The Cardiovascular Effects of Chronic Hypoestrogenism in Amenorrhoeic Athletes
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- O’Donnell, E. & De Souza, M.J. Sports Med (2004) 34: 601. doi:10.2165/00007256-200434090-00004
In premenopausal women, the most severe menstrual dysfunction is amenorrhoea, which is associated with chronic hypoestrogenism. In postmenopausal women, hypoestrogenism is associated with a number of clinical sequelae related to cardiovascular health. A cardioprotective effect of endogenous oestrogen is widely supported, yet recent studies demonstrate a deleterious effect of hormone replacement therapy for cardiovascular health. What remain less clear are the implications of persistently low oestrogen levels in much younger amenorrhoeic athletes. The incidence of amenorrhoea among athletes is much greater than that observed among sedentary women. Recent data in amenorrhoeic athletes demonstrate impaired endothelial function, elevated low- and high-density lipoprotein levels, reduced circulating nitrates and nitrites, and increased susceptibility to lipid peroxidation. Predictive serum markers of cardiovascular health, such as homocysteine and C-reactive protein, have not yet been assessed in amenorrhoeic athletes, but are reportedly elevated in postmenopausal women. The independent and combined effects of chronic hypoestrogenism and exercise, together with subclinical dietary behaviours typically observed in amenorrhoeic athletes, warrants closer examination. Although no longitudinal studies exist, the altered vascular health outcomes reported in amenorrhoeic athletes are suggestive of increased risk for premature cardiovascular disease. Future research should focus on the presentation and progression of these adverse cardiovascular parameters in physically active women and athletes with hypoestrogenism to determine their effects on long-term health.
Unprecedented numbers of women are now participating in physical activity and sport on a regular basis.The physiological benefits of regular exercise are well documented. However, between 2–46% of athletic women have reported experiencing amenorrhoea (absence of menstruation for 3 consecutive months or more) at any given time, compared with 2–5% of eumenorrhoeic sedentary women.A chronic deficit in energy intake relative to energy expenditure is the likely cause of exercise-associated amenorrhoea,[3–5]although insufficient oxidisable metabolic fuel,and psychological stresshave also been indicated.
The harmful effects of hypoestrogenism upon bone health in amenorrhoeic athletes has previously been reported.[8,9]Specifically, a medical condition termed ‘the female athlete triad’ defines the combined or independent existence of disordered eating, amenorrhoea and low bone mass in athletes.[2,10]Since a cardiovascular role for oestrogen (E2) has been identified, impaired cardiovascular health has been suggested as an additional clinical sequelae of hypoestrogenism in female athletes.This hypothesis can be related to studies that show chronic hypoestrogenism exerts unfavourable effects upon serum lipids,[12,13]endothelial function,haemostatic parameters,blood flow,homocysteine[17,18]and antioxidant status.However, models of hypoestrogenism and cardiovascular health are typically derived from observations of postmenopausal women. Although endogenous E2 may be deemed cardioprotective, it is important to acknowledge that recent data from the Women’s Health Initiative trial of hormone replacement therapy (HRT) have identified a significantly increased risk of adverse cardiovascular events.Therefore, it is prudent to acknowledge that administration of exogenous hormones to postmenopausal women is likely to confer different vascular health outcomes when compared with endogenous E2 in younger premenopausal women.
In amenorrhoeic athletes, recent data identifies impaired endothelial function,elevated low-density and high-density lipoprotein levels,reduced metabolites of nitric oxide (nitrates and nitrites),and increased susceptibility to lipid peroxidationas potential cardiovascular consequences, presumably due to the hypoestrogenism. Although the clinical significance of these consequences remains undetermined, these findings are also suggestive of a potentially increased risk of premature cardiovascular disease (CVD).
This purpose of this paper is to bring together the relevant data surrounding exercise-associated amenorrhoea using both traditional and novel markers of CVD. Due to the dearth of information on amenorrhoeic athletes and cardiovascular health, data derived from postmenopausal women, animal models, and in vitro studies are drawn upon to provide an indication of how E2 status may impact such markers. While these studies offer insights into the effects of the hypoestrogenic milieu, there are obvious limitations when extrapolating these findings to amenorrhoeic athletes, i.e. the postmenopausal woman is likely to present with multiple risk factors that are coincidental with aging, whereas the amenorrhoeic athlete will likely have few, if any, risk factors. Thus, extrapolations are intended simply to assist our ability to understand the effects of hypoestrogenism per se on key cardiovascular health parameters. We also utilise comparisons with anorexic women because of similarities in age, menstrual status, and often exercise behaviours. Consideration of the combined and independent effects of amenorrhoea, exercise and diet upon serum lipids, lipid peroxidation, nitric oxide and endothelin are examined in this review. Due to the predictive value of recently identified novel markers to assess risk of future CVD or cardiovascular events, endothelial function, homocysteine and C-reactive protein (CRP) are also discussed. We performed searches on Medline and PubMed for English language studies (English language abstracts from foreign language studies were also considered), that were published between January 1, 1966, and November 1, 2003, and were indexed with key words including amenorrhoea, athlete, CRP, E2, cardiovascular, endothelin, homocysteine, lipids and nitric oxide. We also reviewed the bibliographies of retrieved articles and conference proceedings to obtain additional citations.
1. Total Cholesterol
Cholesterol, a waxy, fat-like substance, is present in every cell in the body. Functionally, cholesterol is important to cellular membrane structure, as well as to synthesise vitamin D, the adrenal gland hormones, and the steroid hormones, namely E2, progesterone, and androgens.Cholesterol also plays a key role in the formation of the bile secretions that emulsify fat during digestion.Cholesterol is derived from dietary sources and is synthesised de novo, predominantly by the liver and intestines, in the cytoplasm and microsomes from the two-carbon acetate group of acetyl-coenzyme A (CoA).The level of cholesterol synthesis is regulated, in part, by 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, the rate-limiting enzyme in cholesterol biosynthesis.Dietary fat intake, specifically saturated fatty acids, also regulates cholesterol levels by increasing the hepatic sterol pool and by down-regulating low-density lipoprotein (LDL) receptors, the principle route of clearance of LDL-cholesterol (LDL-C),and the primary cholesterol-containing atherogenic lipoprotein (60–70% cholesterol).The other two major classes of lipoprotein particles found in fasted serum are: (i) high-density lipoproteins (HDL), considered the atheroprotective lipoprotein (20–30% cholesterol); and (ii) very low-density lipoproteins (VLDL), primarily a triglyceride-containing lipoprotein (10–15% cholesterol).
The link between blood lipid levels and CVD is well recognised.[26,27]Desirable serum total cholesterol (TC) levels are identified as <5.18 mmol/L,while elevated TC (>5.18 mmol/L) is acknowledged as an independent risk factor for the development of coronary heart disease and cerebral vascular diseases.Factors shown to affect TC levels include age, sex, heredity, HRT, sedentary lifestyle, cigarette smoking, excessive alcohol intake, being overweight, obesity, diabetes, diet and thyroid dysfunction.[27,29–32]To avoid repetition, the impact of hypoestrogenism and exercise on lipid metabolism is not discussed here, but in the relevant sections of this paper.
1.1 Postmenopausal Women
Longitudinal data observing the effects of menopause on lipid parameters report increased TC, mainly caused by increased LDL-C and triglycerides, and decreased HDL-cholesterol (HDL-C).Such alterations in TC are predictive of mortality due to CVD,and are associated with agingand E2 deficiency.Studies have consistently shown that postmenopausal women receiving unopposed[36–38]and opposed[38–41]HRT exhibit reduced TC levels, typically by lowering LDL-C, with some data also showing an increase in HDL-C, and no consistent effect observed for triglycerides. The mechanisms of E2 regulation upon LDL and HDL-C, and triglyceride metabolism are detailed in sections 2, 3 and 4 of this paper.
1.2 Amenorrhoeic Athletes
The majority of studies reporting TC levels in amenorrhoeic and eumenorrhoeic athletes[23,42–44]show no significant group differences. Table I lists these studies. However, one study, which was the only study to have adequate statistical power to detect group differences, reported elevated TC levels in amenorrhoeic compared with eumenorrhoeic athletes,with borderline high levels of TC observed in the amenorrhoeic athletes (5.49 ± 0.16 mmol/L), [compared with published standards of the National Cholesterol Education Program, Adult Treatment Panel IIIclassification system]. Contributing factors to this elevation were not only associated with increased LDL-C, as seen in postmenopausal women, but also with elevations in HDL-C and triglycerides. Possible mechanisms for these individual lipoprotein elevations are discussed in sections 2, 3 and 4 of this paper. The LDL : HDL ratio, identified as a strong predictor of cardiac health in men,was reported as 1.7 in both amenorrhoeic and eumenorrhoeic athletes,well below that considered a CVD risk factor.The TC : HDL ratio, recently shown to be the best predictor of CVD at any TC level in women,can also be calculated as low (2.8) for both amenorrhoeic and eumenorrhoeic athletes.Again, this risk prediction calculation does not place these athletes in an ‘at-risk’ category for CVD.
Amenorrhoeic athletes have often been reported to consume less dietary fat compared with eumenorrhoeic athletes,[21,44,46]although several studies have failed to show this.[11,42]As a percentage of total energy intake, several studies report dietary fat intake of ≤30% in amenorrhoeic athletes.[21,44,46–48]Type of dietary fat in these studies has typically not been identified. Since saturated fat has a positive and unsaturated fat a negative correlation with serum TC,identification of type of dietary fat may be an important consideration when performing a TC assessment. Similarly, dietary fat intake of ~30%has also been documented in anorexia nervosa patients, with some reports of elevatedand others of normalTC levels. However, it is important to understand that anorexia nervosa patients typically consume less total calories per day (~1150 kcal/day) compared with amenorrhoeic athletes (~1700 kcal/day[21,44]), and that despite normal relative dietary fat intake, i.e. 30%, anorexia nervosa patients consume very low absolute dietary fat quantities (~32 g/day) compared with amenorrhoeic athletes (~50 g/day[21,44]). Where increased TC is observed in anorexia nervosa, it has been attributed primarily to increased LDL-C levels.The paradoxical increase in serum TC in anorexia nervosa patients, despite very low to normal dietary fat intake, is related to an abnormality in cholesterol metabolism.More specifically, increased TC has been linked with increased synthesis of triglyceride-rich lipoproteins together with an unchanged cholesterol synthesis rate.Contrary to this observation, amenorrhoeic athletes do not exhibit increased VLDL-C (triglyceride-rich lipoproteins) when compared with eumenorrhoeic athletes,and it is not known if the cholesterol synthesis rate is altered in amenorrhoeic athletes.
Nutritional status may be an important factor in cholesterol metabolism. This postulate can be directly related to findings in anorexia nervosa patients, in that those who present with the poorest nutritional status, as defined by low circulating levels of transthyretin, a biological marker of malnutrition, also present with the greatest elevations in TC.Nutritional deficit in the form of either restricted food intake and/or increased exercise energy expenditure has frequently been observed in amenorrhoeic athletes[21,46,47,54]compared with their eumenorrhoeic counterparts. However, it remains unclear if those amenorrhoeic athletes with the largest nutritional deficits, i.e. energy deficits induced by food restriction, exhibit the lowest fat intakes and/or the greatest alterations in cholesterol metabolism.
Strikingly similar to anorexia nervosa, although less marked, amenorrhoeic athletes also exhibit decreased plasma glucose, insulin, leptin and insulin-like growth factor-1, as well as elevated growth hormone and cortisol concentrations when compared with their eumenorrhoeic counterparts.The impact of these metabolic aberrations on TC metabolism in amenorrhoeic athletes is not established.
The clinical significance of altered TC levels in amenorrhoeic athletes is not clear. Elevated LDL-C with concomitantly increased HDL-C may preserve the cardiovascular risk profile. Whether TC concentrations are impaired due to the hypoestrogenic state per se is not known, but it is reasonable to postulate that similar TC metabolism alterations may exist between anorexia nervosa patients and amenorrhoeic athletes because of a great number of similar metabolic, and to a lesser extent dietary, aberrations. Further studies will help identify the extent of these possible similarities.
2. Low-Density Lipoproteins
The LDL particle is heterogeneous in composition and variable in cholesterol content, with small, dense particles having greater atherogenicity than larger, more buoyant LDL particles.Functionally, LDL plays a key role in the transportation of cholesterol to all tissues, but primarily to adipose cells and the liver. Figure 1 shows the key regulators steps for LDL-C concentrations, namely, LDL-C production rate, level of hepatic receptor activity, and the affinity of LDL-C for the LDL receptor.Approximately 60–80% of circulating LDL-C is taken up by the liver via receptor-dependent mechanisms.[57,58]Apolipoprotein B, the major protein moiety of LDL and other non-HDL atherogenic lipids, acts as the ligand to the LDL receptor.[56,59]Hyperlipidaemia, diabetes mellitus (types I and II) and hypothyroidism influence circulating levels of LDL-C.[31,60]In addition, dietary cholesterol and fatty acids also influence circulating levels of LDL-C, mediated by altering either hepatic LDL receptor activity, LDL-C production rate, or both.When dietary cholesterol intake is increased, expansion of the pools of sterol, which can be synthesised de novo, occurs within liver cells.This results in down-regulation of the LDL receptors, causing a plasma increase in the concentration of LDL-C.Elevated serum levels of LDL-C (>3.37 mmol/L) are recognised as an independent risk factor for CVD,[27,61,62]and are also associated with abnormal vasodilatory function in response to flow-mediated dilatation, facilitating the development of atherosclerosis.
2.1 Postmenopausal Women
Premenopausal women have lower LDL-C levels compared with age-matched men.After menopause, LDL-C levels increase, frequently surpassing those of age-matched men, with a trend toward smaller, more dense and subsequently more atherogenic particle sizes.The menopausal increase in cardiovascular risk is associated with unfavourable elevations in TC and triglycerides.[13,56]In an extensive review, Schwertz and Penckoferidentified that 25–50% of the potential cardioprotective effect of E2 is associated with its effect on blood lipids and lipoproteins. The impact of E2 on serum lipids derives primarily from E2-receptor-mediated effects on the hepatic expression of apolipoprotein genes.[66–68]Endogenous E2and E2 replacement and HRT, both transdermal and oral,[70,71]reduce circulating LDL-C levels. This E2-associated lowering effect has been attributed to an increased catabolic rate of hepatic cholesterol into bile acidsand increased expression of LDL receptors on cell surfaces.[73,74]In contrast to this, the postmenopausal, hypoestrogenic milieu causes reduced LDL receptor activity,contributing to the well documented elevation in plasma LDL-C concentrations in this population.
2.2 Amenorrhoeic Athletes
Studies observing LDL-C levels[21,23,42,43,46]and LDL particle sizein amenorrhoeic athletes have been reported, but findings are equivocal, due, in part, to variable methodologies and small sample sizes. These data are shown in table II. LDL-C levels have been reported to be significantly elevated,[21,46]whereas other studies report non-significant differences in amenorrhoeic compared with eumenorrhoeic athletes.[35,42]LDL particle sizeand apolipoprotein B levels[43,44]have also been documented to be similar between the two groups. It is not clear whether the reported elevation in LDL-C levels seen in amenorrhoeic athletes[21,46]is of clinical significance.
Interestingly, studies that report elevated LDL-C levels in amenorrhoeic athletes[21,46]also show statistical significanceor a strong trendfor both reduced caloric intake and dietary fat intake. The paradoxical increase in circulating LDL-C despite reduced dietary fat intake is consistent with findings in chronically hypoestrogenic anorexia nervosa patients.Normal or elevated levels of TC in anorexia nervosa has predominantly consisted of increased LDL-C levels, despite typically very low dietary cholesterol intake and a normal cholesterol synthesis rate.Mechanisms for this phenomenon in this patient group are associated with the known down-regulatory effect of altered thyroid hormones, i.e. reduced total triiodothyronine (T3), and lowered E2 levelson the cellular number of hepatic LDL receptors,[73,75]thereby contributing to increased plasma LDL-C levels.Similarly, but less marked, hypoestrogenic amenorrhoeic athletes have also displayed low total T3 status,[76,77]as well as lower caloric intake,and perhaps most notably, significantly less calories derived from dietary fat[21,46,78]when compared with their eumenorrhoeic counterparts. These findings lend credence to the possibility that, although less severe than those observed in anorexia nervosa patients, the metabolic aberrations observed in amenorrhoeic athletes may, in part, explain the reported elevations in their LDL-C levels. This avenue of potential application merits further exploration.
It is possible that along a continuum of dietary restriction, the amenorrhoeic athletes that demonstrate the greatest nutritional aberrations will also demonstrate the least favourable LDL-C profiles. Furthermore, down-regulation of LDL receptors due to hypoestrogenism and altered thyroid status may likely play an important role in cholesterol metabolism in amenorrhoeic athletes. These possibilities have not yet been explored. In addition, hypoestrogenism may prove to have a more deleterious effect upon LDL-C metabolism in amenorrhoeic athletes as a function of time, i.e. the longer the episode of amenorrhoea the greater the risk of elevated LDL-C. This potential long-term effect of hypoestrogenism has yet to be confirmed.
3. High-Density Lipoproteins
HDL particles correlate inversely with the risk of CVD,and are acknowledged as being anti-atherogenic.The cardioprotection afforded by HDL in the prevention of CVD originates from the role of reverse cholesterol transport whereby HDL is postulated to scavenge surplus cholesterol from peripheral tissues for delivery to and disposal by the liver for excretion via bile.[80,81]Important to cellular cholesterol homeostasis, reverse cholesterol transport is proposed to be a result of apolipoprotein A-I promotion of cholesterol efflux from the cells via receptor (scavenger receptor B type I), and non-receptor (passive diffusion) mediated mechanisms.Apolipoproteins A-I and A-II are the two major proteins associated with HDL.
In addition to the above recognised properties, HDL-C has also demonstrated anti-thrombotic,and favourable vascular toneeffects, including enhanced endothelial function.The antioxidant properties of HDL can be directly related to studies that show HDL attenuates LDL oxidation,inhibits the atherogenic effect of oxidised LDL-C,as well as increasing the half-life of endothelial nitric oxide,all of which are recognised processes that contribute to healthy endothelial function.
Taken together, these data endorse the postulation that HDL and HDL-C may in some way protect against the development of atherosclerosis and heart disease via both cholesterol-dependent and -independent mechanisms.
3.1 Postmenopausal Women
Although consistently higher in women than men during all life stages after puberty,HDL-C levels tend to decrease in postmenopausal compared with premenopausal women,with significant reductions effected as a consequence of menopause.Bilateral oophorectomy also results in decreased circulating levels of HDL-C.Diminished levels of circulating E2 play a key role in these observations, in part due to the known E2 stimulatory effect on apolipoprotein-A1.Evidence supporting a beneficial role of E2 upon HDL-C metabolism has also been shown.[90,91]Following oestrogen replacement therapy, Pickar and colleaguesobserved that HDL-2, the main HDL subfraction found to increase in response to HRT, resulted in enriched HDL phospholipids, thereby promoting an elevation in apolipoprotein AI production while maintaining similar metabolic clearance of HDL-C. Thus, E2 not only modifies HDL-C levels, but also its lipid composition and distribution, thereby augmenting the plasma capacity to execute cholesterol efflux.
3.2 Amenorrhoeic Athletes
Several investigators have evaluated serum HDL-C levels in amenorrhoeic athletes (table III). Findings are variable, including significantly elevated,similar[23,42,43,46]and non-significant trends toward lower[23,42]HDL-C concentrations in amenorrhoeic compared with eumenorrhoeic athletes. However, consistent with the observation that endurance-trained female athletes possess much higher HDL-C levels compared with sedentary women,eumenorrhoeic and amenorrhoeic athletes also demonstrate significantly increased HDL-C levels compared with eumenorrhoeic sedentary controls.[43,46]Elevated HDL-C levels are considered a negative risk factor, negating the presence of another single risk factor.Friday and coworkerspostulated that it is likely the extra 4 hours of weekly exercise participation in the amenorrhoeic compared with eumenorrhoeic athletes in their study contributed to the observed greater elevation in HDL-C. In support of this postulate, similar findings of elevated HDL-C levels have also been reported in female runners, irrespective of menstrual status, with the highest levels observed in those running the greatest distances (>64 km/week).The observed improvements in HDL-C concentrations with increased running distance demonstrates the favourable effect of endurance exercise on cardiovascular risk in amenorrhoeic and eumenorrhoeic runners, and are suggestive of oestrogen-independent mechanisms effecting the HDL-C increase. Indeed, it has been postulated that the primary mechanism for the exercise-induced elevation in HDL-C is due to increased skeletal muscle and/or adipose lipoprotein lipase activity, resulting in accelerated breakdown of triglyceride rich lipoproteins, facilitating plasma clearance and provision of free fatty acids as a fuel source for muscle metabolism or adipocyte storage.However, lipoprotein lipase activity was not assessed by Friday and coworkersand can only be speculated to be elevated as a consequence of a greater volume of endurance activity in the amenorrhoeic versus eumenorrhoeic athletes. Despite being mechanistically unclear, the aforementioned observations demonstrate a robust relationship between endurance exercise and HDL-C, independent of menstrual status.
Women with anorexia nervosa have also been reported to have reduced circulating HDL-C levels, a consequence hypothesised to be a product of their previously discussed hypometabolic profile.This observation can be explained in part by the finding that total T3 is a potent mediator of apolipoprotein-I gene expression.Consequently, when T3 is reduced, apolipoprotein-I gene expression is also reduced, diminishing HDL-C levels. Although not evaluated simultaneously in any of the studies measuring HDL-C levels in amenorrhoeic athletes, Loucks et al.and others[76,98]have documented significant reductions in T3 levels in amenorrhoeic compared with eumenorrhoeic athletes. It is reasonable to suggest that the presence of reduced T3 in amenorrhoeic athletes may serve as one pathway by which circulating apolipoprotein A-I levels can be negatively impacted. However, apolipoprotein A-I levels have been shown to be comparable in amenorrhoeic and eumenorrhoeic athletes,[21,44]and when comparing amenorrhoeic athletes with eumenorrhoeic sedentary controls.It would be interesting to evaluate all of these parameters in a single study.
Triglycerides are esterified fatty oils that form the core of chylomicrons and VLDL-C, and are comprised of a glycerol and three free fatty acid molecules.Triglyceride metabolism is met by two pathways, the exogenous and endogenous cycle. The exogenous cycle is responsible for processing dietary fat, while the endogenous cycle involves internal production of triglyceride-rich lipoprotein particles that are manufactured in the liver.Recent recommendations for TC from the National Cholesterol Education Programpropose that triglycerides are an independent risk factor for CVD, with desirable levels being <1.7 mmol/L. The association between elevated triglyceride levels and CVD is not well defined and remains somewhat controversial. This is, in part, due to the close inverse metabolic relationship between triglycerides and HDL-C,and the documented co-existence of elevated triglyceride levels with other CVD risk factors such as hypertension and abdominal obesity.These associations have made isolated assessment of elevated triglycerides as a predictor of CVD problematic. Furthermore, triglyceride measurement may not accurately reflect CVD risk status because of the lack of information on the specificity of the triglyceride-rich lipoproteins that are present in plasma.[99,101]For example, some triglyceride-rich lipoproteins are highly heterogeneous in terms of size and lipid composition, such as chylomicrons and large VLDL, and these are thought to be unable to enter the arterial wall, and are therefore considered non-atherogenic.Conversely, small VLDL and intermediate-LDL-C can enter into the arterial intima, and as such, are considered highly atherogenic.[99,101]As such, VLDL is the most readily obtainable measure of atherogenic remnant lipoproteins.
A number of factors contribute to higher than normal triglyceride levels (>1.7 mmol/L) in the general population, including obesity, being overweight, physical inactivity, cigarette smoking, excess alcohol intake, high-carbohydrate diets (>60% of energy intake), and diseases such as type II diabetes, chronic renal failure and nephrotic syndrome, and certain drugs including corticosteroids, E2, retinoids, higher doses of β-adrenergic blocking agents, as well as heredity factors.
4.1 Postmenopausal Women
Triglyceride profiles undergo unfavourable changes in menopause and are reported to be elevated in postmenopausal women.This increase can be linked to a strong relationship between excess visceral fat accumulation (android adiposity) and triglyceride levels in postmenopausal women.This type of fat distribution among women, postulated to be due to hypoestrogenism,is associated with unfavourable alterations in the lipid profile, such as increased LDL-C and TC and decreased HDL-C concentrations.Such findings support the postulate that the co-existence of other CVD risk factors is associated with elevated triglycerides.
At the level of the liver, orally administered HRT increases triglyceride levels.[103,104]This is augmented by the hepatic synthesis of VLDL, particularly of the large triglyceride-rich particles, as well as the inhibition of hepatic triglyceride lipase.[15,105]Despite this HRT-associated elevation in triglyceride levels, concomitant favourable effects of HRT administration include decreased LDL-C and TC, as well as elevated HDL-C levels.It is generally accepted that the anti-atherogenic effect of HRT upon TC, LDL-C and HDL-C outweigh the deleterious effect of elevated triglyceride levels.
4.2 Amenorrhoeic Athletes
Studies measuring triglyceride levels in amenorrhoeic athletes report non-significant[23,42,44]and significantelevations compared with eumenorrhoeic athletes. Table IV illustrates these studies. Despite the majority of these findings being equivocal, observation of consistently greater triglyceride concentrations in amenorrhoeic compared with eumenorrhoeic athletes represents what seems to be part of a ‘trend’ in the lipid profiles of these athletes. To date, no studies have reported assessments of triglyceride-rich lipoproteins, such as VLDL, for the determination of atherogenic versus non-atherogenic triglyceride particles in amenorrhoeic athletes.
Since triglycerides are frequently associated with other CVD risk factors such as low HDL-C, elevated LDL-C and low TC, it is interesting to note that the one study to report significantly elevated triglyceride in amenorrhoeic athletesalso reported significantly elevated LDL-C and TC. However, the reported level of triglyceride for amenorrhoeic athletes by Friday and colleaguesdoes not represent a value outside of the normal range (>1.7 mmol/L) as stipulated by the National Cholesterol Education Program.That triglyceride has been shown to be elevated in amenorrhoeic athletes compared with their sedentary counterparts[43,44]is consistent with data from anorexia nervosa patientsbut not consistent with studies that show triglyceride concentrations are frequently lower in endurance athletes when compared with sedentary controls,suggesting a possible oestrogen-dependent and exercise-independent mechanism in triglyceride metabolism in amenorrhoeic athletes and anorexia nervosa patients. Conversely, cross-sectional data identify similar triglyceride concentrations in female athletes, irrespective of self-reported menstrual status and weekly training distance (0–139km),indicating exercise- and E2-independent mechanisms in triglyceride metabolism. Collectively, these data are not conclusive as to the combined effect of hypoestrogenism and endurance training on triglyceride levels in female athletes. However, more subtle elevation and alterations to triglyceride metabolism, including a shift from non-atherogenic to atherogenic triglyceride lipoprotein metabolism may be evident in amenorrhoeic athletes, potentially necessitating closer examination of the triglyceride composition rather than the absolute presence of triglycerides per se. More studies to better allude to the independent and combined effects of exercise and menstrual status upon triglyceride metabolism are warranted.
As previously mentioned, low T3 is associated with a pro-atherogenic lipid profile,suggesting that nutritional status may be an important consideration when assessing triglyceride levels in amenorrhoeic athletes. Indeed, a high-carbohydrate (~69%; ~631 g/day)/low-fat (~15%; ~50 g/day) diet with a total energy intake of ~3360 Kcal/day has been shown to increase fasting triglyceride levels in male and female endurance trained athletes.Although markedly less in total energy intake, some,but not others,[21,46,75]have reported that amenorrhoeic athletes consume similar dietary macronutrient percentages of total energy intake. However, it is not known whether high carbohydrate diets affect fasting triglyceride levels in amenorrhoeic athletes. Conversely, a very low carbohydrate (~10%; ~43 g/day)/high-fat (~60%; ~118 g/day; ) diet with a total energy intake of ~1791 Kcal/day has recently been demonstrated to significantly reduce fasting triglyceride levels as well as favourably impact HDL-C and the TC/HDL ratio in normal weight normolipidaemic women.That allocation of dietary macronutrient calories can have such a notable effect on fasting triglyceride levels indicates that future research efforts examining the interactions between T3, dietary behaviour, and exercise in amenorrhoeic athletes is worthy of investigation.
The physiological significance of the reported elevated concentrations of triglyceride in amenorrhoeic athletes is not clear. The possible mechanism underlying this observation is not known. The inclusion of specific assessment of triglyceride-rich lipoproteins may yield more pertinent information with regard to atherogenic compared with non-atherogenic triglyceride particles.
5. Lipid Peroxidation
Elevated free radical production can negatively influence the oxidative status of circulating LDL particles.[110,111]Oxidative modification of LDL vastly elevates its atherogenicityand has been implicated in the initiation and progression of atherosclerosis.Increasing evidence supports the theory that oxygen-derived free radicals, namely reactive oxygen species (ROS), are associated with destructive biological processes, including DNA and cellular membrane damage.Chronic age-related disease states such as diabetes and carcinogenesis, as well as strenuous physical exercise, have been implicated in this process.[115–117]ROS incorporates hydrogen peroxide, and the less stable, superoxide- and hydroxyl-free radicals.[23,118]The magnitude of the oxidative stress is determined by the capacity of the antioxidant defences to detoxify ROS.Intracellular enzymatic antioxidant defences include glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase,[114,120]all of which reduce the susceptibility of the cell to potentially harmful free radicals.In addition, non-enzymatic extracellular antioxidant defences also exist, including vitamin E (α-tocopherol), vitamin A (beta carotene), and vitamin C (ascorbic acid).It is the balance between ROS production and antioxidant defences that determines the degree of oxidative stress.
5.1 Postmenopausal Women
Research surrounding lipid peroxidation and the effect of exogenous and endogenous E2 demonstrate inconsistencies. Antioxidant effects of E2 on LDL in vivo[122,123]and in vitro[113,124–126]have been demonstrated. Conversely, no effect for combined HRT on LDL oxidisability (the susceptibility of LDL to oxidation) and oxidative stress has been documented,although Lawler and colleagueshave demonstrated a significant beneficial effect for combined exercise and E2 replacement therapy upon markers of HDL oxidation compared with exercise or replacement therapy alone. Differences in model utilisation, subject demographics, E2 administration, dosage, time course and type have, in part, contributed to the varied findings reported in this field.
E2 may protect from atherosclerosis by inhibiting lipid peroxidation.[128,129]Endogenous E2 has free radical-scavenging abilities, with up to 2.5 times the activity of vitamins C and E.The ability of E2 to form moderately stable radicals from less stable radicals by donating a hydrogen atom is consistent with antioxidant function.This antioxidant capability has been evidenced in premenopausal women who have significantly higher 17β-estradiol levels and lower lipid peroxide concentrations, as well as significantly higher glutathione peroxidase activity when compared with postmenopausal women.In addition, significant increases in endometrial glutathione peroxidase have been observed during the high oestrogen E2 phase of the cycle.Such data support a beneficial effect for endogenous E2 on both intracellular antioxidant enzyme activity and free-radical scavenging abilities against lipid peroxidation.
5.2 Amenorrhoeic Athletes
There is no single biomarker that is considered the ‘gold standard’ of lipid or protein oxidation.However, evidence for oxidative stress during and after exercise can be obtained from measurement of free radicals, the assessment of damage to lipids and from measurement of antioxidant redox status, particularly glutathione.Considering the reported antioxidant effect of endogenous E2and exercise training,surprisingly few studies[23,42,120]have investigated the effects of hypoestrogenism on lipid peroxidation and oxidative status in amenorrhoeic athletes. These studies, highlighted in table V, report significantly decreased LDL diene conjugation, i.e. a decreased ability of LDL to resist peroxidation,and a greateras well as similarmagnitude of change for lipid peroxidation potential, that is, the susceptibility of LDL to peroxidation, post-exercise in amenorrhoeic compared with eumenorrhoeic athletes. Kanaley and Jialso report that at rest and post-exercise, amenorrhoeic athletes demonstrate significantly elevated glutathine peroxidase compared with eumenorrhoeic athletes, and that malondialdehyde, an indirect indicator of lipid peroxidation, is similar in both groups. In addition, oxysterol formation, derived from the enzymatic and non-enzymatic oxidation of cholesterol,is increased post-exercise in amenorrhoeic athletes only.Collectively, these findings are conflicting and do not provide answers as to whether exercise in the face of hypoestrogenism affects antioxidant status. This can be attributed, in part, to the fact that each study utilised a different exercise protocol, including a maximal oxygen uptake test,as well as a 30 minuteand a 90 minute submaximal running bout.It is also possible that the exercise intensity and/or duration may have been insufficient to stimulate an oxidant response because of the high level of aerobic fitness of the athletes. Training status of the athletes was also not consistently reported.
An important factor to consider when assessing antioxidant defence mechanisms is the training status, as well as the adaptive status, of the athlete.In studies observing the antioxidant status of amenorrhoeic athletes, training status was reported as mileage per week,[23,120]duration of trainingor not reported at all.None of these studies reported the adaptive status of the athletes, thereby further compounding the lack of clarity regarding interpretation of E2 status on antioxidant status. However, despite conflicting findings, the elevated glutathione peroxidase levels in amenorrhoeic compared with eumenorrhoeic athletesis indicative of an enhanced antioxidative status in the amenorrhoeic athlete, despite a hypoestrogenic environment, suggesting alternative antioxidant mechanismssuch as training status. Indeed, research has shown that well trained and well adapted athletes demonstrate an augmented antioxidant system and a reduction in lipid peroxidation,as well as greater resistance to exercise-induced or -imposed oxidative stress.[111,138,139]Consistent with this finding is the observation that total antioxidant status is positively correlated with peak oxygen consumption in runners,supporting the theory of a stress-tolerance mechanism, whereby enzymatic antioxidant defences are enhanced due to exercise training per se.
In contrast to the adaptive oxidative status of well trained athletes, higher performance and training levels are associated with greater elevations in exercise-induced lipid peroxidation,resulting in an antioxidant system that can become overwhelmed and unable to cope with the increased free-radical production.This occurrence can be linked with overtraining,exercise training intensity,and/or the nutritional status of the athlete,whereby susceptibility to antioxidant deficiency may occur.However, since no studies to date have confirmed an effect of E2 upon antioxidant status in humans in response to exercise,it is likely that the antioxidant status in amenorrhoeic athletes is reflective of the training and/or adaptive status, the nutritional status, and/or the hypoestrogenic milieu.
Consistent with previous research reporting the effect of an acute bout of strenuous exercise on antioxidant defences,a decreased capacity to detoxify ROS after a maximal bout of exercise, as demonstrated by unfavourably altered oxysterol formation and increased lipid peroxidation levels in amenorrhoeic athletes,has been observed. These findings imply a compromised antioxidant status in amenorrhoeic athletes, and that an increased potential risk for premature atherosclerosis may be present. However, (i) a single maximal bout of exercise is not necessarily reflective of a regular training session for the athletes, and thereby not representative of the characteristic oxidative stress these athletes might otherwise incur; and (ii) it is normal for oxidative stress to be elevated after strenuous exercise,and as such, recovery data rather than immediate post-exercise data might provide more relevant information about antioxidant status.
In addition, since non-enzymatic antioxidants are negatively correlated with lag time of diene conjugates,assessment of dietary supplementation may also be an important aspect of oxidative stress determination, particularly in the amenorrhoeic athlete.
The impact of sustained hypoestrogenism in the amenorrhoeic athlete upon the oxidative system is not clear, but data indicate that the antioxidant defences of the amenorrhoeic athlete may be compromised after strenuous aerobic exercise. Inclusion of training, adaptive and dietary status will help minimise confounding factors. Data suggest that in well trained individuals, exercise training elicits favourable effects upon the oxidative milieu.Since higher exercise intensity appears to effect a greater oxidative response in amenorrhoeic compared with eumenorrhoeic athletes after maximal exercise,studies to determine the oxidative stress response to high exercise intensity training in amenorrhoeic athletes are worthy of further investigation. Mechanism(s) underlying the combined E2- and exercise-mediated protection of lipid peroxidation need to be determined. In addition, methodological consistency in measurement of antioxidant enzymes needs to be established.
6. Nitric Oxide
The endothelium is a multifunctional interface between the circulating blood and various tissues and organs of the body,and is recognised as a metabolically active organ that is vital to vascular homeostasis.[147,148]Through the combined release of vasoactive substances, such as nitric oxide and endothelin, endothelial haemostatic function is realised. Nitric oxide is a potent vasodilatorthat also maintains a low resting arterial tone in the peripheraland pulmonarycirculations. Nitric oxide also inhibits platelet aggregation, suppresses smooth-muscle cell proliferation and acts as an antiatherogenic factor.[152,153]Endothelial-derived nitric oxide is produced by the endothelial isoform of nitric oxide synthase upon the conversion of the substrate L-arganine to L-citrulline.The production of this free radical messenger has been identified as effecting a protective role on the endothelium.Impaired release and/or bioavailability of nitric oxide has been linked with hypertension,hypercholesterolaemia,diabetes,tobacco use,established coronary artery diseaseand E2 deficiency.Consequently, factors that decrease nitric oxide production and/or bioavailability may promote atherosclerosis.
In endothelial cells, gene expression of nitric oxide synthase, despite being constitutively activated, can also be up-regulated by both receptor-mediated (i.e. acetylcholine, serotonin, thrombin, bradykinin) and receptor-independent (i.e. shear stress) mechanisms.A number of pathways leading to the release of vascular nitric oxide have been identified, including: (i) basal endothelial release that maintains low vascular tone; (ii) mechanical stimuli, that is, increased shear stress; (iii) dilating factors (i.e. prostaglandins) and metabolites (i.e. adenosine) released from contracting skeletal muscle; (iv) nitroxidergic; and (v) cholinergic nerve stimulation; and (vi) nitric oxide release from skeletal muscle.The multiplicity of pathways to attain vascular nitric oxide release identifies this free radical messenger as a key modulator of vessel function.
6.1 Postmenopausal Women
A plethora of data regarding the effect of E2 and HRT upon endothelial function in women has been published. Most,[14,162,163]but not allreports support a role for E2 increasing endothelium-dependent flow-mediated vasodilation. In vitro data also show an endothelium-independent vasodilatory effect, that is, a direct smooth muscle-relaxing effect, of 17β-estradiol upon the coronary arteries in human females.Blunted circulating levels of nitrite/nitrate,in addition to increased plasma levels of endothelin-1and impaired endothelium-dependentand -independentfunction, occurs in postmenopausal women not receiving E2 therapy or HRT. Recent data also demonstrate that acute (7 days) E2 deficiency due to ovariectomy in humans is associated with unaffected endothelial function,as well as impaired endothelium-dependent, but not -independent vasodilationin response to flow-mediated dilation. Taken together, these data suggest that reduced circulating levels of E2 may effect impaired endothelial-dependent and -independent function, and is associated, in part, with reduced nitric oxide production and/or bioavailability.Furthermore, 17β-estradiol may have an important regulatory role in coronary arterial tonedue to possible direct effects upon endothelium and smooth muscle cells (see figure 2).
6.2 Amenorrhoeic Athletes
Despite the well documented E2/nitric oxide association, only one study documenting nitric oxide levels in amenorrhoeic athletes has been reported. Stacey et al.,observed significantly decreased levels of plasma nitrate/nitrite production despite significantly elevated dietary ingestion of nitrates in amenorrhoeic athletes compared with sedentary eumenorrhoeic women. Since chronic aerobic exercise enhances blood flow and shear stress,and strenuous exercise can incur striking increases in plasmaand urinarynitric oxide metabolite concentrations, it is interesting that despite participating in chronic endurance activities, the amenorrhoeic athletes did not demonstrate increased nitric oxide concentrations. Consequently, the reduced plasma nitrite/nitrate levels are hypothesised to be related to the chronically low E2 status of the amenorrhoeic athletes.The clinical consequence or physiological relevance of this observation is not known.
Although no other studies report nitric oxide levels in amenorrhoeic athletes, some data alluding to the E2/nitric oxide association through observation of vascular response to flow-mediation dilation, an endothelium-dependent, and therefore a predominantly nitric oxide-mediated event, does exist. Zeni Hoch and coworkerswere the first to demonstrate that, like postmenopausal women, young amenorrhoeic athletes (21.9 ± 1.2 years) also present with endothelium-dependent, but not -independent, dysfunction compared with their oligomenorrhoeic and eumenorrhoeic counterparts. Resting heart rate, mean arterial blood pressure and baseline brachial arterial diameter were similar between the groups, yet mean percentage change from baseline brachial artery diameter in response to flow mediated dilation in amenorrhoeic and eumenorrhoeic athletes was 1.08 ± 0.91% and 6.38 ± 1.38%, respectively.Alarmingly, the magnitude of impaired brachial endothelium-dependent vasodilation in amenorrhoeic athletes is comparable to data previously reported in otherwise healthy postmenopausal womenand older patients (60 ± 2 years) with coronary artery diseaseafter a similar flow-mediated stimulus. Since endothelial dysfunction is a predictor of future coronary events,the finding from Zeni Hoch and colleagues,is suggestive of increased risk for accelerated CVD development in amenorrhoeic compared with eumenorrhoeic athletes.
Secondary to chronically diminished circulatory E2 levels, reduced circulatory nitric oxidemay be a contributing factor to the observed endothelial dysfunction in amenorrhoeic athletes. However, Zeni Hoch and colleaguesdid not specifically attribute the impaired endothelial dilatory response in amenorrhoeic athletes to E2 deficiency, perhaps because each group presented with similar E2 levels at the time of testing. However, chronically suppressed versus cyclical fluctuations of E2 levels would likely be better identified with daily urinary analysis of metabolites of E2 rather than a single blood draw. In addition, a single blood draw assessment cannot distinguish the possibility of an effect of E2 exposure across time upon endothelium-dependent function in active women with less severe menstrual disturbances, such as anovulation or oligomenorrhoea. That oligomenorrhoeic and eumenorrhoeic athletes showed similar responses for flow-mediated dilationis suggestive of a “critical threshold of E2 exposure”, and that some E2, as oligomenorrhoeic athletes are likely exposed to, is better than none, i.e. the chronically low levels amenorrhoeic athletes are exposed to.
An additional demonstration of the importance of E2 to vascular function in amenorrhoeic athletes can be realised from other preliminary data from Hoch and colleagues.Resumption of menses in previously amenorrhoeic athletes restored endothelial-dependent function to levels observed in eumenorrhoeic athletes.This reversibility in endothelial-dependent function with return of ovarian function is consistent with data that show improved endothelial function in postmenopausal women after HRT.Mechanisms of non-genomic vasodilatory effects of E2 include stimulation of the opening of calcium-activated potassium channels,and activation of endothelial nitric oxide synthase, the precursory enzyme of endothelium-derived nitric oxide,via E2 receptor-α mediated activation,with heat shock protein 90 acknowledged as a key requirement for this activation.Dependent pathways include E2-receptor mediated mitogen-activated protein kinaseand phosphatidylinositol 3-kinase/Akt.The impact of hypoestrogenism on these pathways, however, is not well understood, but it is reasonable to expect that amenorrhoeic athletes may demonstrate impairment in one or both of these pathways.
Recently, data from our own laboratory is also consistent with altered flow-mediated endothelial-dependent vasodilation.Following a flow-mediated endothelial-dependent vasodilation at rest and following peak ischaemic blood flow assessed by strain-gauge plethysmography, amenorrhoeic athletes had lower resting (2.2 ± 0.1 vs 4.8 ± 0.4, p < 0.001) and peak ischaemic (42.8 ± 2.1 vs 52.9 ± 2.0; p = 0.004) blood flow responses (mL/dL/min). Amenorrhoeic athletes also had lower resting supine heart rate (50.5 ± 4.8 vs 58.8 ± 1.9; p = 0.07), and supine resting systolic blood pressure (90.4 ± 5.7 vs 106.8 ± 2.0mm Hg; p = 0.004), compared with their eumenorrhoeic counterparts. These findings could not be attributed to differences in aerobic capacity since both groups were similar (p > 0.05). Thus, our data also support the finding of diminished flow-mediated blood flow in amenorrhoeic athletes, and that lower resting heart rate and systolic blood pressure may be indicative of altered autonomic regulation, similar to that seen in anorexia nervosa patients.
There is a positive correlation between cardiovascular fitness and endothelial function,suggesting that endothelial dysfunction may limit exercise capacity, either via central or peripheral mechanisms.Conversely, exercise is efficacious in restoring dysfunction of the vascular endothelial nitric oxide system.The exact mechanisms for this effect are not yet understood. Decreased endothelial nitric oxide may also be rate-limiting to oxygen delivery and exercise performance.Data show, however, a lack of correlation between cardiovascular fitness, exercise capacity and endothelial function in amenorrhoeic athletes, despite being demographically similar to their eumenorrhoeic counterparts, including weekly mileage, duration of training, and 5km race time.These findings imply that exercise-associated amenorrhoea does not impact athletic performance, but may be implicated as a potential negator of the known cardioprotective benefits of aerobic exercise.Impaired endothelial function, despite chronic endurance training in amenorrhoeic athletes, suggests that hypoestrogenism may exert a greater negative effect than the positive effect of aerobic exercise per se.
Mechanisms explaining the difference in endothelial function of eumenorrhoea and amenorrhoea are not yet known, but are likely to be related to hormonal regulation of endothelial function which may be the result of receptor-dependent or -independent mechanisms.Progesterone, androgen and E2 receptors have been identified in human vascular endothelium.Two functionally different receptor subtypes for E2 actions exist, E2 receptor-α and -β, although the importance of these subtypes in the vasculature remains unclear.Expression of E2 receptor-α has been observed in human endothelium and vascular smooth muscle cells.E2 receptor-β expression in vascular tissue is less well distinguished, but has been detected predominantly in vascular smooth muscle cells,identifying a potential endothelium-independent role for E2. Several target genes for E2 have also been identified, including those encoding proteins that modulate lipid clearance, cardiac contractility, cell proliferation and, specifically, vascular tone.Consequently, the presence of vascular E2 receptors is associated with protection against coronary atherosclerosis, and their expression is directly impacted by the level of circulating oestrogens.
The effect of sustained, impaired endothelial function upon atherosclerotic development or vessel integrity in amenorrhoeic athletes awaits investigation. To date, the clinical and health implications of prolonged E2 deficiency upon the vasculature in amenorrhoeic athletes is not clear. Further, the time-course of decline in endothelial function, a potential ‘critical threshold of E2 exposure’, and changes in endothelial function with resumption of menses, or perhaps exogenous hormones, should be explored in future research.
Endothelial integrity, essential for normal functioning of blood vessels, is preserved not only by endogenous vasodilatory substances, but also by vasoconstrictive substances such as endothelins (ET-1, ET-2, ET-3) [for review, see Alonso and Radomski]. Alteration of the nitric oxide and endothelin system is augmented by, and is associated with, many CVDs.The vasoconstrictive production of endothelin however, can be inhibited by nitric oxide.Endothelins (ET-1, ET-2, ET-3) are biologically active peptides that oppose the effects of nitric oxide through vasoconstrictive and mitogenic action.[195,196]Of the three endothelin isoforms, only ET-1 is constitutively produced by the endothelial cells, making ET-1 a key vascular regulatorand, consequently, an important focal point for cardiovascular regulation research. Endothelin receptors, endothelin-A (ET-A) and endothelin-B (ET-B), are located on both vascular smooth muscle and endothelial cells. Via endothelin smooth-muscle receptors ET-A and ET-B, and via endothelin endothelial-cell receptor ET-B, endothelium-derived ET-1 predominantly facilitates vasoconstriction,although stimulation of ET-B receptors on the endothelium can also oppose ET-A and ET-B mediated vasoconstriction by stimulating nitric oxide formation.This vasodilatory response is due to increased intracellular calcium, resulting in upregulation of endothelial nitric oxide synthase.In addition to vasoconstrictive and vasodilatory properties, endothelins also increase monocyte adhesion, macrophage activation, and vascular smooth muscle cell proliferation and migration through ET-A and ET-B.As a result of the observation that altered expression and/or activity of ET-1 can lead to the development of vascular diseases,ET-1 has been implicated in the progression of atherosclerosis.
7.1 Postmenopausal Women
Research surrounding ET-1 and postmenopausal women is less plentiful than that found for nitric oxide. Typically, ET-1 levels have been shown to be elevated in postmenopausal women, and are significantly reduced with administration of HRT.[198,199]Long-term (6 months) E2 therapy results in decreased levels of ET-1, as well as an increased ratio of nitric oxide to ET-1.In addition, ET-1 mediated arterial constriction has been shown to be reduced after 1 month of treatment with 17β-estradiol (2 mg/day) in older postmenopausal patients when compared with placebo.This reduction is suggestive of a 17β-estradiol effect on endothelium-dependent vasoconstrictive responses, which may incorporate nitric oxide and/or prostaglandins.Interestingly, long-term (3 months) oral 17β-estradiol administration (2 mg/day) resulted in a loss of the E2-inhibitory effect upon ET-1 mediated arterial constriction, implying possible tachyphylaxis, (i.e. an acute loss of response) due to sustained high doses of 17β-estradiol.
One potential explanation by which E2 may impart favourable anti-vasoconstrictive properties can be related to data that report 17β-estradiol inhibition of ET-1 synthesis,possibly through E2 receptor-dependent pathways.In an in vitro study, Dubey et al.,treatment of porcine coronary artery endothelial cells with varying concentrations of 17β-estradiol and estradiol metabolites, 2-hydroxyestradiol and 2 methoxyestradiol, dependently inhibited basal and serum tumour necrosis factor-α, angiotensin II and thrombin induced ET-1 synthesis. As compared with 17β-estradiol, estradiol metabolites were shown to be more potent in inhibiting ET-1 secretion.Confirmation of such findings in vivo is yet to be reported.
7.2 Amenorrhoeic Athletes
No studies to date have reported on the ET-1 levels of young amenorrhoeic athletes (18–35 years) or in anorexia nervosa patients. However, in women of reproductive age, ET-1 levels are elevated during the menses phase when compared with the follicular and luteal phases of the menstrual cycle,suggesting that endogenous E2 also appears to favourably reduce circulating ET-1. These findings lend credence to the hypothesis that ET-1 levels might be expected to be elevated in amenorrhoeic athletes. However, it is known that long-term exercise training causes an increase in production of nitric oxideand a decrease in production of ET-1 in humans,[152,204]which may produce beneficial effects on the cardiovascular system. Further, a significant negative correlation between plasma nitrite/nitrate concentration and plasma ET-1 concentrations after chronic exercise training has been demonstrated.
The effect of chronic exercise upon ET-1 concentrations in amenorrhoeic athletes is unknown. Since previous data demonstrate diminished levels of nitrites/nitrates despite chronic training and elevated dietary nitrate intake in amenorrhoeic athletes,it can only be speculated that, as in postmenopausal women, ET-1 levels may be elevated, possibly due to decreased sensitivity to ET-1 and/or decreased circulating nitric oxide levels.
Homocysteine is an intermediate sulphur-containing amino acid produced in the metabolism of the essential amino acid, methionine.[205,206]There are two major metabolic outcomes of homocysteine, the first being reversible trans-methylation.This leads to the reformation of methionine, as during methionine deficiency, and ensures a sufficient supply of methionine for protein synthesis.The second is irreversible trans-sulfuration which results in the eventual excretion of homocysteine as sulphate in urine.[205,206]
Homocysteine has been identified as an independent, modifiable risk factor for CVD.[207,208]It is a recognised marker of systemic inflammation,[13,209]and is an important mediator of atherosclerosis.Mechanisms by which elevated homocysteine causes vascular disease include direct toxic effects on the endothelium caused by the generation of hydrogen peroxide during homocysteine metabolism.[205,206,210]This effect of increased free radical generation and subsequent lowered glutathione formation is suggested to be the principal cause of accelerated atherosclerosis.Other factors important to the atherosclerotic process that are also affected by elevated homocysteine levels include proliferation of vascular smooth muscle cells,promotion of thrombosis,elevated collagen production in smooth muscle cells,stimulation of oxidisation of LDL and anticoagulant inhibition.Normal concentrations of fasting plasma homocysteine are somewhat varied, and range from 5 to 15 µmol/L,or ≤12 µmol/L.Hyperhomocysteinaemia may derive from either nutritional (i.e. inadequate dietary intake of folate, vitamin B12 or B6) or genetic (i.e. homocystinuria) origins.[205,206]Additional factors that can contribute to elevated homocysteine are age, sex (in as much as E2 appears to have a lowering effect on homocysteine levels), impaired kidney and liver function and certain medications.[17,205,206,209]
8.1 Postmenopausal Women
The homocysteine-lowering effect of E2 is well documented.[17,18,210,217]E2 status is a non-genetic factor that affects homocysteine metabolism.Decreased levels of homocysteine have been reported in pregnant women,premenopausal women[207,217,219]and postmenopausal women who are on E2 and HRT therapy[17,18,48]compared with age-matched men, surgically menopausal women, and postmenopausal women who are not on E2 or HRT therapy.
8.2 Amenorrhoeic Athletes
There are no data available to date regarding homocysteine levels in amenorrhoeic athletes. However, concerns regarding the homocysteine level of the amenorrhoeic athlete can be related to studies that show nutritional macro- and micronutrient intake of most female athletes to be less than might be anticipated based on their training load.[220–222]Intake of iron, calcium, vitamin B12 and zinc have also been reported to be below the recommended daily allowances among female athletes.Since low folate concentrations have been identified as one of the key mediators of higher homocysteine levels,and reductions as great as 40% in plasma homocysteine concentrations with folate supplementation have been reported,the recommendation of sufficient dietary intake of folic acid may be particularly important for the amenorrhoeic athlete. Interestingly, adolescent anorexia nervosa patients exhibit significantly increased homocysteine levels.Further, young premenopausal women also exhibit menstrual phase-dependent homocysteine concentrations, with the lowest concentrations coinciding with elevated E2 levels.As a result of the identification of an E2- and folate-lowering effect on homocysteine, it is possible that the amenorrhoeic athlete may demonstrate subtle, but potentially significantly sustained elevated levels of homocysteine. Since atherosclerosis is accelerated with even mild increments in homocysteine,this concern will be interesting to address.
9. C-Reactive Protein
Atherosclerosis has recently been identified as an inflammatory disease.Characteristic of most forms of inflammation or tissue damage is the elevated serum concentration of acute-phase reactants, such as CRP.[228,229]As a recognised surrogate marker of low-grade systemic inflammation, CRP reflects heightened levels of pro-inflammatory cytokines.Prospective studies have shown that CRP is a strong independent risk factor for CVD,[13,230]as well as a predictive tool of relative risk for future events such as stroke.Specifically, data indicate that CRP predicts vascular events among low-risk groups of women with no readily apparent markers for disease,and even in women with LDL-C levels below 3.37 mmol/L.
Plasma CRP usually exists at very low concentrations, with 90% of individuals having a CRP <3.0 mg/L,but can be elevated several hundred-fold in response to infection.It is important to note that as a non-specific acute phase response protein,CRP can be influenced by a number of factors, such as bacterial infection and inflammatory diseases,prolonged exercise, smoking and age.[235–237]It is also positively associated with body mass index.Despite the many influencing factors, CRP is still more precise than other markers of the acute-phase response, and is therefore considered an extremely useful marker of ongoing inflammation and/or tissue damage.
9.1 Postmenopausal Women
The reported effects of E2 and HRT on CRP levels have recently been reviewed.Most,[232,238,239]but not all, studiesdemonstrate an E2-mediated effect of CRP. Indeed, a recent clinical trial showed an 85% average increase in CRP over 3 years with HRT when compared with placebo.This E2-increasing effect has been confirmed in cross-sectionaland prospectivestudies and, at first glance, suggests that HRT may be pro-inflammatory.However, the increase in CRP due to exogenous HRT is postulated to be metabolic rather than inflammatory.Observation of decreased inflammatory markers such as interleukin-6 (IL-6) and E-selectin, in the presence of elevated CRP, supports a possible link with the hepatic first-pass effect of oral hormone therapies on CRP plasma concentrations.This postulate makes sense when considering that plasma CRP is produced solely by hepatocytes.That chronic transdermal HRT treatment does not elevate plasma CRP levelsfurther supports a metabolic rather than pro-inflammatory effect of oral HRT. The clinical relevance of these findings, however, is not known.
9.2 Amenorrhoeic Athletes
Studies observing the effects of exercise on CRP in amenorrhoeic athletes have not been reported. Further, data observing CRP levels in female athletes are sparse. Fallon et al.,observed that an acute phase response, as determined by CRP, did not occur as a result of the levels of training typical of elite female athletes participating in court and field sports. These findings are consistent with data that demonstrate that strenuous endurance training is associated with an exercise-lowering effect on CRP concentrations.[241,242]The concept that training itself may attenuate the acute phase response, possibly by maintaining a ‘balance’ between response and anti-inflammation, has been demonstrated via long-term training studies that reveal a diminished acute phase reaction as a result of regular endurance exercise in men.The decrease of the CRP baseline level after chronic training suggests that intensive endurance exercise training may have a systemic anti-inflammatory effect, which has been postulated to be linked with an enhanced exercise-associated antioxidative defence mechanism.In contrast to these findings, exercise has also been allied with an inflammatory reaction in the blood.Cytoskeletal damage due to strenuous exercise can result in substantial tissue injury and clinical signs of transient immunosuppression.That is, the anti-inflammatory response itself is also immunosuppressive, and can result in increased susceptibility to viral infections,a major stimulus of the CRP acute phase response.
Both endurance running and downhill running generate muscle damage,and unaccustomed eccentric-biased exercise, whether due to unfamiliarity of the exercise or the intensity or duration of the exercise, incurs muscle and tissue trauma that subsequently activates an acute inflammatory response.Increased free-radical production due to tissue injury can further heighten the inflammatory response.In animal models, however, E2 has been reported to influence post-exercise muscle damage by maintaining membrane stability and limiting creatine kinase leakage from damaged muscle, thereby reducing the inflammatory response.Running, ballet and gymnastics are sports typically associated with amenorrhoea,and are associated with eccentric-biased activities that provide a high potential for muscle and tissue damage. If an enhanced oxidative defence mechanism due to the presence of E2[122,246]and a well adapted training statedoes indeed confer generalised anti-inflammatory benefits, it might be projected that amenorrhoeic athletes participating in eccentric-biased activities may demonstrate a compromised anti-inflammatory capacity. Together, these factors may subsequently counter the long-term exercise-lowering effect on the acute phase response. Elevated levels of CRP in the amenorrhoeic athlete after training could, therefore, be apparent. Conversely, chronically low E2 levels together with accustomed endurance activities may attenuate or even lower CRP levels. However, since the effect of hypoestrogenism in younger athletes upon CRP levels has not yet been explored, these hypothetical outcomes remain to be discerned.
In addition to measurement of CRP as a method of determining increased risk of CVD, IL-6 has also been hypothesised as a good marker of cardiovascular health in apparently healthy individuals.Indeed, synthesis of CRP in the liver is predominantly modulated byand strongly correlated withthe cytokine IL-6, although IL-6 and CRP are independently related to several clinical cardiovascular risk factors in women.IL-6 not only regulates immune system responses,but also increases fibrinogen, blood viscosity, platelet numbers and activity.Overtrainingand nutrient statushave been shown to impact pro-inflammatory cytokine levels. Consequences of a low calorie diet together with strenuous exercise stress may induce elevated IL-6 and cortisol levels in female athletes.The nutrient status of the amenorrhoeic athlete frequently reveals suboptimal energy and nutrient intake which is linked with compromised immune responses,although data show no relation between susceptibility to infections and menstrual status in recreationally active women.Since cytokine levels, particularly IL-6, increase with strenuous endurance activities,this finding suggests that exercise intensity and duration rather than menstrual status impacts on the immune response. Interestingly, in malnourished individuals such as anorexia nervosa patients, there is an atypical, unexpected finding of lack of viral infections, or minimal symptoms in response to minor viral infections despite poor nutrient status.The mechanisms behind this response have not been ascertained, but it is suggested to be a protective adaptation which is lost during refeeding.Decreased IL-6 levels in anorexia nervosa patientssuggest that a lowered CRP status may be present. Whether hypoestrogenism per se in these patients imparts an effect on IL-6 or CRP is not clear.
In addition to decreased IL-6, persistently elevated levels of cortisol, which ordinarily reduces cytokine responses in healthy individuals, does not convey reduced inflammatory responses in anorexia nervosa patients.This response identifies that the feedback mechanism is somehow rendered ineffective, impairing the ability to establish an acute-phase response.Amenorrhoeic athletes also demonstrate elevated cortisol levels compared with their eumenorrhoeic peers,but it is not known whether this has any impact on acute phase protein concentrations. As a result of the similarity between amenorrhoeic athletes and anorexia nervosa patients, the presence of similar immune cell responses may be revealed.
Because of a lack of data surrounding the cardiovascular status in amenorrhoeic athletes, it is difficult to provide recommendations for useful testing or screening criteria. Since nutrition is a major contributing factor to lipid metabolism, and the prevalence of eating disorders among female athletes has been estimated to range between 15% and 62%,it may be important for athletes and coaches to be aware that prolonged very low dietary fat intake (≤15% total caloric intake) together with high dietary carbohydrate intake (≥70% total caloric intake) may contribute to deleterious lipid profile outcomes over time. Further, the presentation of amenorrhoea in conjunction with eating disorders, or indeed, subclinical disordered eating, which has no clinical endpoint but is associated with restricted energy intake, whether consciously or subconsciously, to control bodyweight, may compound the cardiovascular risk. Until more research is conducted in this area, recommendations regarding practical guidelines for the possible prevention of early onset CVD in amenorrhoeic athletes will remain minimal, except to strongly recommend increased caloric intake to encourage resumption of menses, and restored E2 hormonal environment.
The synthesis of findings secondary to the effects of hypoestrogenism in young female amenorrhoeic athletes upon cardiovascular outcomes discussed in this paper lend credence to the postulate that these women may be at an increased risk of premature CVD, extending the clinical sequelae of the Female Athlete Triad to cardiovascular concerns. Despite several inconsistencies with regard to statistical significance and cardiovascular outcomes, largely due to small sample sizes, it can not be dismissed that sustained unfavourable alterations to markers of cardiovascular health may prove to have long-term deleterious subclinical and/or clinical consequences. Unfavourable changes in LDL-C, lipid peroxidation potential, TC, triglyceride and endothelial function, despite a physically active lifestyle, suggest that amenorrhoeic athletes may potentially be susceptible to increased cardiovascular risk. This risk should be acknowledged as part of the already recognised sequelae of the ‘female athlete triad’ associated with amenorrhoea in athletes, as depicted in figure 3. More studies, specifically longitudinal and prospective studies need to be performed to help discern the long-term effects of hypoestrogenism on cardiac and vascular function in young athletic women and to determine if the aforementioned unfavourable changes observed represent a clinically significant increase in CVD risk.
The authors wish to acknowledge the Arthur Thornton Cardiopulmonary Fund. The authors have no conflicts of interest directly relevant to the content of this review.