Factor V Leiden mutation accelerates the onset of
natural menopause
Kristel M. van Asselt, MD,
1,2,3
Helen S. Kok, MD,
1,2,3
Petra H. M. Peeters, MD, PhD,
1
Mark Roest, PhD,
1,4
Peter L. Pearson, PhD,
2
Egbert R. te Velde, MD, PhD,
3
Diederick E. Grobbee, MD, PhD,
1
and Yvonne T. van der Schouw, PhD
1
ABSTRACT
Objective: Smoking is consistently associated with a younger age for menopause. Although this
may be because of the direct toxic effects of tobacco smoke on follicles, we hypothesize that there
may also be a relationship between smoking and a vascular origin of early menopausal onset.
Several lifestyle factors have been investigated, but never factors of the clotting cascade. The
objective of this study, then, was to determine the effect of factor V Leiden mutation and smoking
with respect to age at menopause.
Design: Data were used from a subset of 373 postmenopausal participants of a Dutch population-
based cohort, born between 1911 and 1925. All women had experienced natural menopause, with-
out use of hormone replacement therapy.
Results: Female carriers of the factor V Leiden mutation (n = 14) reported the onset of meno-
pause at an earlier age than noncarriers (n = 359; difference, 3.1 years; 95% CI: 0.3, 5.9). Smoker
carriers (n = 5) were 4.3 years younger at menopause than smoker noncarriers (n = 92; 95% CI:
0.9,7.6). In nonsmokers, this relationship was less strong.
Conclusions: We found that the factor V Leiden mutation was related, but not statistically sig-
nificant, to an earlier age at menopause; smoking possibly enhances this effect. The mutation can
be one of the genetic determinants of menopausal age operating through a vascular mechanism.
Keywords: Age at menopause – Determinants – Factor V Leiden – Smoking.
T
he onset of natural menopause varies between
40 to 60 years, with a median age of 50 to 51.5
years,
1,2
and may be caused by the exhaustion
of the oocyte/follicular reserve.
3
Early meno-
pause has been associated with several chronic diseases
later in life. An annual risk reduction of 2% (95% CI:
1%, 3%) each year that menopause is delayed has been
reported for cardiovascular mortality.
4
Also, osteopo-
rosis is associated with an earlier age at menopause;
5
Japanese women who experience menopause after age
50 have a decreased risk of low bone density (odds ratio
0.70; 95% CI: 0.50, 0.99).
6
On the other hand, an early
end of reproductive life might lower the risk of breast
cancer; for each year increase in age at menopause, the
relative risk of breast cancer is 1.03 (95% CI: 1.02,
1.03).
7
Several determinants of menopausal age, including
various lifestyle factors, have been investigated. How-
ever, these factors only explain less than 3% of the
large variation in age at menopause.
2
Genetic factors
could well be responsible for the remaining variation
in age at menopause, as twin and sibling studies
have shown heritabilities varying between 50% and
85%.
8-10
The most important lifestyle factor affecting age at
menopause discovered so far is smoking. Women who
Received October 3, 2002; revised and accepted December 23, 2002.
From the
1
Julius Center for Health Sciences and Primary Care, the
2
De-
partment of Biomedical Genetics, the
3
Department of Reproductive
Medicine, and the
4
Research Laboratory of the Department of Clinical
Chemistry, University Medical Center, Utrecht, The Netherlands.
Financial support was provided by N. V. Organon, The Netherlands, the
University Medical Center Utrecht and the Netherlands Heart Foundation.
Address correspondence to: K. M. van Asselt, University Medical Cen-
ter Utrecht, Location Academic Hospital Utrecht, Heidelberglaan 100,
Hp Str. 6.119, 3684 CX Utrecht, The Netherlands. E-mail: k.m.
vanasselt@jc.azu.nl.
Menopause: The Journal of The North American Menopause Society
Vol. 10, No. 5, pp. 477-481
DOI: 10.1097/01.GME.0000056040.51813.1A
© 2003 The North American Menopause Society
嘷
⬁
Text printed on acid-free paper.
Menopause, Vol. 10, No. 5, 2003
477
smoke reach menopause approximately 0.8 to 2 years
earlier than nonsmokers.
11-13
Enhanced follicu-
lar depletion from a toxic effect may explain some
of this effect of smoking. Recent observations on
the effect of the biologically active ingredients of
cigarette smoke in mice demonstrate an up-regulation
of the pro-apoptosis genes in ovaries, which results in
an increased depletion of the ovarian reserve.
14
Smoking is a well-established risk factor for vascular
disease, which led us to hypothesize that a vascular
pathway might also be involved in ovarian aging.
Smoking has been identified as one of the most impor-
tant determinants of fibrinogen levels,
15
and fibrinogen
levels in turn have been associated with occlusive
thrombotic disease.
16
Although findings are still specu-
lative in this field, it may be interesting to test whether
thrombosis also affects highly vascularized organs like
the ovaries. Factor V Leiden mutation is known for its
association with thrombotic events.
17
Heterozygosity
for the factor V Leiden mutation is found in 2% to
4% of the people in Western Europe, including the
Dutch population. The purpose of this study was to ex-
amine the individual and combined effects of the factor
V Leiden mutation and smoking on age at natural
menopause.
METHODS
Population
Women were recruited from participants of an ex-
perimental breast cancer screening program (DOM,
Diagnostic Investigation Mamma cancer), carried out
between December 1974 an October 1980. Participants
were born between 1911 and 1925 and resided in the
city of Utrecht, The Netherlands. Women were invited
for repeated examinations at 1-, 2-, and 4-year inter-
vals. The source population consisted of 12,239 women
who attended at least the first and second screening be-
cause, at the second exam, a questionnaire on lifestyle
factors, including smoking, was administered. During
the first visit, urine samples were collected and stored
at −20°C.
A random sample of 650 women was selected to de-
termine the factor V Leiden polymorphism. All women
gave informed consent to use their data and urine for
future scientific research. The Institutional Review
Board of the University Medical Center Utrecht, The
Netherlands, approved the study to determine factor V
Leiden.
Genotyping
DNA was isolated from 50 mL urine.
18
Factor V
Arg/Gln506 genotype was determined from each DNA
fraction using Polymerase Chain Reaction and hybrid-
ization with antigen specific oligonucleotides. The
antigen specific oligonucleotide for factor V Arg506
was ‘3-TGGACAGGAAGGAATAC-5’ and for Factor
V Gln506 ‘3-GGACAGGCGAGGAATAC-5’. Dots
were visualized on x-ray films (DuPont, Brussels,
Belgium) after overnight radiation. For 47 women, no
urine was stored; and in 41 women, either DNA iso-
lation or genotyping for factor V Leiden was not
successful.
Determinants
Smoking status was assessed at the second screening
visit, and women were defined as smokers if they re-
ported being current smokers at that time. Menopausal
status was self-reported and questioned at the first and
fifth screening visit. The first mention of menopausal
age was included in the analysis. Menopause was de-
fined as at least 12 consecutive months of amenorrhea.
Women were excluded if they were still premenopausal
at the fifth examination (30 women) or if they had un-
dergone either a hysterectomy or bilateral ovariectomy
(103 women). Women who did not menstruate after
cessation of oral contraception or who reported having
used hormone replacement therapy (HRT) were also
excluded (47 women), and 8 women were excluded be-
cause data on menopausal status were missing. The re-
sulting study population consisted of 374 women with a
natural menopause.
Statistical analysis
Descriptive analysis was used to describe the study
population. Mean age at menopause was computed for
factor V Leiden carriers and noncarriers. A t test with
nonequal variances was used to compare mean meno-
pausal ages of carriers and noncarriers. Stratification
according to smoking was performed to determine the
presence of effect modification. Interaction was tested
using a linear regression model, with menopausal age
as a dependent variable and smoking, factor V Leiden,
and the multiplicative factor of smoking and carriership
included as independent variables.
All statistical analyses were performed using SPSS
10.1 for Windows.
RESULTS
The allele frequency of the factor V Arg506Gln
polymorphism was 4%. One homozygote was found
VAN ASSELT ET AL
Menopause, Vol. 10, No. 5, 2003478
but was left out of the analyses because of the small
number. The mean age of the remaining 373 women at
enrollment was 58.0 (SD 4.1) years; it was equal for
carriers and noncarriers.
All women with the factor V Leiden mutation re-
ported a postmenopausal status at the first screening
visit. For noncarriers, 21 women were premenopausal
at the first exam but reported to be postmenopausal at
the fifth examination.
The mean age at menopause for the whole group was
50.2 years (SD 3.7, range 39-60). The smoking percent-
age was 35.7% in carriers and 25.7% in noncarriers
(P = 0.4). Results are shown in Table 1.
Smoking was associated with a slightly decreased
age at menopause, although this was not statistically
significant. Women carrying the mutation had meno-
pause earlier than noncarriers (mean age at menopause
of 47.3 years v 50.4 years; 95% CI for the difference in
means: 0.3, 5.9). Stratified for smoking, we observed
that this relationship was just significant in nonsmokers
(difference 2.4 years, 95% CI: 0.0, 4.8) but was strong
and statistically significant in smoking women (differ-
ence 4.3 years, 95% CI: 0.9, 7.6). Modification by
smoking of the association between factor V Leiden
carriership and menopausal age was also tested in a lin-
ear regression model but was nonsignificant (P = 0.4).
DISCUSSION
This study suggests that the factor V Leiden muta-
tion was associated with an earlier age at menopause,
and that smoking may possibly enhance this effect. De-
spite the small number of individuals investigated, the
phenotypic effect of a reduction in age at natural meno-
pause of 3 years in carriers of factor V Leiden was
substantial. The finding suggested modification by
smoking, as the effect was strong in smokers and bor-
derline significant in nonsmokers. However, the inter-
action term in the regression model was not significant,
which may have been due to the small numbers in the
strata.
One limitation of our study is that smoking behavior
was reported at study recruitment, when most women
were already postmenopausal. However, from demo-
graphic studies it is known that the quitting rate of
women 35 to 50 years of age in the ‘70s was about 1%
to 3%, depending on educational level.
19
This small
percentage suggests that smoking behavior at study re-
cruitment probably still reflects the smoking status at
menopausal transition. Although some misclassifica-
tion of smoking behavior during menopausal transition
may have occurred, this is most likely nondifferential,
ie, women with factor V Leiden did not stop smoking
more often than noncarriers or the reverse. Nondiffer-
ential misclassification predominantly leads to under-
estimation of the true association.
The association between smoking and menopausal
age has earlier been studied in the DOM population,
and a significant relationship was found; smoking
decreased menopausal age by 1.2 years (P < 0.001).
20
Our sample was apparently too small to replicate this
finding.
Because menopausal status is almost always mea-
sured retrospectively and by questionnaires, this inher-
ently can give problems with validation and reproduc-
ibility. However, these aspects have been reported
previously in this cohort.
21
The reproducibility was
studied in 4,892 women who attended the first and the
fifth screening, with an interval of 7.5 years. For 80%
of the sample, the two reports differed by at most
1 year. The findings were similar to those of the
Nurses’ Health Study.
22
Furthermore, the number of
years since menopause decreases the validity of report-
ing menopausal age; the percentage of women report-
ing their true age at menopause was higher when less
TABLE 1. Menopausal age according to smoking status and factor V Leiden mutation in 373 women
Number Mean age (SE) 95% CI
a
P-value
a
Nonsmokers 276 50.3 (0.22) −0.6, 1.1 0.55
Smokers 97 50.1 (0.38)
Noncarriers’ factor V Leiden 359 50.4 (0.19) 0.3, 5.9 0.035
Carriers’ factor V Leiden 14 47.3 (1.29)
Nonsmoking
Noncarriers’ factor V Leiden 267 50.4 (0.22) 0.0, 4.8 0.05
Carriers’ factor V Leiden 9 48.0 (1.69)
Smokers
Noncarriers’ factor V Leiden 92 50.3 (0.38) 0.9, 7.6 0.013
Carriers’ factor V Leiden 5 46.0 (2.07)
CI, confidence interval; SE, standard error.
a
Two-sided t-test.
FACTOR V LEIDEN MUTATION AND MENOPAUSE ONSET
Menopause, Vol. 10, No. 5, 2003 479
time had passed since menopause. Both under-as over-
estimations occurred when more time had passed since
menopause, but the mean of the self-reported meno-
pausal age was higher than the true menopausal age.
21
The average time since menopause in our study was
7 years for noncarriers and 10 years for carriers. This
suggests that carriers may be less accurate in self-
reported menopausal age than noncarriers, but it also
suggests that our estimate could even be an underesti-
mation of the real association because the true meno-
pausal age of the carriers may have occurred earlier
than they reported.
In general, blood samples are used to isolate DNA.
However, when blood is unavailable, frozen urine
samples can be considered as a useful and valid method
of obtaining DNA in large cohort studies, as published
recently.
18
There is currently no biological explanation for why
factor V Leiden would lead to an early menopause, but
we hypothesize a possible vascular pathway. The factor
V Leiden mutation is responsible for a reduced factor
Va inactivation, leading to a reduced inhibition of pro-
thrombin. It is known that this mutation leads to an in-
creased risk of venous thrombosis.
17
Furthermore, in
two pooled analyses, factor V Leiden mutation was
also significantly associated with the risk of myocardial
infarction.
23,24
Moreover, the risk of premature ischemic stroke has
been reported to be higher for female carriers of the
factor V Leiden mutation than for male carriers.
25
This
suggests the possibility of an interaction of this geno-
type with female sex hormones. Estrogens are known
to further lower the inactivation rate of factor Va,
which could explain the interaction with sex.
26
Estrogen levels in the follicular fluid can cyclically
reach high levels. If estrogen and factor V interact, then
the effect must be most pronounced locally in and near
the ovaries. Ovaries are highly vascularized organs,
and angiogenesis of developing follicles is known to
play an important role in both the follicular and luteal
phase.
27
Enhanced activation of factor V, as exhibited
by the factor V Leiden mutation and by high estrogen
levels,
26
may lead to formation of ovarian micro-
thrombi and subsequently affect the vascularization of
the follicular system and reduce oxygen levels of the
microenvironment of the follicles. Infarction of devel-
oping follicles could lead to accelerated depletion of
the oocyte/follicle pool that would be translated into an
earlier onset of menopause.
Miscarriages have been reported to occur more fre-
quently in female carriers of the factor V Leiden muta-
tion.
28
This also may be explained by adverse effects of
the microenvironment of follicles because oocytes
from severely hypoxic follicles have been associated
with abnormalities in chromosomes.
27
The relationship between thrombosis and smoking is
still controversial;
29
nevertheless, fibrinogen levels are
raised in smokers, possibly increasing the susceptibil-
ity of smokers to thrombosis.
30
Moreover, in premeno-
pausal women, the combination of smoking and carri-
ership of the factor V Leiden mutation leads to a much
higher risk of myocardial infarction compared with
smoking or carriership of factor V Leiden mutation
alone.
31
Early menopause has been associated with cardio-
vascular disease, but some risk factors of cardiovascu-
lar disease, like smoking or factor V Leiden mutation,
may affect menopausal age. If this effect is proven in
future studies, underlying cardiovascular disease might
predispose a woman to early menopause.
Another less likely explanation would be that factor
V Leiden may be in linkage disequilibrium with an-
other gene involved in ovarian aging. This then implies
that this gene must be located very near to the factor V
Leiden mutation on chromosome 1.
If subsequent studies confirm our results, factor V
Leiden would be the second genetic factor shown to
contribute to variation in age at natural menopause. The
first was the estrogen receptor 1 (ESR1) polymor-
phism, in which the minor allele frequency is 47% and
responsible for a 1-year decrease in age at natural
menopause in homozygotes.
32
The factor V Leiden
mutation has a much smaller mutant allele frequency,
but its relative impact on menopausal age is much
larger. Future research should also include ESR1 poly-
morphism to test interaction between estrogen sensitiv-
ity and a disabled clotting system.
CONCLUSION
The factor V Leiden mutation may be associated
with an earlier age at menopause. The mutation can be
one of the genetic determinants of menopausal age. Our
finding suggests new lines of investigation of the pos-
sible role of clotting factor polymorphisms in determin-
ing age at natural menopause. More studies are needed
to accept this prospect with confidence.
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