European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72

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European Journal of Obstetrics & Gynecology and

Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb

Polymorphic variants of folate and choline metabolism genes and the risk
of endometriosis-associated infertility
Malgorzata Szczepańska a, Adrianna Mostowska b, Przemyslaw Wirstlein a,b, Margarita Lianeri b,
Piotr Marianowski b,c, Jana Skrzypczak a, Paweł P. Jagodziński b,*
a
Department of Obstetrics, Gynecology and Gynecological Oncology, Division of Reproduction, Poland
b
Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, 6 Świe˛cickiego St., 60-781 Poznań, Poland
c
I Clinic of Obstetrics and Gynecology Medical University of Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Article history: Objective: Endometriosis has been considered an epigenetic disease. Single nucleotide polymorphisms
Received 6 December 2010 (SNPs) located in genes encoding enzymes of the folate and choline metabolism may affect DNA
Received in revised form 12 January 2011 methyltransferase activity.
Accepted 23 February 2011
Study design: We studied 16 SNPs in 12 folate and choline metabolism genes, including BHMT
(rs7356530 and rs3733890), BHMT2 (rs625879), CBS (844ins68), CHDH (rs893363 and rs2289205), CHKA
Keywords: (rs7928739), MTHFD1 (rs2236225), MTHFR (rs1801133), MTR (rs1805087), MTRR (rs1801394), PCYT1A
Endometriosis
(rs712012 and rs7639752), PEMT (rs4244593 and rs4646406) and TCN (rs1801198) in one hundred and
DNA methylation
Folate
sixty-three infertile women with minimal endometriosis and one hundred and fifty fertile women.
Choline Results: There were no significant differences between genotype and allele frequencies of these gene
Polymorphism variants in infertile women with endometriosis (n = 163) and controls (n = 150). The lowest, but not
statistically significant, p values of the trend test were observed for the CBS 844ins68 and MTR rs1805087
(ptrend = 0.0527 and ptrend = 0.0771, respectively) polymorphisms. However, the exhaustive multifactor
dimensionality reduction analysis revealed an epistatic interaction between rs1801133 of MTHFR and
rs4244593 of PEMT in endometriosis-associated infertility (p = 0.0240).
Conclusions: Our results showed moderate evidence for the contribution of SNPs located in genes
encoding folate and choline metabolism enzymes to infertility in women with endometriosis.
ß 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction methylation is carried out by DNA methyltransferases (DNMTs)

Endometriosis is a complex gynecological condition, diagnosed
in approximately 10% of reproductive age women, characterized
by the presence and growth of endometrial-like tissue outside the
uterus [1,2]. Endometriosis results in disability in reproductive age
women, due to dysmenorrhea, pelvic pain, and inflammation, and
accounts for 30–50% of their infertility [2,3]. The risk factors for
endometriosis include aberrations in estrogen production and its
metabolism, immunological abnormalities, exposure to environ-
mental pollution and toxins, and genetic background [4–7]. The
etiopathogenesis and pathophysiology of endometriosis associat-
ed with below normal fertility are, however, still poorly under-
stood [2].
The abnormal methylation of several genes has been associated
with infertility in women with endometriosis [8–11]. DNA
* Corresponding author. Tel.: +48 61 854 65 13; fax: +48 61 854 65 10.
E-mail address: pjagodzi@am.poznan.pl (P.P. Jagodziński).
and increased levels of DNMT1, DNMT3A and DNMT3B have been
demonstrated in endometriosis [12]. DNMTs exploit S-adenosyl-
methionine (AdoMet) as the donor of a methyl group for DNA
methylation, followed by conversion of AdoMet to S-adenosylho-
mocysteine (AdoHcy) [13]. DNMT activity can be regulated by the
intracellular AdoMet/AdoHcy ratio, which is strongly related to
folate and choline metabolism (Fig. 1) [14]. AdoMet is biosynthe-
sized by methionine adenosyltransferase from methionine [15].
Cellular availability of methionine may be determined by
homocysteine remethylation by 5-methyltetrahydrofolate-homo-
cysteine methyltransferase (MTR) or betaine-homocysteine
methyltransferase (BHMT) [16,17]. MTR and BHMT use 5-
methyltetrahydrofolate or betaine, a choline oxidization product,
respectively, as methyl group donor (Fig. 1).
Many polymorphic variants have been found in the genes
encoding enzymes of the folate and choline metabolism, which
may determine the cellular ratio AdoMet/AdoHcy and DNMTs
activity (Fig. 1) [18,19]. Most of these investigations involve
polymorphisms of 5,10-methylenetetrahydrofolate reductase

0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ejogrb.2011.02.003
[()TD$FIG]
68 M. Szczepańska et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72

Fig. 1. Localization of 16 SNPs in 12 folate and choline metabolism genes. Enzyme names and reference number(s) of studied SNPs are stated in grey boxes. Enzyme
abbreviations: BHMT, betaine-homocysteine methyltransferase; BHMT2, betaine-homocysteine methyltransferase 2; CBS, cystathionine-beta-synthase; CHDH, choline
dehydrogenase; CHKA, choline kinase; DNA methyltransferases (DNMTs), MTHFD1, methylenetetrahydrofolate dehydrogenase 1; MTHFR, 5,10-methylenetetrahydrofolate
reductase; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase; MTRR, methionine synthase reductase; PLD, phospholipase D; PCYT1A, choline-phosphate
cytidylyltransferase A; PEMT, phosphatidylethanolamine N-methyltransferase; TCN2, transcobalamin II. Substrate abbreviations: AdoHcy, S-adenosylhomocysteine;
AdoMet, S-adenosylmethionine; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 5-CH3-THF, 5 metylotetrahydrofolate; CDP-Cho, CDP-choline; DAG, diacylglycerol; DMG,
dimethylglycine; HCY, homocysteine; SMM, S-methylmethionine; MET, methionine; PC, phosphatidylcholine; PtdCho, phosphatidylcholine; PtdEth,
phosphatidylethanolamine; SM, sphingomyelin; THF, tetrahydrofolate.

(MTHFR), MTR, methionine synthase reductase (MTRR) and
methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) (Table 2
and Fig. 1) [19]. Our study also included polymorphic variants of
genes encoding the vitamin B12 transporter transcobalamin II
(TCN2), and cystathionine-beta-synthase (CBS), which consumes
homocysteine and BHMT2 and is an enzyme responsible for
homocysteine remethylation with the use of S-methylmethionine.
We also assessed polymorphisms of genes encoding enzymes
helping to use choline as a methyl donor: choline dehydrogenase
(CHDH) and BHMT. Our studies also included polymorphic variants
of genes encoding enzymes of the CDP-choline pathway: choline
kinase (CHKA) and choline-phosphate cytidylyltransferase A
(PCYT1A), which is a regulatory enzyme of this pathway.
Additionally, we also studied polymorphisms of the gene encoding
phosphatidylethanolamine N-methyltransferase (PEMT), involved
in biosynthesis of phosphatidylcholine in the liver.
2. Materials and methods
2.1. Patients and controls
Peripheral blood samples from infertile women with endome-
triosis and control women were collected from the Gynecologic
and Obstetrical University Hospital, Division of Reproduction at
Poznan University of Medical Sciences and I Clinic of Obstetrics and
Gynecology Medical University of Warsaw, Poland. Chronic pelvic
pain and suspected pelvic endometriosis were investigated
laparoscopically. The patients were divided into two groups:
one hundred and sixty-three women were included in the infertile
with minimal endometriosis group, and one hundred and fifty
women were used as fertile controls (Table 1). Visualization of
endometriotic lesions and histopathologic criteria were used to
diagnose minimal endometriosis in infertile women. The stage of
endometriosis was assessed according to the revised classification
of the American Society for Reproductive Medicine [20]. Infertile
women with endometriosis exhibited regular menses, no anatom-
ical changes in the reproductive tract, and a minimum 1 year of
infertility with a current desire for conception, without contribu-
tion of male factor infertility. Fertile control women were
individuals that had chronic pelvic pain without any pelvic
abnormalities determined by laparoscopy. Pelvic floor varicose
veins were diagnosed in the control women, but with no signs of
past or present inflammation. The control women had at least one
child born no more than 1 year before laparoscopy, regular menses,
and no anatomical changes in the reproductive tract (Table 1).
Patients and controls were matched by age and were all
caucasian of Polish descent (Table 1). Written informed consent
was obtained from all participating individuals. The procedures of
 M. Szczepańska et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72 69

Table 1 in the coding or putative regulatory regions, and minor allele

Clinical characteristics of infertile women with minimal endometriosis and fertile
frequency (MAF) > 0.1 in the caucasian population. The genotyp-
women.
ing of 14 SNPs was performed by PCR followed by the appropriate
Characteristic Patients Controls restriction enzyme digestion (PCR-RFLP) according to manufac-
Numbers 163 150 turer’s instructions (Fermentas, Vilnius, Lithuania). DNA fragments
Age (years) 31.8 (4.2)a 32.5 (5.9)a were separated in 2% agarose gels and visualized by ethidium
31 (23–40)b 32 (18–40)b bromide staining. The BHMT rs3733890 and MTRR rs1801394
Parity NA 1 (1–3)b
polymorphic variant frequencies were determined by high-
Duration of infertility (years) 3.9 (1.9)a NA
rASRM (stage)c Stage I (n = 80) NA resolution melting curve analysis (HRM) on the LightCycler 480
Stage II (n = 83) system (Roche Diagnostics, Mannheim, Germany). Detection of the
NA-not applicable. 68-bp insertion in exon 8 of CBS was carried out by PCR followed by
a
Mean (SD). agarose gel electrophoresis. Primer sequences and conditions for
b
Median (range). PCR-RFLP and HRM analyses are presented in Supplementary Table
c
Revised American Society for reproductive medicine classification (rASRM).
2S. Genotyping quality was evaluated by repeated genotyping of
10% randomly selected samples.
the study were approved by the Local Ethical Committee of Poznan
University of Medical Sciences. 2.3. Statistical analysis

2.2. Genotyping The distribution of genotypes in patients and controls was

Genomic DNA was isolated from peripheral blood leucocytes by
salt extraction. DNA samples were genotyped for 16 single
nucleotide polymorphisms (SNPs) in 12 folate and choline
metabolism genes: BHMT, BHMT2, CBS, CHDH, CHKA, MTHFD1,
MTHFR, MTR, MTRR, PCYT1A, PEMT and TCN2 (Supplementary Table
1S). SNPs were selected with the use of the genome browsers of the
International HapMap Consortium (http://www.hapmap.org/
index.html.en), UCSC (http://genome.ucsc.edu) and dbSNP data-
base (http://www.ncbi.nlm.nih.gov/projects/SNP/). SNPs were
chosen based on several criteria: functional significance, location
tested for deviation from Hardy–Weinberg equilibrium by Chi-
square analysis. Differences in allele and genotype frequencies
between infertile women with endometriosis and controls were
evaluated by standard Chi-square and Fisher exact tests. SNPs
were individually tested for association with infertility using
the Cochran–Armitage trend test. The odds ratio (OR) and
95% confidence intervals (95% CI) were also determined.
Statistical significance was interpreted as p values < 0.05.
The exact positions of SNPs were determined using the UCSC
genome browser, February 2009 human reference sequence
(GRCh37).

Table 2
Associations of folate-metabolizing gene SNPs and risk of infertility in women with endometriosis.

Gene rs no. Genotype Cases (frequency) Controls (frequency) Odds Ratio (95% CI) pa ptrendb

CBS 844ins68 WWc 144 (0.88) 122 (0.81) Referent – 0.0527

Wins 19 (0.12) 26 (0.18) 0.619 (0.327–1.173) 0.1389
insins 0 (0.00) 2 (0.01) 0.170 (0.008–3.568) 0.2131d
Wins + insins 19 (0.12) 28 (0.19) 0.575 (0.306–1.080) 0.0828
Minor allele frequency 0.06 0.10 – –

MTHFD1 rs2236225 CC 60 (0.37) 51 (0.35) Referent – 0.6709

CT 75 (0.46) 70 (0.47) 0.911 (0.555–1.495) 0.7114
TT 28 (0.17) 27 (0.18) 0.881 (0.461–1.684) 0.7024
CT + TT 103 (0.63) 97 (0.65) 0.903 (0.567–1.437) 0.6657
Minor allele frequency 0.40 0.42 – –

MTHFR rs1801133 CC 70 (0.43) 77 (0.52) Referent – 0.1101

CT 81 (0.50) 63 (0.43) 1.414 (0.891–2.244) 0.1407
TT 12 (0.07) 8 (0.05) 1.650 (0.637–4.273) 0.2987
CT + TT 93 (0.57) 71 (0.48) 1.441 (0.921–2.254) 0.1091
Minor allele frequency 0.32 0.27 – –

MTR rs1805087 AA 95 (0.59) 101 (0.68) Referent – 0.0771

AG 60 (0.37) 43 (0.29) 1.483 (0.916–2.401) 0.1077
GG 7 (0.04) 4 (0.03) 1.861 (0.528–6.561) 0.3688d
AG + GG 67 (0.41) 47 (0.32) 1.516 (0.950–2.416) 0.0799
Minor allele frequency 0.23 0.17 – –

MTRR rs1801394 AA 52 (0.32) 54 (0.37) Referent – 0.2008

AG 84 (0.51) 77 (0.52) 1.133 (0.694–1.850) 0.6181
GG 27 (0.17) 17 (0.11) 1.649 (0.805–3.377) 0.1693
AG + GG 111 (0.68) 94 (0.63) 1.226 (0.767–1.961) 0.3943
Minor allele frequency 0.42 0.38 – –

TCN2 rs1801198 CC 45 (0.28) 41 (0.28) Referent – 0.7184

CG 85 (0.52) 81 (0.55) 0.956 (0.568–1.610) 0.8660
GG 33 (0.20) 26 (0.17) 1.156 (0.594–2.251) 0.6687
CG + GG 118 (0.72) 107 (0.72) 1.005 (0.611–1.653) 0.9850
Minor allele frequency 0.46 0.45 – –
a
Chi-square analysis.
b
Cochran–Armitage trend test.
c
W, wild allele; ins, allele with insertion.
d
Fisher exact test.
70 M. Szczepańska et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72

High order gene–gene interactions among all tested polymor- 3. Results

phic loci were studied by the multifactor dimensionality reduction
(MDR) approach (MDR version 2.0 beta 5). A detailed explanation
on the MDR method has been described elsewhere. Because
missing data are not allowed in MDR, samples with missing
genotypes were excluded from the analysis. Based on the obtained
testing balanced accuracy and cross-validation consistency values,
the best statistical gene–gene interaction models were established.
A 1000-fold permutation test was used to assess the statistical
significance of MDR models (MDR permutation testing module
0.4.9 alpha).
The genotype frequencies of all studied SNPs were consistent
with the Hardy–Weinberg equilibrium among both cases and
controls (p > 0.05), except for the PEMT rs4244593 (p = 0.016) in
infertile women with endometriosis.
The MAF for all markers (calculated from the control samples)
was above 16%, except for the CBS variant, with MAF equal to 10%
(Table 2). Genotype counts, OR, and 95% CI calculations for the 16
studied SNPs in 12 genes involved in folate and choline metabolism
are presented in Tables 2 and 3. There were no significant

Table 3
Associations of choline-metabolizing gene SNPs and risk of infertility in women with endometriosis.

Gene rs no. Genotype Cases Controls Odds Ratio (95% CI) pa pb

(frequency) (frequency)

BHMT rs7356530 AA 60 (0.37) 53 (0.35) Referent – 0.6370

AG 76 (0.47) 71 (0.48) 0.945 (0.579–1.545) 0.8231
GG 25 (0.16) 26 (0.17) 0.849 (0.438–1.646) 0.6285
AG + GG 101 (0.63) 97 (0.65) 0.920 (0.579–1.461) 0.7231
Minor allele frequency 0.39 0.41 – –

BHMT rs3733890 GG 85 (0.52) 71 (0.47) Referent – 0.6573

AG 60 (0.37) 66 (0.44) 0.759 (0.474–1.216) 0.2513
AA 17 (0.11) 13 (0.09) 1.092 (0.497–2.402) 0.8261
AG + AA 77 (0.48) 79 (0.53) 0.814 (0.522–1.270) 0.3647
Minor allele frequency 0.29 0.31 – –

BHMT2 rs625879 GG 60 (0.37) 56 (0.37) Referent – 0.5448

GT 80 (0.49) 66 (0.44) 1.131 (0.694–1.845) 0.6207
TT 22 (0.14) 28 (0.19) 0.733 (0.376–1.429) 0.3611
GT + TT 102 (0.63) 94 (0.63) 1.013 (0.640–1.604) 0.9569
Minor allele frequency 0.38 0.41 – –

CHDH rs893363 TT 71 (0.43) 68 (0.45) Referent – 0.9509

CT 68 (0.42) 58 (0.39) 1.123 (0.693–1.820) 0.6381
CC 24 (0.15) 24 (0.16) 0.958 (0.497–1.846) 0.8974
CT+CC 92 (0.57) 82 (0.55) 1.075 (0.688–1.679) 0.7522
Minor allele frequency 0.36 0.35 – –

CHDH rs2289205 GG 83 (0.51) 86 (0.58) Referent – 0.2586

AG 67 (0.41) 53 (0.35) 1.310 (0.819–2.096) 0.2598
AA 13 (0.08) 10 (0.07) 1.347 (0.560–3.241) 0.5049
AG + AA 80 (0.49) 63 (0.42) 1.316 (0.841–2.058) 0.2287
Minor allele frequency 0.29 0.24 – –

CHKA rs7928739 AA 56 (0.34) 52 (0.35) Referent – 0.7742

AC 77 (0.47) 71 (0.48) 1.007 (0.613–1.655) 0.9779
CC 30 (0.19) 25 (0.17) 1.114 (0.581–2.138) 0.7446
AC + CC 107 (0.66) 96 (0.65) 1.035 (0.648–1.652) 0.8854
Minor allele frequency 0.42 0.41 – –

PCYT1A rs712012 TT 68 (0.42) 63 (0.42) Referent – 0.6208

CT 66 (0.41) 67 (0.45) 0.913 (0.563–1.479) 0.7105
CC 28 (0.17) 20 (0.13) 1.297 (0.665–2.531) 0.4451
CT + CC 94 (0.58) 87 (0.58) 1.001 (0.638–1.570) 0.9965
Minor allele frequency 0.38 0.36 – –

PCYT1A rs7639752 GG 53 (0.32) 40 (0.27) Referent – 0.4775

AG 70 (0.43) 71 (0.48) 0.744 (0.439–1.260) 0.2709
AA 40 (0.25) 37 (0.25) 0.816 (0.445–1.497) 0.5110
AG + AA 110 (0.68) 108 (0.73) 0.769 (0.471–1.254) 0.2911
Minor allele frequency 0.46 0.49 – –

PEMT rs4244593 CC 39 (0.24) 48 (0.32) Referent – 0.4666

AC 99 (0.61) 75 (0.50) 1.625 (0.967–2.728) 0.0656
AA 25 (0.15) 27 (0.18) 1.140 (0.572–2.270) 0.7100
AC + AA 124 (0.76) 102 (0.68) 1.496 (0.910–2.460) 0.1112
Minor allele frequency 0.46 0.43 – –

PEMT rs4646406 TT 43 (0.27) 48 (0.33) Referent – 0.4783

AT 89 (0.55) 73 (0.49) 1.361 (0.813–2.278) 0.2402
AA 29 (0.18) 27 (0.18) 1.199 (0.616–2.335) 0.5934
AT + AA 118 (0.73) 100 (0.67) 1.317 (0.807–2.151) 0.2701
Minor allele frequency 0.46 0.43 – –
a
Chi-square analysis.
b
Cochran–Armitage trend test.
 M. Szczepańska et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72
Table 4
Results of gene-gene interactions analyzed by MDR method.
Genes and rs numbers
PEMT_rs4244593
MTHFR_rs1801133, PEMT_rs4244593
MTHFR_rs1801133, PEMT_rs4244593, PCYT1A_rs712012
MTRR_rs1801394, MTHFR_rs1801133, PEMT_rs4244593, PCYT1A_rs712012
Significant results are highlighted in bold font.
a
Testing balanced accuracy is the accuracy of classification of cases and controls in the testing dataset calculated as (sensitivity + specificity)/2.
b
Cross validation consistency is the number of times the model was selected as the best model after 10-fold cross-validation runs.
c
Significance of accuracy (empirical p value based on 1000 permutations).
differences between genotype and allele frequencies of any of the
investigated gene variants. The lowest, but not statistically
significant, p values of the trend test were observed for CBS
844ins68 and MTR rs1805087 (ptrend = 0.0527 and ptrend = 0.0771,
respectively).
The results of the exhaustive MDR analysis evaluating two- to
four-loci combinations of all tested SNPs for each comparison are
summarized in Table 4. The best combination of possibly
interactive polymorphisms was observed for rs1801133 of MTHFR
and rs4244593 of PEMT (testing balanced accuracy = 63.33%, cross
validation consistency of 10 out of 10, permutation test
p = 0.0240). None of these polymorphisms, when considered
separately, was significantly associated with endometriosis-
associated infertility (Tables 2 and 3). The 3- and 4-locus
combinations did not reach statistical significance in predicting
susceptibility to infertility.
4. Comment
Endometriosis is a complicated disorder resulting from the
interplay between genetic and environmental factors. Cohort
studies and increased rates of endometriosis in relatives of women
with this disorder have confirmed the significance of the genetic
background in the susceptibility to endometriosis [21–25]. The
polymorphic variants of genes encoding inflammatory mediators,
proteins involved in sex hormone activity, enzymes involved in
glucose homeostasis, and proteins involved in vascular function
and tissue remodeling have been considered as risk factors for
endometriosis [26].
During the various phases of the menstrual cycle, the
expression of hundreds of genes in the endometrium is under
the control of estrogen and progesterone [27]. The analysis of
the transcriptome profile during the window of implantation in
healthy women and women with endometriosis has demon-
strated the defective expression of many genes regulated by
progesterone [28,29] in the women with endometriosis. The
expression of HOXA10, progesterone receptor (PR-B), steroidogenic
factor-1 (SF-1) and estrogen receptor 2 (ESR2) genes is
changed by their promoter methylation level in the eutopic
endometrium in women with endometriosis [8–11]. In women
with endometriosis, hypermethylation of HOXA10, resulting in
its reduced expression, is associated with some defects in
uterine receptivity during the midsecretory phase of the
menstrual cycle [8,30]. The PR-B promoter is also hypermethy-
lated in women with endometriosis, providing a possible
explanation for the decreased levels of this receptor’s isoforms,
associated with a loss of progesterone response [9]. By contrast,
SF-1 and ESR2 promoters exhibited hypomethylation in women
with endometriosis, which may account for their overexpression
and increased estrogen activity [10,11]. DNA methylation is
also involved in the regulation of the immune response,
which may also play a role in the immunopathology of
endometriosis [31].
71
Testing balanced accuracya Cross validation consistencyb p valuec
0.4918 6/10 0.9080
0.6333 10/10 0.0240
0.6095 5/10 0.0680
0.4892 3/10 0.9200
To date, a correlation has been found between endometriosis-
associated infertility and genetic variants of ESR1, ESR2, 17b-
hydroxysteroid dehydrogenase type 1 and luteinizing hormone beta-
subunit gene [32–35]. Moreover, some polymorphisms in folate
pathway genes have been determined to be the cause of
unexplained infertility in women [36,37]. We did not, however,
observe a significant association of polymorphic variants of genes
encoding folate metabolism enzymes with endometriosis-associ-
ated infertility, when analyzed separately. These differences
between our findings in endometriosis-associated infertility and
those of other researchers regarding unexplained infertility can
result from the distinct complex etiology of this disorder,
differences in the racial structure of the studied populations, or
too small population samples. It is also possible that the number of
selected polymorphisms was insufficient to fully cover the
investigated genes and some associations may have been missed.
In our study we also did not determine a contribution of
separate polymorphic variants of genes encoding choline
metabolism enzymes to endometriosis-associated infertility.
However, we observed an interaction between the MTHFR
Ala222Val and rs4244593 of PEMT polymorphic variants in
women with endometriosis-associated infertility. The MTHFR
Ala222Val polymorphism has already been associated with
unexplained infertility in women [36,37]. PEMT is an important
enzyme in the biosynthesis of endogenous choline and
membrane phospholipids [38]. PEMT expression in humans
and mouse hepatocytes is up-regulated by estrogen, and
estrogen production is abnormal in women with endometriosis
[39]. Moreover, the dietary choline requirements in women can
be modulated by estrogen levels and PEMT genetic variants [40].
We may presume that rs4244593 of PEMT can be in linkage
disequilibrium with an unknown functional PEMT polymor-
phism, which may modulate the production of choline or
phospholipids and, together with MTHFR Ala222Val, contribute
to infertility in women with endometriosis.
Our study did not show an association of the 16 studied SNPs in
12 folate and choline metabolism genes, namely BHMT, BHMT2,
CBS, CHDH, CHKA, MTHFD1, MTHFR, MTR, MTRR, PCYT1A, PEMT and
TCN2, with endometriosis-associated infertility. We observed an
interaction, however, between MTHFR Ala222Val and PEMT
rs4244593 polymorphic variants in infertile women with
endometriosis. Our genetic studies were conducted on a limited
number of infertile women with endometriosis and controls,
therefore the role of these polymorphisms should be further
explored in other populations. Moreover, it would also be
interesting to study the prevalence of these gene variants in a
large number of women with endometriosis and women with
infertility, separately.
Acknowledgement
Supported by grant no. 502-01-01124182-07474, Poznan
University of Medical Sciences.
72 M. Szczepańska et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 157 (2011) 67–72
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejogrb.2011.02.003
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