| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Departments of Obstetrics and Gynecology (A.R., A.L.H.), Clinical Physiology (M.J.E.), and Cardiology (K.S.-G.), Karolinska University Hospital, SE-17176 Stockholm, Sweden
Address all correspondence and requests for reprints to: Anette Rickenlund, M.D., Ph.D., Department of Clinical Physiology, N101, Karolinska University Hospital, Box 140, SE-17176 Stockholm, Sweden. E-mail: anette.rickenlund{at}karolinska.se.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Estrogen has been suggested to have protective effects on the cardiovascular system (10, 11, 12). The effects are mediated by estrogenic receptors-
and -ß and involve direct rapid effects due to activation of endothelial nitric oxide (NO) and long-term effects due to changes in gene and protein expression of NO (10, 11, 12). Increased NO synthase activity enhances endothelial-dependent vasodilatation (10). Estrogen also has a positive effect on the serum lipoprotein profile and both positive and negative effect on the coagulation-fibrinolytic system (12). Furthermore, estrogen is known to decrease low-density lipoprotein (LDL) oxidation and the accumulation of oxidized LDL in the intima (10), which are crucial steps in the atherosclerotic process. However, it has been shown that apolipoproteins are better risk indicators of coronary disease than the lipoproteins alone (13) and therefore should be included in the lipid analysis.
Previous studies have demonstrated that decreased levels of endogenous estrogen unfavorably modify the lipid profile and vascular function in postmenopausal women (10, 12, 14). Elevated lipid levels have also been reported in premenopausal women with hypoestrogenic conditions due to caloric deficiency, i.e. anorexia nervosa and in amenorrheic binge-eating patients (15, 16, 17). Friday et al. (18) also described elevated LDL levels in amenorrheic athletes in comparison with regularly cycling athletes.
Over the past years, a noninvasive ultrasound technique has been developed to determine flow-mediated dilatation (FMD), which is considered to be an endothelium-dependent function (10, 19, 20, 21, 22). The FMD response is associated with many risk factors for atherosclerosis and has been regarded as an early risk marker of cardiovascular disease (19, 20, 23). There is evidence that the magnitude of FMD relates to endogenous estradiol (E2) (12). Kawano et al. (24) demonstrated that in premenopausal women with variant angina, the frequency of myocardial ischemia was highest and FMD was lowest from the end of the luteal phase to early follicular phase when endogenous E2 levels were lowest. In healthy young women, FMD varies during the menstrual cycle in relation to E2 levels (25). Recently, an association between endothelial function and markers of endothelial inflammatory activation has been reported in healthy individuals (26). Thus, endothelial dysfunction, unfavorable lipid profile, and increased levels of inflammatory markers may be associated with menstrual disturbance and estrogen deficiency.
The aim of this study was to evaluate whether endothelial function measured as FMD of the brachial artery, blood lipids, and blood markers of endothelial inflammatory activation are related to menstrual disturbance in young female endurance athletes.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Female athletes in endurance sports, such as medium and long-distance running, marathon, orienteering, cross-country skiing, and triathlon, were recruited from universities and high schools specializing in sports and at public sports events and championships all over Sweden. The different sports were all characterized by being weight bearing of the lower extremities. Detailed information about these subjects has previously been reported (5, 6). Briefly, they were healthy, nulliparous, nonsmoking women aged 16–35 yr with body mass index 18–24 kg/m2. Endurance training criteria were defined as a minimum of 6 h of aerobic weight-bearing training of the legs or a minimum of 70 km of running weekly for a period of at least 6 months. Detailed information about the pattern of menstrual periods during the last year was provided from the athletes’ sport diaries. Amenorrhea was defined as no bleeding for the last 3 months, oligomenorrhea as periods at an interval exceeding 6 wk and five to nine periods the last year, and regular menstruation as periods with an interval of 22–34 d. A family history of cardiovascular disease and dyslipidemia among first-degree relatives was excluded. No medications, including oral contraceptives and asthma medications, were allowed. Intake of minerals/vitamins or nutritional supplements was accepted. None of the supplements were reported to include anabolic steroids.
Inactive women were recruited from universities and high schools and the staff at the Karolinska University Hospital. They were screened using the same criteria as for the athletes, except that the amount of training was restricted to 1 h of light aerobic training per week. The local committee for medical ethics approved the study protocol, and all women gave their informed consent to participate.
The study subjects were divided into four groups on the basis of endurance training and menstrual status: 14 amenorrheic athletes (AM), nine oligomenorrheic athletes (OM), 12 regularly menstruating athletes (RM), and 12 regularly menstruating sedentary controls (CTR).
Experimental design
All subjects were examined in the morning at the Women’s Health, Clinical Research Unit at the Department of Obstetrics and Gynecology, Karolinska University Hospital. Body weight, height, and blood pressure were measured, and a physical examination was performed. Fasting blood samples were collected from a peripheral vein in a resting state. After centrifugation of blood samples, sera were stored at –20 C until assayed. Menstruating subjects were examined in the early follicular phase (menstrual cycle d 1–5).
Serum levels of FSH, LH, E2, TSH, free T4 (fT4), and prolactin (PRL) were determined by time-resolved fluorescence immunoassay, using commercial kits (Autodelfia, Wallac OY, Turku, Finland). Serum concentrations of testosterone (T) and SHBG were determined with RIA in untreated serum, using commercial kits (Coat-a-Count Testosterone, Diagnostic Products Corp., Los Angeles, CA; and SHBG, Eurodiagnostics AB, Malmö, Sweden) according to the manufacturers’ protocols. Apparent concentrations of free testosterone (fT) were calculated from values of total T, SHBG, and a fixed albumin concentration of 40 g/liter by successive approximation using a computer program based on an equation derived from the law of mass action (27). The hormonal detection limits and within- and between-assay coefficients of variation were for FSH, 0.05 U/liter, 2 and 3%; LH, 0.05 U/liter, 2 and 2%; E2, 50 pmol/liter, 5 and 8%; TSH, 0.005 mU/liter, 3 and 5%; fT4, 2 pmol/liter, 5 and 4%; PRL, 0.04 µg/liter, 2 and 4%; T, 0.1 nmol/liter, 6 and 10%; and SHBG, 0.05 nmol/liter, 4 and 8%, respectively.
Fat mass and bone mineral density (BMD, grams per square centimeter) were determined by dual-energy x-ray absorptiometry using Lunar model DPX-L equipment (Lunar Radiation, Madison, WI). Maximal oxygen uptake was determined with the treadmill test (Cardionics AB, Stockholm, Sweden), using the leveling-off criterion.
Endothelial function
Endothelial function was determined in the afternoon, 3–5 h after a light meal. Brachial artery (BA) flow velocity, FMD, and endothelium-independent nitroglycerin (NTG)-induced dilatation were examined according to the method described by Celermajer et al. (21). The measurements were made noninvasively using a high-resolution scanner (model 128 XP/10c, Acuson, Mountain View, CA) with a 7-MHz linear array transducer. The left BA was scanned longitudinally 1–10 cm above the elbow, at which a clear image was found with the artery placed horizontally across the screen. Baseline measurements of blood flow and the inner diameter of BA were performed at rest. Reactive hyperemia was obtained by distal forearm artery occlusion with a 12.5-cm blood pressure cuff inflated to 300 mm Hg for 4.5 min. Blood velocity was measured immediately after cuff release, and the diameter of the artery was measured 50–60 sec after deflation. The BA diameter was measured again after a 10-min rest followed by administration of 0.4 mg sublingual NTG. Four minutes after NTG, blood velocity and the diameter measurements were repeated. To minimize variability, one experienced operator performed all investigations.
All analyses of the BA diameters were performed off-line by one investigator unaware of the subjects’ group and the sequence of the ultrasound scan. Three consecutive late-diastolic frames taken coincidentally with the R-wave on the electrocardiogram were analyzed at rest (baseline) and subsequently to different stimulations. The average diameter of the three frames was calculated. Blood flow was calculated from Doppler velocity, vessel diameter, and heart rate. The increase in blood flow after reactive hyperemia is presented as a percentage of the basal flow values. The within-individual variations between two determinations of FMD performed during the same day and between the determinations made on separate days in the laboratory are 0.88 ± 0.82 and 3.3 ± 2.7%, respectively, as previously reported (23).
Serum lipids and inflammatory markers
Serum lipids [triglycerides (TGs), total cholesterol (Chol), high-density lipoprotein (HDL), apolipoprotein (Apo) A, and Apo B] were determined by enzymatic methods using kits from Beckman Coulter Inc. (Fullerton, CA; SYNCHRON LX Systems). LDL was determined using the Friedewald formula (28). Lipoprotein (a) [Lp(a)] was immunochemically determined with kinetic nephelometry (Beckman Coulter). High sensitive C-reactive protein (CRP) was immunonephelometrically measured with a kit (Dade Behring, Marburg, Germany). IL-6, TNF, and soluble vascular cell adhesion molecule-1 (VCAM) were measured by immunoassays (R&D Systems Inc., Minneapolis, MN). Detection limits within and between assay coefficients of variation were for TG, 0.1 mmol/liter, 2.3 and 3.1%; Chol, 0.13 mmol/liter, 1.1 and 1.6%; HDL, 0.13 mmol/liter, 3 and 4.5%; Apo A, 0.25 g/liter, 5.0 and 6.3%; Apo B, 0.35 g/liter, 2.0 and 3.9%; Lp(a), 0.02 g/liter, 2 and 4%, (nondetectable values were set to 0.01); high sensitive CRP, 0.2 mg/liter, 3.4 and 2.1%; IL-6, 0.04 pg/ml, 7 and 7%; TNF, 0.12 pg/ml, 6 and 13%; and VCAM, 2.0 ng/ml 4.3 and 8.5%.
Statistical analysis
Normally distributed values are given as the mean and SD, whereas other values are given as the median and quartile range (P25-P75). Results were analyzed using a one-way ANOVA (between-groups design for four levels: AM, OM, RM, and CTR) or Kruskal-Wallis test according to distribution. A significant F value was followed by t test post hoc analysis, whereas the significant Kruskal-Wallis was followed by the Kruskal-Wallis-post hoc analysis based on mean ranks. The P values in the post hoc tests were corrected according to the Bonferroni procedure. Correlations were assessed using Spearman’s rank-order correlation. P < 0.05 was considered statistically significant. Analysis of covariance was used to adjust for effects of prognostic factors. Power analysis for the primary outcome variable FMD revealed a sample size of at least seven in each group to detect a difference between groups at a significance level of 0.05 with 80% power. The observed power analysis for FMD and lipoproteins showed an appropriate power to determine whether there were significant or no significant differences between groups for all variables with the possible exception of HDL. Software used was Statistica (6.1, StatSoft Inc., Tulsa OK).
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
. Age at menarche was significantly higher in the oligomenorrheic athletes than all other groups, whereas the amenorrheic athletes did not differ from the regularly menstruating groups. The average number of menstruations the year before the study was 1.9 ± 1.8 in the AM group and 4.8 ± 2.0 in the OM group. The amenorrheic athletes had the lowest percentage of fat mass and BMD in spine of all groups. There were no differences in onset of training, amount of specific endurance training, and maximal oxygen uptake among the athlete groups. Levels of FSH, LH, E2, total T, and SHBG were not different among the groups. However, fT was significantly increased in the OM group, compared with all other groups. The three athlete groups had significantly lower fT4 levels than controls, and the AM group displayed the lowest PRL levels.
|
FMD was impaired in the amenorrheic athletes, compared with all other groups (Fig. 1). FMD was also decreased in the OM group, compared with regularly menstruating athletes but not controls. The percentage change in flow and the NTG-induced vasodilatation did not differ among the groups (Table 2). The difference in FMD among the four groups remained significant when FMD was adjusted for vessel size.
|
|
The results from the analyses of lipids and inflammatory markers are presented in Table 3. The AM group displayed the highest levels of Chol and LDL of all groups and significantly higher levels, compared with the other athlete groups. The AM group also had the highest levels of Apo B and the lowest IL-6 levels. The OM group had the lowest levels of Chol, LDL, and Apo B, although significant only in comparison with the AM athletes. Oligomenorrheic athletes also had the lowest Lp(a) levels, which were significant, compared with the RM athletes. There were no differences among groups in levels of other lipids or inflammatory markers.
|
In the athlete groups with menstrual disturbance (AM + OM, n = 23), we found negative correlations between the number of menstruations the last year and lipid levels, including LDL (rs = –0.48, P < 0.05) and Apo B (rs = –0.53, P < 0.05). Correlation between the number of menstruations and results from FMD was not significant (rs = 0.33, P = 0.14). However, there were negative correlations between FMD and lipids and several endothelial cell markers in the athletes with menstrual disturbance. FMD was inversely correlated with levels of total Chol, LDL, HDL, Apo B, and Lp(a) (rs = –0.59, P < 0.01; rs = –0.58, P < 0.01; rs = –0.51, P < 0.05; rs = –0.56, P < 0.01; and rs = –0.60, P < 0.01, respectively) in these athletes. In the whole material, decreased FMD was also correlated with high LDL (rs = –0.31, P < 0.05) and high Apo B (rs = –0.31, P < 0.05). Correlations between the number of menstruations the last year vs. LDL and Apo B and correlations between LDL or Apo B vs. FMD in athletes with menstrual disturbance are shown in Fig. 2.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Levels of VCAM, one of the endothelial adhesion molecules, were negatively correlated to endothelial vascular function expressed as FMD in the athletes. The higher VCAM levels among those with impaired FMD might suggest endothelial inflammatory activation. One possible pathway of action could be an inhibition of NO production by oxidative modified LDL, which increases the expression of endothelial adhesion molecules and thereby participates in the recruitment and activation of inflammatory cells (10). The production of VCAM is also increased by cytokines like IL-6 and TNF. The significantly lower levels of IL-6 in amenorrheic athletes may be explained by estrogen deficiency because estrogen increases cytokines such as IL-6 and TNF. However, there were no correlations between FMD and CRP, IL-6, or TNF in our study. On the one hand, estrogen increases the expression of endothelial adhesion molecules via increased cytokines; on the other hand, estrogen enhances the production of NO, leading to decreased expression of endothelial adhesion molecules (10). Additional investigations are needed to elucidate the relationships between inflammatory markers and estrogen status or FMD, respectively.
The oligomenorrheic athletes displayed values of FMD representing an intermediate between the amenorrheic and regularly menstruating subjects but had the most favorable lipid profile with lowest levels of total Chol, LDL, Apo B, and Lp(a) of all groups. The OM group differed from the other groups by having the highest levels of fT. This hormonal pattern may indicate essential hyperandrogenism, which has been suggested to be an alternative mechanism for menstrual disturbance in athletes (5, 6). Clinical and/or biochemical signs of hyperandrogenism and the typical finding of polycystic ovaries (PCOs) on ultrasound are used as diagnostic criteria for the PCO syndrome (PCOS) according to the Rotterdam consensus of 2003 (29, 30). However, the ultrasound picture of PCO is not included in the former National Institutes of Health criteria of PCOS (29, 30). In our study, clinical symptoms of hyperandrogenism were not systematically evaluated. However, PCO on ultrasound was retrospectively investigated, and three of nine had PCO in the OM group, whereas one subject had PCO in the AM, RM, and CTR groups, respectively. PCOS with overweight is associated with the metabolic syndrome including an impaired FMD (31, 32). However, there is no clear association between decreased FMD and PCOS in normal-weight patients (32, 33). There are no previous data on endothelial function and blood lipid profile in hyperandrogenic women who are lean and well trained. We found no correlations between fT and lipid profile or FMD in the athletes. This may indicate that hyperandrogenism plays a less important role than hypoestrogenism in the regulation of vascular endothelial function and blood lipid profile, at least in young, lean, and extensively exercising female athletes.
The main cause of amenorrhea in athletes seems to be an inhibition of the hypothalamus-pituitary-ovarian axis due to caloric deficiency, supported in this study by the lowest percentage of fat mass and BMD in this group. Low levels of PRL and fT4 are also endocrine changes associated with energy deficiency (5, 6). Although the amenorrheic athletes are hypoestrogenic, they are young, physically active, and otherwise healthy women with no known risk factors for cardiovascular disease. Usually we consider a physically active lifestyle to be protective against the development of atherosclerosis. What are the clinical implications of endothelial dysfunction and an impaired lipid profile in young athletic women with amenorrhea? This question cannot be answered due to lack of longitudinal studies in this group of women. Whether the condition of endothelial dysfunction and impaired lipid profile in young athletic women with amenorrhea is reversible after resumption of menses is not known. Furthermore, we do not know whether this condition may have long-term consequences for the future risk of cardiovascular disease. However, it is noteworthy that the mean levels of Chol and LDL in the amenorrheic athletes exceeded the upper limit of the normal range according to the laboratory methods we used.
In conclusion, amenorrhea in young endurance athletes is associated with endothelial dysfunction and unfavorable lipid profile with increased total Chol, LDL, and Apo B, i.e. recognized risk factors for atherosclerosis. The clinical implications of these findings are not known. Additional studies are needed to elucidate whether there is an association between athletic amenorrhea and long-term cardiovascular morbidity.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
First Published Online November 30, 2004
Abbreviations: AM, Amenorrheic; Apo, apolipoprotein; BA, brachial artery; BMD, bone mineral density; Chol, cholesterol; CRP, C-reactive protein; CTR, regularly menstruating sedentary control; E2, estradiol; FMD, flow-mediated dilatation; fT, free testosterone; fT4, free T4; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Lp(a), lipoprotein (a); NO, nitric oxide; NTG, nitroglycerin; OM, oligomenorrheic; PCO, polycystic ovary; PCOS, PCO syndrome; PRL, prolactin; RM, regularly menstruating; T, testosterone; TG, triglyceride; VCAM, vascular cell adhesion molecule.
Received July 2, 2004.
Accepted November 22, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
rd R, Bäckström T, Shanbhag V, Carstensen H 1982 Calculation of free and bound fractions of testosterone and estradiol-17ß to human plasma proteins at body temperature. J Steroid Biochem 16:801–810[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  B. N. Torgrimson, J. R. Meendering, P. F. Kaplan, and C. T. Minson Endothelial function across an oral contraceptive cycle in women using levonorgestrel and ethinyl estradiol Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2874 - H2880. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. O'Donnell, P. J. Harvey, J. M. Goodman, and M. J. De Souza Long-term estrogen deficiency lowers regional blood flow, resting systolic blood pressure, and heart rate in exercising premenopausal women Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1401 - E1409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. de Ronde, Y. T. van der Schouw, H. A.P. Pols, L. J.G. Gooren, M. Muller, D. E. Grobbee, and F. H. de Jong Calculation of Bioavailable and Free Testosterone in Men: A Comparison of 5 Published Algorithms Clin. Chem., September 1, 2006; 52(9): 1777 - 1784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Rickenlund, M. J. Eriksson, K. Schenck-Gustafsson, and A. L. Hirschberg Oral Contraceptives Improve Endothelial Function in Amenorrheic Athletes J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3162 - 3167. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
