Schlüsselwörter MTHFR-677C>T-Mutation - Infertilität - weibliche Reproduktionssteroide - ovarielle
Follikelflüssigkeit - LC-MS/MS-Analyse
Keywords MTHFR 677C>T mutation - infertility - female reproductive steroids - ovarian follicular
fluid - LC-MS/MS analysis
Introduction
The one-carbon metabolism is an essential biological process that regulates the biosynthesis
of nucleosides and the methylation of proteins, lipids, and DNA. Epidemiological evidence
suggests
that genetic variants of enzymes in the one-carbon metabolism pathway as well as folate
intake influence ovarian function [1 ]
[2 ]
[3 ]. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) plays a key role in
one-carbon metabolism as it catalyzes the reduction of 5,10-methylenetetrahydrofolate
to 5-methyltetrahydrofolate, a methyl donor for the remethylation of homocysteine
to methionine.
The common 677C>T mutation of the MTHFR gene, which results in an alanine-to-valine
substitution at amino acid position 222, leads to a thermolabile variant of the enzyme
with 70% reduced
activity [4 ]
[5 ]. The prevalence of homozygous 677TT and heterozygous 677CT carriers
is about 4–12% and 40–50% in the Caucasian population respectively [6 ]. The 677TT genotype is associated with a mild to moderately
elevated serum homocysteine concentration, particularly in patients with insufficient
folate supply [7 ]
[8 ]
[9 ]
[10 ].
MTHFR gene variants have been correlated with the increased risk of neural tube [11 ]
[12 ] and congenital heart defects [13 ]
[14 ] as well as recurrent
pregnancy losses [3 ]
[15 ]
[16 ]. Interestingly, folic acid supplementation has been reported as being potentially
effective in reducing the risk of such conditions [17 ]
[18 ]
[19 ]
[20 ].
We have previously demonstrated that homozygous 677TT carriers, when undergoing controlled
ovarian hyperstimulation and ovulation induction protocols, show a reduced ovarian
responsiveness as
they require more r-FSH during ovarian hyperstimulation, produce significantly fewer
oocytes, and have lower maximal serum estradiol (E2) concentrations [21 ]. Moreover, we have reported significantly reduced E2 in follicular fluid and reduced
E2 synthesis in granulosa cells (GC) of homozygous 677TT individuals compared with
heterozygous 677CT and homozygous 677CC carriers [22 ].
In contrast to our previous study [22 ] Rosen et al. reported that in his US-American study group, the C677T mutation was
not associated
with diminished response to ovarian stimulation. According to Rosen this could be
due to a higher intake of folic acid in addition to the recommended 400 µg of folic
acid per day. This appears
likely because foods such as breads and cereals in the United States are fortified
with folic acid.
The exact mechanism of folic acid action has not been entirely clarified. It has been
shown, that folic acid supplementation can reduce elevated concentrations of homocysteine
and compensate
for effects of the MTHFR 677C<T mutation [24 ]. We propose that increased folic acid supplementation might also reverse the hormonal
changes previously described by our group [21 ]
[22 ]. These studies had been
conducted in patients receiving a folic acid supplementation of 400 µg daily which
was recommended at that time in order to reduce the risk of neural tube defects [25 ]. By the time the current study was designed, a daily administration of at least
800 μg of folic acid was proposed to be effective in NTD
prevention [26 ]
[27 ]. Thus, we altered our clinical practice accordingly. Since
then, all of our patients receive a daily folic acid supplementation of 800 µg. The
prospective investigation of the effect of a daily folic acid administration of 400 μg
vs. 800 μg on
homozygous MTHFR 677TT would require a recruitment of women supplemented with low
folic acid daily doses. However, according to the present guidelines, a low folic
acid supplementation would
be considered unethical. Therefore, we tested our hypothesis in a retrospective analysis
of DNA and follicular fluid probes stored from infertility patients who had received
400 µg or 800 µg
folic acid daily while undergoing oocyte retrieval after controlled ovarian stimulation
(COH) in our institution in 2006 and after 2008. This retrospective case control study
forms the basis
of this report.
Subjects and Methods
Patients
Due to internal protocols, infertility patients treated in 2006 had received daily
folic acid supplementation doses of 400 µg. This group was designated Group-400 and
we chose 10 infertile
patients from the Centre for Reproduction, Munich, that met the inclusion criteria
(below) and had stored FF available. From 2008 onward, all patients took 800 µg folic
acid daily per os.
This group was designated Group-800. Exclusion criteria involved
signs or symptoms of anomalies such as uterine fusion defects or submucosal fibroids,
and acute inflammation,
an abnormal hormone profile (values for cycle day 3 FSH, LH, estradiol, testosterone,
DHEA-S, prolactin, SHBG, and TSH), and
hepatitis B/C and HIV infections.
In all patients, protective titers against rubella virus were also confirmed. Individuals
in Group-400 and Group-800 were matched according to their ovulation induction management
and
efficacy. As a result, we selected infertile Group-800 patients from the same center
with frozen FF available, that matched age, total dose of FSH, days of stimulation
and number of oocytes
retrieved (n = 28). Additionally, both groups were selected, so that half of the patients
per group was wildtype 677CC and the other half was homozygous 677TT and no significant
difference
were present between wildtype 677CC and homozygous 677TT carriers ([Table 1 ]).
Table 1
Main clinical features of the patients included in this study. Patients were matched
regarding their ovulation induction management and efficacy. As a
result, no significant difference was noted between the wildtype 677CC and the homozygous
677TT carriers.
MTHR 677C>T mutation status
Wildtype CC
Homozygous TT
p
NS: not significant
Group-400 (n = 10)
Age
34.80 ± 1.39
36.40 ± 1.20
NS
BMI
24.20 ± 2.03
27.80 ± 2.17
NS
Total FSH (IU)
1900.00 ± 269.72
2210.00 ± 152.80
NS
Days of stimulation
10.20 ± 1.59
10.80 ± 1.62
NS
Oocytes retrieved
14.40 ± 2.71
12.40 ± 2.71
NS
Group-800 (n = 28)
Age
36.21 ± 0.94
35.143 ± 0.94
NS
BMI
22.15 ± 0.55
21.95 ± 0.64
NS
Total FSH administered
1779.64 ± 100.86
2021.429 ± 89.86
NS
Days of stimulation
10.14 ± 0.32
10.42 ± 0.30
NS
Oocytes retrieved
14.57 ± 1.42
11.85 ± 1.18
NS
MTHFR 677C>T genotyping and the use of follicular fluids was approved by the Ethics
Committee of the University of Munich (LMU), and written informed consent was obtained
from all
participants. The study was carried out according to the guidelines of the Declaration
of Helsinki (clinical trial registration number: 178/0).
Folic acid supplementation
During 2006, all women were supplemented with 400 μg folic acid per os daily according
to the existing recommendations [25 ]
[28 ]
[29 ]. Treatment was initiated at least one month prior to any ART procedure and
continued at least up to pregnancy detection. Due to the recommendations [26 ]
[27 ] of 2008, the daily folic acid dose was increased to 800 μg. Thus, the current study
had two arms: Group-400 refers to women supplemented with 400 μg folic acid/day,
while Group-800 refers to women receiving 800 μg daily.
Treatment protocol
The “long” protocol was used, as described elsewhere [30 ]. Starting between day 20 and 23 of the pre-treatment cycle, patients
received the gonadotrophin-releasing hormone agonist nafarelin (Synarela, Pharmacia
GmbH, Karlsruhe, Germany) by twice-daily nasal spray applications for at least 10
days. When pituitary
down-regulation was confirmed (LH < 5 mIU/ml, E2 < 50 pg/ml) and double endometrial
width was less than 5 mm, ovarian stimulation was started, using recombinant (r-)
FSH (Puregon;
Essex Pharma, Munich, Germany) with a daily subcutaneous dose of 150–200 IU. FSH doses
were maintained or adjusted according to transvaginal sonographic folliculometry at
stimulation day 8.
When > 3 follicles reached a mean diameter of at least 17 mm, 250 µg of recombinant
chorionic gonadotrophin (Ovitrelle, Serono, Unterschleißheim, Germany) were administered
subcutaneously. Oocyte retrieval was performed under general anaesthesia by transvaginal,
ultrasound-guided aspiration 36 hours later by a single person (U.N.) blinded to the
MTHFR 677C>T
status.
DNA isolation and analysis of the MTHFR 677C>T mutation
All of our infertility patients were routinely genotyped for the MTHFR 677C>T mutation.
EDTA blood was drawn und genomic DNA was extracted from leukocytes with the QIAmp
DNA blood mini
kit (QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. Samples were
genotyped by employing the 5’ nuclease assay for allelic discrimination on an ABI
PRISM 7700 sequence
detection system (Applied Biosystems, Foster City, CA). The fluorogenic, allele-specific
oligonucleotide probes and PCR primers were provided by Applied Biosystems (TaqMan
genotyping assay
ID: C_1202883_20). PCR was performed in 96-well plates in a total volume of 10 µL
containing 5 µL TaqMan Universal PCR Master Mix (Applied Biosystems), 0.5 µL primer/TaqMan
probe mix, and
10 ng genomic DNA. The PCR profile included an initial denaturation step at 95 °C
for 10 min and 40 cycles at 92 °C for 15 s and 60 °C for 1 min. For allelic discrimination,
a
post-amplification evaluation was done using the ABI Sequence Detection Software supplied
with the instrument.
Follicular fluid collection, storage, and analysis
After transvaginal aspiration, all retrieved oocytes were removed and denuded immediately
for the subsequent ICSI procedure. FF from all follicles of the same patient were
pooled and
centrifuged immediately at 1000 xg for 10 min. Supernatants were aliquoted and stored
at −70 °C until further analysis. Samples were retrieved, thawed, and used for further
analysis, after
individual groups (i.e. 400 versus 800) and subgroups (i.e. MTHFR 677CC versus 677TT)
had been designated according to the criteria described above.
Determination of the folic acid concentration in the follicular fluid
The folic acid content in the FF of Group-400 and Group-800 individuals was compared
by off label use of a competitive chemiluminescent enzyme immunoassay kit on an automated
Immulite 2000
immunoassay system. According to the manufacturer’s (DPC, USA) protocol, this assay
is intended for quantitation of folic acid in human serum and plasma.
Determination of steroid hormones by isotope dilution liquid chromatography-photospray
ionization tandem mass spectrometry
Aliquots of frozen FF were shipped overnight on dry ice to the Georgetown University
Medical Center (GUMC) Bioanalytical Mass Spectrometry Core Laboratory, Washington,
DC. The research
staff of the GUMC was blinded to the MTHFR 677C>T genotype. Isotope Dilution Liquid
chromatography-tandem mass spectrometry (LC-MS/MS) was used in order to simultaneously
measure the
following steroid hormones: 17β estradiol, estrone and estriol in the estrogen profile
method [31 ] and, cortisol, progesterone, 17-OH
progesterone, testosterone, androstenedione, aldosterone, DHEA, and DHEA-S in the
state of the art steroid profile method [32 ]
[33 ].
An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with
a photospray source and a HPLC system (Shimadzu Scientific Instruments, Columbia,
MD) was used for the
determination of the steroid profiles, employing isotope dilution with a deuterium-labelled
internal standard (IS) for each analyte. Estrogen measurements were performed with
the same
instrument using an ESI ionization source.
300 µl of IS solution diluted in acetonitrile was added to a serum sample volume of
200 µl for deproteinization. After centrifugation at 13000 rpm for 10 min, 450 µl
of the supernatant was
mixed with 900 µl of water, and a 1000-µl aliquot was transferred into the LC-APPI-MS/MS
system onto a C8 column. After 3 min of washing with 3% methanol in ammonium acetate
followed by a
steroid elution with a water/methanol gradient, the sample was injected into the mass
spectrometer which continuously recorded two MRMs, one for each analyte and one for
each deuterated
internal standard corresponding to the labeled and the non-labeled hormones.
Statistical analysis
We performed an observational study using matched controls. Results are expressed
as mean ± standard error of the mean (SE). Statistical significance was assessed by
using the nonparametric
Mann-Whitney U test. Analysis of the data was performed with the Statistical Program
for Social Sciences 18.0.1 (SPSS Inc., Chicago, IL). Significance was assessed by
two-sided exact p and
was considered to be reached at an exact P value < 0.05.
Results
Effects of increased folic acid dosage on the folic acid concentration in the follicular
fluids
Folic acid contents were compared in the FF of Group-400 and -800 and this confirmed
significantly increased folic acid contents in the FF of Group-800 versus Group-400
patients (23.44 ± 1.38
vs. 11.91 ± 1.62 ng/ml respectively, exact p < 10−
3 ). This difference was confirmed also, when Group-800 and Group-400 patients carrying
the 677CC genotype
(24.06 ± 1.54 vs. 13.63 ± 1.86 ng/ml respectively, exact p = 0.001) and the 677TT
genotype (22.82 ± 2.34 vs. 10.20 ± 2.62 ng/ml respectively, exact p = 0.005) were
compared ([Fig. 1 ]).
Fig. 1
Folic acid concentration in the follicular fluid of both homozygous 677TT carriers
and wildtype 677CC controls. A significant increase in folic acid concentration was
achieved when administering 800 μg folic acid per os daily (Group-800, n = 28) compared
to the corresponding 400 μg folic acid per os daily supplementation (Group-400, n = 10).
Effect of increased folic acid administration on FF steroid hormones in 677TT and
677CC carriers
In Group-400, FF concentrations of E2 were significantly lower in 677TT than in 677CC
individuals (0.52 ± 0.08-fold, exact p = 0.032). In addition, estriol (E3) concentrations
also were
significantly lower in 677TT individuals (0.54 ± 0.05-fold, p = 0.016). A marginally
non-significant reduction was noted in 17-OH progesterone concentrations of 677TT
carriers
(0.78 ± 0.06-fold, exact p = 0.056) and no differences were observed between 677TT
and 677CC individuals of the Group-400 for FF concentrations of the other measured
steroids ([Fig. 2 ]).
Fig. 2
Hormone profile of the follicular fluid of homozygous 677TT carriers and wildtype
677CC controls under the effect of a daily dose of 400 μg (Group-400) or 800 μg
(Group-800) folic acid. A significant reduction in estradiol and estriol concentrations
was observed in carriers of the homozygous 677TT genotype that received 400 μg folic
acid
(Group-400). We also observed a marginal non significant reduction of 17-OH progesterone
concentrations in these samples (triangle). No MTHFR-dependent differences were found
in patients
receiving daily doses of 800 μg folic acid (Group-800).
Within Group-800, none of the analyzed steroid hormones showed significantly different
concentrations in follicular fluids of 677TT and 677CC individuals ([Fig. 2 ]).
Discussion
We previously reported significantly reduced E2 in serum [21 ], FF, and GC cultures [22 ] of infertile homozygous 677TT individuals when compared with 677CC homozygotes.
These data suggested an influence of the MTHFR 677C>T mutation on ovarian
steroidogenesis. Increased folic acid supplementation having been shown to compensate
for MTHFR 677T-effects [17 ]
[18 ]
[19 ]
[20 ], we previously
suggested this intervention might also restore ovarian steroidogenesis [21 ]
[22 ].
Since 2008, all our infertile patients undergoing assisted reproductive treatments
received a higher dose of folic acid and this prompted this retrospective comparison
of steroid metabolite
concentrations in follicular fluids of patients receiving different doses of folic
acid supplementation. This approach was limited to infertile IVF/ICSI-patients where
we had frozen FF samples
available, resulting in a small number of selected patients. Retrospective design
is an additional limitation of this study. While previous studies had been done by
using immunometric E2
measurement, this study employed liquid chromatography-tandem mass spectrometry (LC-MS/MS),
allowing more specific and sensitive analyses of a wider spectrum of steroids. Compliance
with our
recommendation of supplementing 400 μg or 800 μg of folic acid daily in the 400- or
800-group respectively was supported by significantly higher contents of folic acid
in FF of the 800-group
individuals ([Fig. 1 ]). In women supplemented with 400 μg folic acid daily, we confirmed significantly
reduced E2 concentrations in FF of
homozygous 677T allele carriers. In addition, our mass spectrometric analyses demonstrated
for the first time that 677TT homozygotes also had reduced concentrations of E3 when
compared to
677CC individuals. This is intriguing, as intrafollicular E3 has been shown not to
correlate with E2-concentrations, nor with size or maturity of ovarian follicles.
Indeed, intrafollicular E3
is not believed to be an end-product of E2 metabolism and rather the result of an
autonomous pathway of production from granulosa and theca interna cells [23 ]. Our report for the first time suggests that such pathways also may be affected
by the MTHFR 677TT-genotype in women with inadequate folate supply.
Steroid hormones are derivates of cholesterol, which is converted into pregnenolone,
a precursor for the production of all consecutive steroid hormones. The biosynthesis
of steroids follows
several pathways and therefore requires a battery of enzymes, mainly dehydrogenases
and cytochrome P450 oxygenases. Although our sample size was rather small and the
study was retrospective
and limited to infertile women, our results suggest that the MTHFR 677C>T mutation
may be associated with affected activities of enzymes involved in the steroid metabolism,
especially CYP19
(aromatase), as CYP19-dependent steroids were predominantly reduced in 677TT carriers.
We also observed a marginally non-significant reduction of 17-OH progesterone. It
could thus be
speculated that the MTHFR 677C>T mutation also is associated with an impaired CYP17
(17α-hydroxylase/17,20-lyase)-catalyzed conversion of progesterone into 17-OH progesterone.
Since this
enzyme is present only in theca cells, the MTHFR 677C>T effect might influence also
the metabolism of theca cells, in addition to GC.
The biological background of our observations is not known. MTHFR catalyzes the conversion
of 5,10-methylenetetrahydrofolate (5,10-MTHF) into 5-methyltetrahydrofolate (5-MTHF).
5-MTHF itself,
the biologically most active form of folate, functions as a methyl group donor for
the remethylation of homocysteine to methionine. This is an important step in the
metabolic network that
regulates the biosynthesis of nucleosides and the methylation of proteins, lipids,
and DNA.
On the level of ovarian cell function, the MTHFR 677C>T mutation may interfere with
the complex regulation of steroid biosynthesis and metabolism. To completely understand
the underlying
mechanisms, it may be necessary to study genes, which are involved in the process
of ovarian steroidogenesis and whose methylation may be altered by the MTHFR 677C>T
mutation.
Apart from alterations in DNA methylation patterns, the elevated plasma homocysteine
observed in homozygous T/T subjects might have a direct influence on ovarian steroidogenesis.
Its role in
inducing oxidative damage and stress has been well documented in many studies. Homocysteine
contains a reactive sulfhydryl group, promoting oxidation reactions and the formation
of reactive
oxygen species [34 ] which can result in a significant damage of proteins and the cell structure. Moreover,
there is growing evidence that
homocysteine leads to changes in the concentrations of other plasma aminothiols, generating
a pro-oxidant milieu by lowering the concentration of reduced cysteine [35 ]. In this way, hyperhomocysteinemia might directly affect or modify particular proteins
and/or enzymes involved in ovarian steroid
hormone biosynthesis and thus also lead to reduced steroid concentrations in homozygous
677TT individuals.
Interestingly, the FF hormone profile of 677TT carriers is restored to the normal
values of homozygous 677CC carriers by administering an increased dosage of 800 instead
of 400 µg folic acid.
Such an observation could be of clinical importance.
In fact, some recent clinical studies could demonstrate an association between higher
folate intake with increased fecundability [36 ],
lower risk of anovulation [37 ] and ovulatory [1 ] infertility. In addition, Kadir et
al. in a recent study reported significantly increased antral follicle counts in women
with 0,8 mg of folic acid daily [38 ].
Taking into account that homozygous 677TT carriers are expected to present with low
ovarian responsiveness and thus reduced oocyte output, sufficiently high folic acid
intake appears to
improve ART outcome. Indeed, results of a prospective cohort study suggest increased
higher folate intake was related to a higher probability of live birth among women
undergoing assisted
reproductive technology [39 ].
In conclusion, our findings support previous studies suggesting that folate metabolism
influences synthesis of E2, and potentially also other ovarian steroids, such as estriol
and
17-OH-progesterone. Our observations could help to gain further insights into the
mechanisms related to the MTHFR 677C>T mutation and its effects on folliculogenesis
and ovarian steroid
synthesis.
Capsule
Increased folic acid administration results in the restoration of the follicle fluid
hormone profile which may have an impact on the success of controlled ovarian stimulation
in infertile
women presenting with reduced MTHFR activity.
