Cardiovascular Research 53 (2002) 550–557
www.elsevier.com / locate / cardiores
Review
Hormones, genetic factors, and gender differences in cardiovascular
disease
Jacques E. Rossouw*
Women’ s Health Initiative, National Heart, Lung and Blood Institute, 1 Rockledge Center, Suite 300, 6705 Rockledge Drive, Bethesda,
MD 20892 -7966, USA
Received 6 July 2001; accepted 30 August 2001
Keywords: Gender; Gene therapy
1. Gender differences in age of onset of coronary heart
disease
On average, women develop heart disease some 10–15
years later than men. This raises the question of whether
there is some aspect of ‘femaleness’ which reduces risk, or
whether there is some aspect of ‘maleness’ that raises risk.
To date, most attention has been focused on the hypothesis
that endogenous estrogen is cardioprotective in women [1].
Rising rates of coronary heart disease (CHD) after the
menopause, and after oophorectomy, are among the strands
of evidence in humans that endogenous estrogen may
prevent CHD [2]. However, upon closer examination this
evidence is not persuasive, and in fact the evidence is
amenable to alternative explanations.
During the first 3 decades of adult life, low-density
lipoprotein (LDL) cholesterol levels are lower in women
than men, and this may contribute to the delayed onset of
CHD in women. A more widely held explanation for the
later onset of CHD in women is their higher high-density
lipoprotein (HDL) cholesterol levels, attributed to higher
endogenous estrogen levels in women. However, the
difference in HDL cholesterol between women and men is
an androgen effect, not an estrogen effect. Up to puberty,
young men and women have similar HDL cholesterol
levels. At puberty, concurrent with the rise in endogenous
testosterone levels, the HDL cholesterol levels in young
men decline to the adult level [3,4]. A 20% difference in
HDL cholesterol levels predicts at least a 20% difference
in CHD rates in the short term, and may predict even
larger differences in CHD rates over a lifetime [5]. Thus,
the entire gender difference in CHD risk may indeed be
*Tel.: 11-301-435-6669; fax: 11-301-480-5158.
E-mail address: rossouwj@nih.gov (J.E. Rossouw).
0008-6363 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 01 )00478-3
due to the lifelong difference in HDL cholesterol levels;
however, this difference is a consequence of having the Y
chromosome. During fetal development, the Y chromo-
some directs the formation of testes rather than ovaries,
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and the testes in turn produce testosterone and dihydrotes-
tosterone rather than estrone and estradiol as the primary
sex steroids [6]. The high levels of male sex hormones
drive down the HDL cholesterol levels, with consequent
higher early risk of CHD. Thus, the gender difference in
CHD may well be due the most basic genetic difference
between men and women, which is the presence of the Y
chromosome.
Some of the observational studies in adult males suggest
that higher levels of testosterone are associated with higher
(rather than lower) levels of HDL cholesterol, and with
lower risk of CHD [7]. Intracoronary infusion of testo-
sterone induces coronary artery dilatation and increases
coronary blood flow in men with coronary artery disease
[8]. These observations do not fit the concept that testo-
sterone is harmful to the male cardiovascular system. On
the other hand, androgen deprivation in men is associated
with enhanced endothelium-dependent dilatation in men
successfully treated for prostate cancer, while dilatation is
reduced in genetic females taking high dose androgens
[9,10]. In the laboratory, androgen receptor expression is
greater in macrophages from male than from female
donors, and lipid loading of male (but not female) macro-
phages was increased by dihydrotestosterone [11].
Dihydrotestosterone also increases the adhesion of mono-
cytes to endothelial cells, increases the expression of
endothelial vascular cell adhesion molecule-1, increases
platelet expression of thromboxane receptors, and in-
creases platelet aggregation [12,13]. However, there are
Time for primary review 31 days.
 J.E. Rossouw / Cardiovascular Research 53 (2002) 550 – 557
even more gaps in our knowledge about the real effects of
testosterone than there are for estrogen. As in the case of
estrogen, clinical trials with hard endpoints using an-
drogens, androgen antagonists, or selective androgen re-
ceptor modulators will ultimately be needed to resolve this
issue, but it is difficult to anticipate that such trials will be
done in the near future.
The statement that CHD rates in women rise steeply
after the age of menopause, and the corollary that this is
due to lower levels of estrogen after that age, is also open
to question. In actuality, there is no evidence for an
increase in the year-on-year rate of increase in CHD
around the age of menopause. The linear relationship
between age and CHD incidence as seen on a semilogarit-
hmic plot shows that there is a constant proportional
increase in CHD incidence with age, with no inflection
upward at the average age of menopause [14]. This is
evidence for an age effect, and evidence against an effect
of menopause. The Nurses’ Health Study investigators
have reported that, after controlling for age and smoking
status, the natural menopause is not associated with an
increased risk for CHD [2]. The same investigators have
reported that, in contrast to the natural menopause, bilater-
al oophorectomy is associated with an increased risk for
CHD in women who had never taken estrogen after
menopause. However, the study had very few cases of
CHD in women with oophorectomy, and the increased risk
was no longer significant in the multivariate analysis.
Nonetheless, the use of estrogens appeared to eliminate
this increased risk. It is possible that women who have a
hysterectomy and bilateral oophorectomy (often done for
menorrhagia / metrorrhagia associated with endometrial
hyperplasia) are at higher risk for CHD because of the
co-existence of metabolic risk factors such as central
obesity, high blood pressure, lipid disorders, and glucose
intolerance. The finding of an apparent lower risk in
women who subsequently used estrogen would be subject
to the biases discussed below. In summary, the post-
menopausal increase in risk is most likely due to age and
not the menopause, and the increase in risk in women after
oophorectomy may be due to confounding by other risk
factors.
2. Observational studies of estrogen users
By far the most persuasive evidence in favor of a
protective effect for estrogen comes from the large number
of cohort studies comparing CHD risk in postmenopausal
women currently using estrogen to never-users. These
studies have shown consistently that CHD risk is 35–50%
lower in estrogen users, after adjusting for other risk
factors [15,16]. The lower risk has been found in studies of
estrogen alone, as well as in studies of estrogen in
combination with a progestin [16]. For healthy women, the
551
lower risk is found in those who have recently started
estrogen as well as in long-term users [16].
These findings from observational epidemiology provide
the rationale for clinical trials testing whether and to what
degree current use of postmenopausal hormone therapy
prevents a first heart attack. However, the observational
epidemiology is not sufficient to prove the case, because
even the best studies may be subject to a variety of
systematic biases that could lead to an overestimation of
benefit and an underestimation of harm from hormone
therapy, hence the need for an unbiased estimate from
clinical trials. These biases in observational studies include
healthy user selection bias, compliance bias, surveillance
bias, and survivor bias [14,17].
In combination, these biases will lead to a systematic
overestimation of benefit, and an underestimation of risk in
observational studies. Adjusting for baseline differences in
risk factors will mitigate healthy user selection bias, but
will not correct for compliance, surveillance, or survivor
bias. Thus, the real benefit for CHD may be much less than
predicted by the observational studies, or there may be no
benefit at all [14]. The clinical trials to date have failed to
show overall benefit for CHD over the short term (ongoing
trials will ascertain the long term effects particularly in
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women without prevalent CHD) [18].
3. Biological mechanisms for cardioprotection by
estrogen
The possibility that estrogen may reduce CHD risk has
stimulated a wide variety of studies that attempt to explain
the presumed benefit. Because it was unexpected, fewer
studies have been done to explain the apparent excess risk
early in the course of treatment, found in recent clinical
trials (see below). Effects of estrogen that may predict
benefit include: lowered LDL cholesterol and lipoprotein
(a) levels, raised HDL cholesterol levels, reduced fibrino-
gen levels and enhanced fibrinolysis (reduced plasminogen
activator inhibitor-1 (PAI-1) and increased D-dimer levels),
reduced homocysteine levels, antioxidant properties, and
improved endothelial function (e.g. reduced E-selectin
levels and enhanced flow-mediated dilatation) [1]. Es-
trogen and progesterone reduce lipid accumulation in
macrophages from female, but not male, donors [19]. On
the other hand, several mechanisms that might increase
risk after estrogen administration have been found: tri-
glycerides increase, some coagulation markers increase
(e.g. Factor VII, prothrombin fragments 112, activated
protein C resistance), and the inflammatory marker C-
reactive protein increases [20–23]. Some observational
studies have suggested that certain of the lipid markers
changed by estrogen administration are associated with
higher relative risks for CHD in women than in men,
including HDL cholesterol and triglycerides [23,24]. How-
552 J.E. Rossouw / Cardiovascular Research 53 (2002) 550 – 557
ever, particularly in respect of coagulation and inflamma-
tion, laboratory measurements do not predict whether the
predominant effect will be favorable or unfavorable.
Studies of venous thromboembolism (including clinical
trials) provide unequivocal evidence that the overall effect
is indeed procoagulant [25,26].
Compounding this difficulty in interpreting laboratory
measurements is the fact that progestins counteract some
of the estrogen effects, and that the clinical expression of
metabolic changes may be time-dependent. The early
excess risk for arterial disease observed in the Heart and
Estrogen / Progestin Replacement Study (HERS) may have
been due to an initial procoagulant or inflammatory effect
on susceptible plaques, while the favorable effects in the
survivors may be due to the later assertion of the generally
favorable lipid effects [27]. In HERS, participants with
higher levels at baseline had the largest decrease in
lipoprotein (a) on treatment, and had a more favorable
clinical outcome than participants with lower baseline
levels [28]. The role of estrogen’s direct vascular effect is
unclear, since impaired endothelial function has not yet
been established as a risk factor for CHD. Interestingly,
current estrogen users do not appear to have a lower risk
for angina, as one would expect if direct vascular effects
were important [16]. On the other hand, in a clinical trial
sublingual estrogen relieved exercise-induced angina [29].
Overall, the studies of mechanisms have not resolved the
core issue of whether estrogens protect against CHD.
It is important to realize that almost all the studies of
mechanism used oral estrogen preparations, and may turn
out to have little relevance towards explaining the gender
difference in CHD. Ovarian estrogen directly enters the
systemic circulation through the inferior vena cava, unlike
oral estrogens, which enter the portal vein and undergo
first pass hepatic circulation. Because of extensive metabo-
lism in the liver, in order to achieve similar blood levels
the dose of oral estrogen needs to be approximately ten
times that of non-oral (e.g transdermal) estrogen. These
doses of estrogens profoundly influence the hepatic metab-
olism of a variety of proteins, including lipid apoproteins,
coagulation proteins, and (probably) C-reactive protein
[20–22,30–34]. The large effects on lipids and coagulation
proteins described for oral estrogens are greatly attenuated,
absent, or in the opposite direction with non-oral estrogens.
Non-oral estrogens have very modest effects on lowering
LDL-cholesterol and lipoprotein (a), have no effect or
reduce triglycerides, have no effect on HDL-cholesterol,
and have a modest or no effect on levels of coagulation
proteins [30–34]. Non-oral estrogens retain the ability to
improve endothelial function [35]. Additional mechanistic
studies, as well as epidemiologic studies and clinical trials,
that focus on the role of non-oral estrogen preparations are
needed. Of interest, epidemiologic studies in post-
menopausal women have not shown an association of
endogenous estrogen levels with CHD [36,37].
4. Genetic mechanisms for cardioprotection by
estrogen
The role of the major monogenic disorders such as
familial hypercholesterolemia and familial hyper-
homocysteinemia in arterial disease is well established.
Even within kindreds with familial hypercholesterolemia,
affected females develop CHD later than in males, pre-
sumably because the same (unknown) factors that delay
onset in non-affected females continue to operate in
affected females [38]. Though estrogen administration
lowers LDL cholesterol in women with familial hyper-
cholesterolemia [39], this pharmacologic property does not
necessarily mean that endogenous estrogen has the same
effect. There is no sex difference in the elevated LDL
cholesterol levels of individuals with familial hypercholes-
terolemia [38]. Rather, as in non-affected individuals,
factors that blunt the impact of a given level of LDL
cholesterol in females (or accelerate it in males) need to be
sought.
The importance to arterial disease of polymorphisms in
the genes that code for coagulation proteins and markers of
inflammation is not clear. For example, a variety of
polymorphisms in the fibrinogen gene are known to affect
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circulating fibrinogen levels, and fibrinogen levels have
been associated with arterial disease [40]. However,
studies seeking to quantify the association of a fibrinogen
gene polymorphism with arterial disease have generally
yielded disappointing results. This may be because any
individual polymorphism has a modest influence on fibrin-
ogen levels, and the levels are simultaneously affected by
both other genetic factors, and importantly, by environ-
mental factors [40]. Hence, it is difficult to discern the
direct effect of any individual polymorphism on clinical
disease. In addition to fibrinogen, polymorphisms in the
genes for FVII, FXIII, FV, prothrombin, thrombomodulin,
tissue plasminogen activator, PAI-1, and platelet-mem-
brane glycoproteins have been described [40]. Except for
fibrinogen, the relationships of the phenotypic factors
affected by these polymorphisms with arterial disease are
uncertain. A possible interaction of gender with poly-
morphisms in the FVII gene has been described, in that
plasma activity of FVII in males varies markedly accord-
ing to the presence of three of these polymorphisms, but in
females these polymorphisms are associated with much
smaller changes in FVII activity [41]. Estrogen affects
many of these phenotypic factors [20–22,32–35], but in
general it is not known whether there is a protective or
harmful interaction of estrogen with the polymorphisms as
risk factors for arterial disease.
Both males and females have a- and b-estrogen re-
ceptors in the vascular endothelium, smooth muscle, and
myocardium, though receptor numbers may be higher in
females because estrogen induces their expression [1,42].
One group of investigators found a gender difference for
 J.E. Rossouw / Cardiovascular Research 53 (2002) 550 – 557
the non-genomic effects of estrogen on the response of
coronary arteries to acetylcholine. Males did not show the
reversal of acetylcholine-induced vasoconstriction in cor-
onary arteries after acute estrogen administration found in
females [43]. On the other hand, genetic males receiving
long-term estrogen therapy show enhanced flow-mediated
dilatation [44]. The presence of higher numbers of func-
tional estrogen receptors in females may relate to the
inhibition of injury-induced vascular intimal thickening by
estrogen in female arteries, and the lower prevalence of left
ventricular hypertrophy in women compared to men
[1,45,46]. Estrogen receptor numbers are lower in female
atherosclerotic arteries [46]. There is some potential for
polymorphisms of the estrogen receptors themselves to
affect the clinical outcome of estrogen administration. For
example, the common ER Pvu II and Xbal genotypes are
not associated with differences in HDL cholesterol levels,
but after estrogen administration women who are hetero-
zygous for the genotype have a greater elevation of HDL
cholesterol [47]. The clinical relevance of this finding is
unknown.
5. Evidence of early harm from clinical trials
The evidence emerging from trials of hormone replace-
ment therapy (HRT) to prevent coronary heart disease
(CHD) continues to confound the belief that HRT is
cardioprotective. On the contrary, a fairly consistent
pattern of early harm is emerging from secondary and, to a
lesser extent, from primary prevention trials. Long term
effects of HRT on the cardiovascular system, and also on
the overall health of postmenopausal women, are being
evaluated in the ongoing primary prevention trials [18].
The findings from four clinical trials of HRT in women
are consistent with an early excess of arterial cardiovascu-
lar events: HERS, the Papworth HRT and Atherosclerosis
Survival Enquiry (PHASE), the Women’s Estrogen for
Stroke Trial (WEST), and the Women’s Health Initiative
(WHI) [27,48–50]. In addition, a combined analysis of
short-term trials (typically lasting a year) in healthy
women showed a non-significant 39% excess risk for CHD
[51]. Support for short-term harm comes also from the
early trials in men, and several observational studies of
new users of HRT among women with prior CHD [52–55]
and one of healthy women in the Group Health Co-
operative study [56]. The evidence comes from secondary
and primary prevention trials, trials using conjugated
equine estrogens (CEE) with and without medroxyproges-
terone (MPA), and trials of estradiol (oral and transder-
mal). Many of the trials have weaknesses (and in the case
of WHI the data have not been published); individually
their findings can be rationalized away, but in aggregate
the findings are persuasive. Therefore, the question is no
longer whether the early harm is real, but rather what is
553
causing the harm. It remains possible that the lipid changes
induced by HRT may result in long-term benefit for
women who survive the first years. This question remains
unanswered at this point, but for many women the answer
may be moot if ways of avoiding the early harm are not
found.
6. Mechanisms for explaining an increased risk for
arterial disease shortly after commencing HRT
Hypotheses for explaining early harm center around a
potential interaction of HRT with coagulation and / or
inflammation mechanisms in a subset of women with
vulnerable plaques. According to these hypotheses, expo-
sure to HRT for certain women with a susceptibility factor
in the form of an elevated blood or tissue level, or with an
augmented response to HRT perhaps due to a genetic
mutation, may result in plaque destabilization and throm-
bosis.
HRT induces a wide variety of changes in coagulation
factors in healthy women. Some of these are likely to be
procoagulant, while others are likely to profibrinolytic,
hence the clinical effect cannot be predicted from labora-
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tory studies. Because of an emphasis on a few factors, in
particular reductions in levels of fibrinogen and PAI-1,
many scientists believed that HRT exerted a favorable
effect on coagulation. As noted above, clinical trials and
epidemiologic studies confirm that the net effect of HRT is
to promote coagulation in the venous system [25,26].
There is a growing suspicion that coagulation factors
beyond platelets may be more important in arterial disease
than previously appreciated. Among the coagulation mech-
anisms the Factor V Leiden (FVL) mutation and the
prothrombin G20210A mutation are currently of most
interest for both venous and arterial disease.
FVL produces a coagulation factor V which resists the
action of the major natural anticoagulant, activated protein
C. Hence, the phenotypic expression can be measured as
activated protein C (APC) resistance. The prevalence of
heterozygous FVL in whites is about 4.8% (range 3–7%),
with a much lower prevalence of 0.05% in Blacks and
Asians [57]. FVL is associated with venous thromboembo-
lism (VTE), and is found in about 18% of unselected cases
of VTE and about 40% of selected cases with presumed
familial thrombophilia. The risk for VTE associated with
FVL is amplified by exposure to oral contraceptives (OCs)
and the risk associated with APC resistance is amplified by
HRT use [57–59]. In contrast, the association of FVL with
CHD has not been found in men, and in women it has been
inconsistent [60–63]. In a case–control study of younger
women, FVL was associated with MI in those with other
risk factors for CHD, particularly smoking [62]. However,
two other case–control studies in women failed to show an
association with CHD, or an interaction with HRT use
554 J.E. Rossouw / Cardiovascular Research 53 (2002) 550 – 557
[60,65]. In one cross-sectional study of women referred to
a lipid clinic for management of dyslipidemia, HRT use in
the absence of FVL was associated with a reduced risk for
atherothrombotic arterial disease, while HRT use in the
presence of FVL was associated with an increased risk
[63]. Thus, the status of FVL as a risk factor for CHD is
much less certain than for VTE, and only one study
suggested an interaction with HRT. On the other hand,
there is insufficient data to exclude a possible role for FVL
in CHD in women, particularly in the context of CHD
associated with HRT use.
The prothrombin G20210A mutation is procoagulant
because it increases prothrombin levels. The prevalence is
lower than FVL, being found in about 2.7% of whites
(range 1–4%). As for FVL, the prevalence is much lower
in Blacks and Asians (0.06%) [57]. It is associated with
VTE, being found in 7% of unselected cases and 16% of
selected cases of VTE. In women, but not in men, the
mutation has been found to be associated with CHD
[64–67]. A cross-sectional analysis of women with
dyslipidemia found that the HRT use was associated with
decreased risk for atherothrombotic disease in the absence
of the G20210A mutation, and increased risk in its
presence [66]. A case–control study of non-fatal MI in
women with hypertension (a parallel study of women
without hypertension was negative) found that presence of
the mutation alone was associated with an odds ratio for
MI of 1.5 (95% CI 0.6–1.4) and current use of HRT alone
was associated with an odds ratio of 0.9 (0.3–7.7) [64].
However, the presence of both the mutation and use of
HRT was associated with an odds ratio of 10.9 (2.2–55.2,
P50.002). This supra-multiplicative effect of the com-
bined factors represents true synergism. If confirmed, a
large synergistic effect between HRT and a prothrombotic
factor in the range of 3–5% could explain the pattern of
early harm and late benefit seen in the HERS trial [64].
HRT also affects several markers of inflammation, some
in a potentially unfavorable direction and some in a
potentially favorable direction. HRT increases circulating
levels of C-reactive protein and matrix metalloproteinases
including matrix metalloproteinase-9 (MMP-9) [22,68,69].
On the other hand, HRT decreases circulating levels of cell
adhesion molecules E-selectin, intercellular adhesion
molecule-1, and vascular cell adhesion molecule-1, and in
the short term improves endothelial function [70,71]. An
intriguing hypothesis put forward by Cannon. et al. centers
on the effects of estrogen on MMP-9 [68]. According to
this hypothesis, acute administration may be harmful, and
chronic administration may be beneficial. Estrogen de-
creases PAI-1 levels and increases plasmin activity, which
in turn increases the expression of MMP-9. Local eleva-
tions of matrix metalloproteinases in the vulnerable plaque
are associated with weakening and rupture of the thin
fibrous cap, which is thought to precipitate the thrombotic
occlusion of the vessel [69]. Thus, the acute effect of HRT
might be unfavorable in women with vulnerable plaques.
On the other hand, for women who survive the acute phase
or who do not have vulnerable plaques, the long-term
effect of elevated levels of MMP-9 might be favorable,
since the proteinases might prevent the accumulation of
matrix proteins and improve arterial compliance.
These intriguing observations and hypotheses have great
potential relevance to women, as they affect the future use
of exogenous estrogens in the form of HRT. It is unknown
whether endogenous estrogens affect these markers of
inflammation and coagulation, and therefore unknown
whether these factors have a role in explaining the gender
difference in cardiovascular disease. If anything, the
known and suspected interactions of estrogen with these
markers go against the observation of delayed onset in
women, since they raise the risk for arterial disease in
women. Younger women may have higher rates of VTE
events than men, but it is not known whether endogenous
estrogen or use of oral contraceptives explain that observa-
tion [72].
7. Conclusions
The gender difference in the age of onset of CHD is as
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yet unexplained. A natural starting point is that the
biologic effects of the XX and XY chromosomes are
expressed through sex hormones, and that these explain the
gender difference in CHD. However, the respective roles
of female and male sex hormones in this regard remain
unclear. To date, research has focused overwhelmingly on
estrogen. The substantial evidence in favor of a protective
effect of estrogen in women is being called into question
by recent clinical trial data indicating that HRT is not
protective, and at least in women with existing heart
disease may initially increase risk. Because of the in-
creased in risk in secondary prevention trials, and the
absence of published clinical trial data for primary preven-
tion, HRT is no longer being recommended for prevention
of CHD [73]. It remains possible that HRT given for many
years may be cardioprotective.
Oral estrogen has pronounced effects on blood markers
and cell function, including endothelial cells and mono-
cyte-derived macrophages. Some of these effects may be
favorable, others unfavorable, and thus laboratory studies
cannot predict clinical outcomes. Clinical trials indicate
that the net effect of estrogen is prothrombotic. The
metabolic effects of oral estrogen are very different from
those of non-oral (e.g. transdermal) estrogen; the latter is
more likely to mimic the effects of endogenous estrogen,
and thus may be more informative about the mechanisms
underlying the gender difference in CHD. More studies on
the effects of non-oral estrogens are needed, including
trials with clinical outcomes.
It is possible, but unproven, that in part the gender
difference may be due to adverse effects of androgens in
men, and more studies of androgens, antiandrogens, and
 J.E. Rossouw / Cardiovascular Research 53 (2002) 550 – 557
selective androgen receptor modulators are needed. Males
and females not only differ in their levels of sex hormones,
the effects of those sex hormones also differ by gender. It
appear likely that more research will reveal that the gender
difference has male as well as female components.
In addition to effects of the sex chromosomes, it is
possible that autosomal mutations may influence the risk
for CHD. By definition, autosomal mutations are as
frequent in males as in females, hence the search should be
focused on mutations that modify risk and interact with sex
hormones. This research is in its infancy, but a good
example of its potential is the interaction between prot-
hrombotic mutations and estrogen (in oral contraceptives
and HRT formulations) that underlie a substantial propor-
tion of VTE in families with thrombophilia, and also in
unrelated individuals with VTE. The most pressing sci-
entific challenge of the moment is to find ways of
identifying the women who are at increased risk for CHD
when they commence HRT. The prothrombotic mutations
are being investigated in this regard, as are several other
markers of coagulation and inflammation. If it becomes
possible to identify susceptible women, they could be
counseled against HRT, while the non-susceptible women
would have a higher chance of experiencing benefit from
HRT.
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