In 1972, Congress passed Title IX, the Educational Amendment Act that ensured that women would have equal opportunities for interscholastic sports participation. Since that time, the number of female athletes has risen dramatically, and today almost 3 million young women compete in American high school sports programs. For women, the benefits of an active lifestyle including running are profound and well known. Women who participate in regular sports programs have been found to have higher self-esteem, a reduction in depression, and better body images (31). The physiological benefits of exercise include increased cardiorespiratory fitness, which leads to decreased cardiovascular disease and obesity (25). Teegarden et al. (32) found that active girls who participate in high school sports had a significantly greater bone mineral density, which may help to prevent osteoporosis in the future. However, as the number of women participating in sports grows, we have also discovered an increasing prevalence of exercise-associated menstrual irregularities (amenorrhea, oligomenorrhea, luteal phase dysfunction, and anovulatory cycles). Athletic amenorrhea is a component of the Female Athlete Triad, an interrelated problem of disordered eating, amenorrhea of hypothalamic origin, and osteoporosis. Athletic amenorrhea is a complex, multifactorial condition. Extreme exercise, excessive caloric restrictions, physical and emotional stress associated with exercise/competition, percentage of body fat, and genetics all play a role. However, recent studies have pointed toward dietary factors as the key etiologic component in athletic induced amenorrhea (26).
Although the exact mechanism of athletic amenorrhea is not fully understood, it has been shown to be associated with osteopenia and osteoporosis in several studies (11,12). Athletic amenorrhea is known to have a hormonal profile similar to menopause, characterized by low estrogen levels, which is etiologic in the development of osteoporosis in postmenopausal women. However, the greatest medical consequence of menopause is the associated cardiovascular disease (7). Cardiovascular disease is the number 1 killer of women. Cardiovascular risk increases significantly after menopause, when estrogen levels drop. The earliest sign of cardiovascular disease is reduced endothelial-dependent vasodilation, which is seen as early as 3 months after menopause (5). Knowing that nearly 3 million girls are playing high school sports, and that the prevalence of “athletic amenorrhea” is estimated at 12–59% in competitive athletic women (36), the potential link between abnormal endothelial function as a manifestation of early cardiovascular disease demands further attention. This is important not only for public health and prevention reasons but also for athletic performance and training. Furthermore, abnormal flow-induced dilation may limit central and peripheral oxygen uptake at higher aerobic demands.
A noninvasive method for evaluating endothelial function uses ultrasound to measure endothelial dilation of the brachial artery, which occurs normally in response to an increase in blood flow through the vessel (4). Reduced endothelium-mediated vasodilation measured in this way is one of the earliest manifestations of cardiovascular disease (3). Reduced brachial flow-mediated dilation occurs with all risk factors for coronary artery disease and is strongly correlated with coronary artery endothelial dysfunction (30), justifying it as a marker for cardiovascular disease risk (1). In fact, abnormal brachial flow-mediated dilation is 95% predictive of abnormal coronary endothelial function (1).
We hypothesize that young women with athletic-induced alterations in menses (amenorrhea and oligomenorrhea) show early signs of cardiovascular dysfunction manifested as reduced endothelium-dependent vasodilation of the brachial artery. Because amenorrhea is also associated with disordered eating and low peak bone mass, bone mineral density, energy expenditure, and caloric intake were also measured.
Thirty-two women athletes (age 20.9 ± 0.9 yr) volunteered for this investigation and signed an informed consent in accordance with our Institutional Review Board policy. Inclusion criteria consisted of age 18–35 yr and the presence of primary amenorrhea defined as the absence of menstruation by age 16 yr or secondary amenorrhea defined as the absence of six or more consecutive menstrual cycles after menarche and oligomenorrhea defined as cycles greater than 38 d for at least 2 yr or eumenorrhea defined as menstrual cycles every 28–30 d for at least the last 12 months. Subjects had to run at least 25 miles·wk−1 to qualify for study. Subjects were excluded if they had hypertension, diabetes, smoking, pregnancy, pituitary tumor, thyroid disorder, chromosomal abnormalities, known adverse reaction to nitroglycerin, known hypercoagulable state, anorexia nervosa, bulimia nervosa, or oral contraceptive or Depo-Provera use within last 12 months. Subjects were recruited via news media, advertisements at local universities, and from verbal communication with physicians at our medical center.
Nine women met the criteria for secondary amenorrhea, one woman met the criteria for primary amenorrhea, 11 were oligomenorrheic, and 11 were eumenorrheic. All of the subjects were competitive college athletes except two; these women were competitive runners in the community. The physical characteristics and exercise histories of the three groups of runners were similar (Table 1).
All subjects completed a questionnaire establishing exercise and menstrual history, including age at menarche and date of last menstrual period. Medical history was reviewed with each subject. Each subject met with a registered dietitian who conducted a prospective 3-d diet record to assess caloric, carbohydrate, protein, fat, calcium, and phosphate intake. The same dietitian evaluated each diet record. A urine sample was collected to test for pregnancy before entry into the study sample. Fasting whole venous blood samples were obtained the morning of the test. Progesterone was measured by the ACS: 180 Chemiluminescence’s System (Chiron Diagnostics Corporation, East Walpole, MA), using the Chiron Diagnostics ACS: 180 Progesterone assay. The inter-assay coefficient of variation was 5.0–10.0%, the intra-assay coefficient of variation was 7.8–12.0%, and the lower limit of sensitivity was 0.11 ng·mL−1. Cholesterol was measured by the BM/Hitachi 917 analyzer (Boehringer Mannheim, Indianapolis, IN), using the Boehringer Mannheim Cholesterol/HP enzymatic reagent. The inter-assay coefficient of variation was 0.9–1.0%, the intra-assay coefficient of variation was 1.4–2.2%, and the lower level of sensitivity was 3 mg·dL−1. Thyroid stimulating hormone (TSH) was measured by the Abbott AxSYM System (Abbot Laboratories, Abbott Park, IL), using the AxSYM Ultrasensitive hTSH II Microparticle Enzyme Immunoassay (MEIA). The inter-assay coefficient of variation was 3.54–10.41%, the intra-assay coefficient of variation was 5.20–14.86%, and the lower limit of sensitivity was 0.3 μIU·mL−1. Estradiol was measured by the Abbott AxSYM System, using the AxSYM MEIA. The inter-assay coefficient of variation was 3.4–9.5%, the intra-assay coefficient of variation was 4.9–15.2%, and the lower limit of sensitivity was 20 pg·mL−1. Prolactin was measured by the Abbott AxSYM System, using the AxSYM Prolactin MEIA. The inter-assay coefficient of variation was 2.95–3.36, the intra-assay coefficient of variation was 3.68–4.87%, and the lower limit of sensitivity was 0.6 m·mL−1. Follicle-stimulating hormone (FSH) was measured by the Abbott AxSYM System, using the AxSYM FSH MEIA. The inter-assay coefficient of variation was 3.72–5.94%, the intra-assay coefficient of variation was 6.04–8.48%, and the lower limit of sensitivity was 0.37 mIU·mL−1. Hemoglobin was measured by the Beckman/Coulter-GENS Hematology analyzer (Miami, FL), using the modified Cyanmethemoglobin (Spectrophotometric). The inter-assay coefficient of variation was <0.8%, the intra-assay coefficient of variation was 0.4%, and the lower limit of sensitivity was 3 g·dL−1. Blood was drawn one time for the amenorrheic group and two times, separated by 14 d, for the control and oligomenorrheic groups because their hormonal status was expected to be more variable than the amenorrheic subjects. Bone mineral density (g·cm−2) of the lumbar vertebral bodies (L2–L4) and total left hip were measured in each subject by dual energy x-ray absorptiometry (DEXA) using a GE Lunar Prodigy densitometer, version 2.15 (Madison, WI). Each subject was positioned consistently on the scanning bed and measured by the same experienced and certified technologist to help eliminate any positioning variability. Daily calibrations were routinely performed with an aluminum spine phantom. The coefficient of variation of the phantom over a 6-month period was 0.29%. T and Z scores were calculated using the manufacturer’s established database. Data from the 15th International Bone Densitometry Workshop, Monterey, CA, 2002, investigated the short-term reproducibility of replicate scans in 99 women by using the GE software. They found the average root mean square coefficients of variations for the lumbar spine and total hip measurements were 1.6% (90% confidence interval 1.3–2.1) and 0.94% (90% confidence interval 0.8–1.2, respectively). Height (cm) and weight (kg) were measured on the same scale (Scaletronix, Model No. 5005, Wheaton, IL) by the same technologist and combined with whole-body absorptiometry to calculate total body fat percentage (15,16). All data on each woman was collected within a 14-d period.
All subjects were brought to the vascular surgery ultrasound laboratory in a sedentary state after an overnight (8 h) fast. All subjects were studied in the resting supine position. Systemic arterial pressure was periodically monitored in the nondominant arm with an automated sphygmomanometer. Ambient room temperature and relative humidity were recorded before testing each subject. All subjects were tested at approximately the same time of day by a cardiologist (DG or EC). Before testing, the dominant arm was placed in a comfortable position and secured to a custom mechanical restraint device with a stereotaxic support for positioning a high-frequency (10 MHz) ultrasound probe over the brachial artery (ATL Ultramark 7 or GE Logiq 700). A manual sphygmomanometer cuff was placed on the forearm distal to the antecubital fossa. The brachial artery was imaged along the longitudinal axis proximal to the antecubital fossa, in similar fashion to that described by other authors (20). Areas of interest were chosen so that both near and far intimal-medial borders of the arterial wall were distinct over at least a 1.5-cm segment. The ultrasound probe was used to measure either diameter or central flow velocity (pulsed Doppler). At least 10 s of stable diameter recordings were assessed at each point where data was acquired for analysis. All brachial artery images were recorded on a super VHS videotape for subsequent analysis. Image analysis was performed on a dedicated Pentium III computer using a Matrox millennium card for A/D conversion of analog images at high resolution. Diameter was measured from digitized images using an automated edge detection program (Brachial Analyzer and Brachial Imager version 3.2.2) from Medical Imaging Systems (Iowa City, IA). Ten images were obtained each second for 10 s, and the averaged minimal diameter for each cardiac cycle was used for analysis.
Endothelium-dependent vasodilation was measured as the peak percent change in diameter of the brachial artery from baseline and during reactive hyperemia, measured 30 s and 1 and 2 min after release of a 4.5-min forearm occlusion (cuff inflation to 40 mm Hg above systolic pressure). Flow generally returned to baseline within 2 min of cuff release. Brachial artery diameter typically returns to baseline within several minutes of release. Ten minutes after baseline was reestablished, 0.4 mg of sublingual nitroglycerin was administered, and brachial artery diameter and velocity were imaged and averaged each minute for an additional 5 min. Subjects remained supine for at least 15 min after administration of nitroglycerin. Nitroglycerin-induced brachial artery dilation is considered an endothelium-independent process. Brachial flow velocity was measured as the peak systolic velocity from the pulsed-wave velocity signal corrected for probe angulations with respect to the vessel. The hyperemic response was measured as the percent change from baseline to maximal reactive hyperemia after release of the forearm cuff occlusion. Our technique of measuring flow-mediated dilation is patterned after that of Celermajer et al. (4). They found the brachial artery technique to have a reproducibility coefficient of variation of 1.4% and a repeatability coefficient of variation of 2.3% (4). In Celermajer et al.’s (4) and our current study, all measurements were made at end-diastole to avoid possible errors resulting from arterial compliance.
Comparison data between amenorrheic, oligomenorrheic, and control runners were analyzed with repeated measures analysis of variance. For the brachial artery studies, averaged peak individual percent flow-mediated dilation was compared among the three groups. Values are expressed as the mean ± SE. Significant F values were followed with Newman-Keuls post hoc analysis. A P value of < 0.05 was set as the level of statistical significance.
There were no differences among groups in height, weight, percentage body fat, body mass index, bone mineral density, and age of menarche (Table 1). Subjects were amenorrheic an average of 2.3 ± 0.5 yr (range 0.6–5) and oligomenorrheic an average of 6.6 ± 1.0 yr.
Baseline brachial diameters were similar among amenorrheic (3.61 ± 0.18 mm), oligomenorrheic (3.46 ± 0.13 mm), and control groups (3.44 ± 0.22 mm) (Table 2). The brachial artery vasodilator response to reactive hyperemia was significantly lower (P < 0.05) in the amenorrheic runners (1.08 ± 0.91%) compared with the control (6.38 ± 1.38%) and oligomenorrheic runners (6.44 ± 1.28%) (Fig. 1). However, endothelium-independent dilation to nitroglycerin was similar among groups (Fig. 1). The peak change in flow velocity during reperfusion, mean arterial blood pressure, heart rate, relative humidity, and temperature were similar among the groups (Table 2).
There were no differences in estradiol, estrone, total estrogen, progesterone, or FSH among the groups (Table 3). Although the mean estradiol level tended to be slightly lower in the amenorrheic runners (58.6 ± 9.32 pmol·L−1) than the control runners (67.09 ± 12.77 pmol·L−1), the difference was not significant. Prolactin, thyroid stimulating hormone, cholesterol, and hemoglobin were also similar among the groups (Table 3).
The total caloric, calcium, and phosphate intake of the three groups were similar (Table 4). There was no difference in carbohydrate, fat, or protein intake over the 3-d measurement period. All three groups consumed less than the daily-recommended 1500 mg of calcium (26).
This is the first study to demonstrate that young amenorrheic runners have a significant reduction in endothelium-dependent arterial vasodilation. Endothelial dysfunction has been implicated as a key event in the pathogenesis of atherosclerosis, hypertension, and heart failure (3,4). Under normal conditions, the endothelium produces nitric oxide, which is not only a potent vasodilator but also prevents platelet aggregation, leukocyte adhesion, and vascular smooth muscle proliferation and migration, each a key component of the atherosclerotic process (13,24). The endothelium senses and responds to a myriad of internal and external stimuli through complex cell membrane receptors and signal transduction mechanisms (9). Furthermore, work by Schachinger et al. (30) has shown that brachial endothelial dysfunction predicts long-term atherosclerotic disease progression and cardiovascular event rates (cardiovascular death, unstable angina, myocardial infarction, percutaneous transluminal coronary angioplasty, coronary bypass grafting, and ischemic stroke).
The importance of endothelial function in cardiovascular health is evident by the finding that subjects with risk factors for coronary artery disease, such as diabetes, hypertension, hyperlipidemia, tobacco use, and a family history of coronary disease, have reduced endothelial-dependent vasodilation as a heralding factor in the early acceleration of atherosclerotic plaques and cardiovascular events (5,17,18).
The postmenopausal state is associated with vascular changes consistent with enhanced risk for coronary events. Cardiovascular disease, primarily the consequence of atherosclerosis, is the leading cause of death in women. As with other risk factors, endothelial dysfunction is evident shortly after the onset of menopause (27). But just as estrogen treatment after menopause can reduce the risk of cardiovascular events, such treatment can also restore endothelium-dependent vasodilation (21). Lieberman et al. (21) have shown in previous studies of postmenopausal women that 1 mg of estradiol per day can improve brachial artery endothelial dilation in 9 wk. Estrogen is considered cardioprotective through its actions to reduce cholesterol and low-density lipoprotein levels, increase high-density lipoprotein levels, and stimulate release of nitric oxide. In our current study, we have demonstrated that athletic amenorrhea is also associated with endothelial dysfunction. The clinical implications of this dysfunction are twofold. First, loss of flow-mediated vasodilation in conduit arteries (an endothelium-dependent process) will restrict exercise-induced dilation of these vessels and limit maximum perfusion available to the tissue supplied. This could reduce exercise capacity by restricting flow to critical muscles involved in exercise. Second, chronic impairment of endothelial function leads to accelerated development of atherosclerosis in animal models and humans (2,6,8) and increased cardiovascular events in humans (30). Loss of endothelial function in athletes may hasten the development of cardiovascular disease and events by years or decades.
Exercise-induced amenorrhea is a complex physiological condition. The exact etiology of hypothalamic athletic amenorrhea is not known, and the diagnosis remains one of exclusion. The pathophysiology appears to be a result of dysfunction at the hypothalamic level. Gonadotropin-releasing hormone, which normally pulses every 60–90 min, is decreased. This leads to reduced levels of luteinizing hormone and ultimately to decreased estrogen production and amenorrhea (35). Excessive exercise (10), anorexia (34), low body fat, and psychogenic and physical stress (19) have all been implicated in decreasing gonadotropin-releasing hormone and resulting amenorrhea. However, increasing evidence suggests that nutritional restrictions and the resulting endocrine and metabolic changes are a critical initiator of hypothalamic-induced athletic amenorrhea and osteoporosis (35). Whether nutritional deficiencies are etiologic in endothelial dysfunction has not been studied.
Hypothalamic athletic amenorrhea is also a significant risk factor for osteoporosis and osteopenia (12). The original study by Drinkwater et al. (12) in 1984 revealed that young amenorrheic runners had lower vertebral body bone mineral density (1.12 ± 0.04 g·cm−2) compared with eumenorrheic runners (1.30 ± 0.03 g·cm−2). Our study confirmed a similar degree of bone demineralization (1.12 ± 0.04 g·cm−2). However, the control runners in this study had a lower vertebral body bone mineral density (1.22 ± 0.03 g·cm−2) compared with control runners (1.30 ± 0.03 g·cm−2) in the study by Drinkwater et al. (12). The most important bone density finding in our study is that our amenorrheic subjects were as equally demineralized as the subjects (1.12 ± 0.04 g·cm−2) in the study by Drinkwater et al. (12). Interestingly, von der Recke et al. (33) found that low bone mineral content at menopause is a risk factor for increased cardiovascular mortality later in life. It will be important to address this relationship further in future studies.
Drinkwater et al. (12) and Rencken et al. (28) have previously demonstrated that athletic amenorrhea is associated with estrogen deficiency, which appears to contribute to premenopausal osteoporosis in athletes. Estrogen receptors have been found on osteoblast-like cells, and the hypoestrogenic state seems to increase bone resorption in postmenopausal women (14). The current study found a trend toward a reduction in estrogen levels in the amenorrheic group, (58.6 ± 29.47 pmol·L−1) compared with the control group (67.09 ± 42.36 pmol·L−1). One limitation of this study is the relatively low number of subjects, which decreases the power to detect small differences in serum hormone levels between groups. Multiple studies by Warren et al. (35,37) did not find a difference in estradiol levels in 14 amenorrheic subjects (137.24 ± 155.04 pmol·L−1) compared with controls (207.63 ± 131.43 pmol·L−1), unlike our current study. They measured the amenorrheic group one time at random and the eumenorrheic group one time in the early follicular phase (days 3–7), suggesting that the hypoestrogenic state does not fully explain the cause of bone demineralization (35,37). Two previous studies (12,28) observed reductions in estradiol levels in amenorrheic subjects (38.58 ± 7 pmol·L−1 vs 106.99 ± 9.8 pmol·L−1) and (151.9 ± 32.7 pmol·L−1 vs 517 ± 54.3 pmol·L−1). In contrast to our protocol, Drinkwater et al. (12) and Rencken et al. (28) performed four blood draws separated by 7 d for both studies, which may have contributed to their ability to detect differences in average estradiol levels.
The American College of Sports Medicine published a position stand in 1997 describing in detail the inter-related problems of disordered eating, amenorrhea and osteoporosis, commonly known as the “Female Athlete Triad” (26). Our study subjects all had evidence of “disordered eating” characterized by a significant caloric restriction. According to calculations based on the World Health Organization (38) report on energy requirements, baseline caloric intake for the amenorrheic runners is 1725 kcal·d−1, and with the addition of running 35 miles·wk−1, they should consume at least 2190 cal·d−1 to stay in a positive energy balance; therefore, they were in a 465-calorie deficit (23). In addition, the average bone mineral density of the amenorrheic runners was 1.12 g·cm−2. This is similar to reference values established for 50-yr-old women (22). Two women had bone mineral densities less than 0.965 g·cm−2, which is considered below the fracture threshold (29). Based on the results of this study, further large-scale research studies are needed to confirm whether endothelial dysfunction is a consistent component of the Female Athlete Triad (Fig. 2).
Sports participation by women has increased significantly in the last few decades. This is due to a combination of factors including legislation creating new opportunities for women, physician endorsement, research confirming the benefits of exercise and team competition, societal changes, and media attention (36). However, we have also learned over the decades that sports participation by women has its own set of unique risk factors. This study implicates endothelial dysfunction with the attendant predisposition to early vascular disease as one of those risk factors for women who develop exercise-associated amenorrhea. In conclusion, young women with athletic amenorrhea have decreased endothelium-dependent vasodilation compared with oligomenorrheic and eumenorrheic runners. Endothelial dysfunction is a precursor of early cardiovascular disease. Although exercise is widely believed to be cardioprotective, excessive exercise to the point of amenorrhea may obviate this benefit and actually accelerate cardiovascular morbidity in young women. A better understanding of the relationship between impaired endothelial function, athletic performance, and the risk of an acute or subacute cardiovascular events in amenorrheic women runners is necessary before specific recommendations can be made about treatment or modification of exercise routines. For the vast majority of women, the health benefits of exercise significantly outweigh the detrimental effects.
The authors would like to thank GE Medical for donating the GE Logiq 700 ultrasound machine for the study and Jane Schimke, A.A.S., for technical assistance.
This study was supported in part by the Physiatric Association of Spine Sport and Occupational Rehabilitation (P.A.S.S.O.R.) and the Cardiovascular Center at the Medical College of Wisconsin.
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Keywords:©2003The American College of Sports Medicine
FEMALE ATHLETE TRIAD; WOMEN’S HEALTH; CARDIOVASCULAR DISEASE; FEMALE ATHLETES