had shown fewer oocytes to be retrieved in 677T allele carriers (8), it appeared possible that the reduced serum E2 concentrations observed in these patients were merely the result of a lower number of follicles, because follicle numbers reportedly correlate with serum E2 concentrations (11). The present study also showed a decreased number of oocytes in 677T carriers, but because of the small number of cycles evaluated, this effect was not sigFertility and Sterility

nificant. The MTHFR 677C/T mutation, however, appears not only to affect the number of follicles but also to reduce the E2 production within tertiary follicles, because we observed significantly decreased E2 concentrations in GC cultures isolated from T/T homozygous ART patients. The stimulatory effect of r-FSH and r-LH on human GCs, in contrast, was not altered by the MTHFR 677C/T mutation. Granulosa cells from homozygous T/T individuals showed no relative differences when stimulated with r-LH or r-FSH, despite a significantly decreased basal E2 production. Hence, our data suggest, that the effects of the MTHFR 677C/T mutation are independent of gonadotropin receptor interaction and signaling functions. The biological background of our observations is not known. 5,10-methylenetetrahydrofolate reductase catalyzes the conversion of 5,10-methylenetetrahydrofolate (5,10MTHF) into 5-methyltetrahydrofolate (5-MTHF), which is the major circulating form of folate. Folate in turn is used in many biochemical pathways, including the remethylation of homocysteine to methionine, which is an important step in the metabolic network that regulates the biosynthesis of nucleosides and the methylation of proteins, lipids, and DNA. A nucleotide exchange at position 677 of the MTHFR cDNA results in a thermolabile enzyme with reduced catalytic activity (2). Individuals with C/T and T/T genotypes have 65% and 30% of the enzymatic activity, respectively, of C/C homozygous subjects (12). The lower MTHFR activity limits the availability of methyl groups from S-adenosylmethionine, resulting in the hypomethylation of many biological molecules, including DNA. Methylation is one of the most important epigenetic features of eukaryotic DNA, because it is involved in the regulation of gene expression and in the maintenance of genome integrity (13). Hypomethylation results in chromosome instability and DNA damage (14), leading to an impaired cell function. Hence, on the level of GC function, the MTHFR 677C/T mutation may interfere with the complex regulation of mechanisms that are involved in steroid biosynthesis and 59

FIGURE 1

FIGURE 1 Continued Estradiol production of cultured human granulosa cells with regard to the MTHFR 677C/T genotype. Box plots show median, interquartile range, outliers (black dots), and extreme cases of individual variables within the 5th and 95th percentiles. Experiments (n ¼ 25 for C/C patients; n ¼ 36 for C/T patients; n ¼ 5 for T/T patients) were performed after 72 hours. Human granulosa cells were stimulated with either 80 IU/L r-FSH (B) or 80 IU/L r-LH (C) for 48 hours or were sham stimulated (A). Androstenedione (106 mol/L) was added to all cultures as a substrate. teinemia (15). All of our infertility patients received folic acid (0.4 mg by mouth, once per d) to prevent fetal neural tube defects. Hence, the negative effects of the MTHFR 677C/T mutation on E2 synthesis potentially could have been alleviated by the use of low-dose folic acid treatment, particularly in our homozygous T/T patients, because this therapeutic regimen has been shown to be most effective in individuals carrying the mutation on both alleles (16). Because homocysteine levels were not analyzed in our study patients, we could not confirm that the presence of the MTHFR 677T allele indeed reduced the methylation of homocysteine to methionine. Nevertheless, chronic alterations of methylation status may explain, at least in part, why we observed significant differences in E2 synthesis in individuals with the three different MTHFR genotypes. Ovaries of these patients were exposed to the effects of the MTHFR 677C/T mutation for a long time span, and apparently,

FIGURE 2 Calculated E2–protein ratio of individual follicular fluids with regard to the MTHFR 677C/T genotype. Box plots show median, interquartile range, outliers (black dots), and extreme cases of individual variables within the 5th and 95th percentiles.

Hecht. Lower estradiol as a result of MTHFR 677T. Fertil Steril 2009.

metabolism. To understand the underlying mechanisms, it may be necessary to identify genes that are involved in the process of steroidogenesis in human GCs and whose methylation may be altered by the MTHFR 677C/T mutation. Increased folate uptake reverses many biological effects of the MTHFR 677C/T mutation, including hyperhomocys60

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Hecht. Lower estradiol as a result of MTHFR 677T. Fertil Steril 2009.

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such consequences are not rapidly reversed by the low–dose, short-term folate fortification prescribed to our infertility patients. Our findings strongly suggest that a woman’s folate metabolism has an influence on the steroid synthesis of her GCs. The obtained data support our hypothesis that the MTHFR 677C/T mutation affects the E2 synthesis in individual follicles and indeed, in individual GCs. It will be interesting to study human GCs under conditions of different concentrations of folate and homocysteine, as well as to analyze steroid metabolites in the culture supernatant. This may help to further define the block of E2 synthesis as well as its mechanism. Our observations therefore could be useful for gaining further insights into the mechanisms related to folliculogenesis and steroid metabolism in human GCs. They also may be of importance for patient management in assisted reproduction. Acknowledgments: The authors thank all patients, who participated in this study. They also are indebted to Helena Angermaier and the team of the Centre for Reproductive Medicine, Munich, Germany, for their assistance.

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