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Plasma corticotropin-releasing hormone, corticotropin, and endorphins at rest and during exercise in eumenorrheic and amenorrheic athletes*
FERTILITY AND STERILITY Copyright c 1988 The American Fertility Society
Vol. 50, No. 2, August 1988 Printed in U.S.A.
Plasma corticotropin-releasing hormone, corticotropin, and endorphins at rest and during exercise in eumenorrheic and amenorrheic athletes*
Hannele Hohtari, M.D.t Risto Elovainio, M.D.t Katariina Salminen, B.Sc.t Timo Laatikainen, M.D.t§
The Finnish Foundation of Exercise and Sports Medicine, and Helsinki University Central Hospital, Helsinki Finland
The hypothalamic-pituitary response to exercise was studied in 12 amenorrheic and in 9 eumenorrheic athletes by comparing the concentrations of corticotropin-releasing hormone (CRH), corticotropin (ACTH), and endorphins ({3-endorphin + {3-lipotropin) in plasma at rest and during an acute exercise on a bicycle ergometer requiring 80% and 100% of the maximal oxygen uptake (V0 2 max). Plasma CRH levels did not change during the exercise, and the mean CRH values did not differ between the amenorrheic and eumenorrheic groups. In both groups, significant increases in the response to exercise were found in the concentrations of ACTH and endorphins. The only significant difference between the groups was a larger mean pre-exercise concentration of endorphins in amenorrheic than in eumenorrheic athletes (4.8 ± 0.8 standard error [SE] and 2.9 ± 0.2 pmol/1, respectively). It is concluded that in amenorrheic athletes the capacity of the anterior pituitary to secrete ACTH and endorphins in response to exercise does not significantly differ from that in eumenorrheic atheletes, although basal endorphin secretion may be increased. Fertil Steril 50:233, 1988
Received February 10, 1988; revised and accepted April 18, 1988. * Supported by a grant from the Academy of Finland. t The Finnish Foundation of Exercise and Sports Medicine. :(:Department I of Obstetrics and Gynecology, Helsinki University Central Hospital. § Reprint requests: Timo Laatikainen, M.D., Department I of Obstetrics and Gynecology, Helsinki University Central Hospital, Haartmanink 2, 00290 Helsinki, Finland.
cretion of luteinizing hormone (LH) in eumenorrheic athletes, 4 •5 as well as in amenorrheic athletes. 6 These findings suggest a hypothalamic disturbance. In the central nervous system, corticotropin-releasing hormone (CRH) and ~-endorphin (~-E) are released in response to stress. 7 CRH and ~-E play a role in the inhibitory control of LH secretion,8 and their activation has been proposed to be a mechanism through which stress inhibits LH secretion. In the peripheral plasma, the concentration of ~- E of pituitary origin increases in response to physical exercise. 9 The presence of CRH has also been reported in the peripheral plasma. 10 Demonstration of a diurnal variation in the plasma level of CRH 11 suggests that changes in central CRH activity are reflected in the peripheral plasma. No data are available on CRH in eumenorrheic and amenorrheic athletes during exercise. The aim of the
Vol. 50, No.2, August 1988
Hohtari et al.
Among female athletes, anovulatory cycles, oligomenorrhea, and amenorrhea occur more often than in the general population. 1 Hard endurance training may suppress the pituitary-ovarian axis 2 •3 and "other factors" such as psychologic stress, decrease in body fat and increase in energy output may contribute. The cause of exercise-associated amenorrhea has not been conclusively established. Acute exercise and training decrease pulsatile se-
Stress response in athletic amenorrhea
233
present study was to investigate possible differences in hypothalamic-pituitary function between eumenorrheic and amenorrheic athletes by comparing plasma concentrations of CRH, corticotropin (ACTH), and endorphins at rest and during acute exercise.
MATERIALS AND METHODS Subjects
Twenty-one healthy female athletes participated in the study. All subjects filled out a questionnaire on physical aspects, reproductive endocrine characteristics, and sports activities. Seven of the athletes were endurance runners, and there were four skiers, four rowers, three orienteers, two trackand-field athletes, and one volleyball player. Twelve of these subjects were amenorrheic, and nine had regular periods. The mean (± standard deviation [SD]) duration of amenorrhea was 13.6 (±15.1) months, the range being from 4 months to 5 years, and the range of the menstrual cycle of eumenorrheic athletes was 21 to 35 days. Other endocrine causes of amenorrhea were excluded in these women. All subjects had trained regularly for an average of 6 years. The average (mean± SD) hours used in training activities weekly at the time of the study were 12.2 ± 2.8 hours in the eumenorrheic athletes and 11.4 ± 3.6 hours in the amenorrheic subjects, and the ranges of weekly distances were 20 to 90 km and 20 to 200 km, respectively. The groups did not show any significant differences in training as compared by means of days/week training, weekly training mileage, power training schedule, or years of training. Some physical and endocrine characteristics in both groups are shown in Table 1. Gynecologic examination of the amenorrheic athletes was carried by one of us (HH). None of the subjects in the study had been on a regimen of contraceptive steroids or on any other hormonal therapy for at least half a year before the study. During the first visit the percentage of body fat and maximal oxygen uptake (V0 2 max) were determined. Body fat was assessed by measuring skinfold thicknesses with a Harpender skinfold caliper (British Indicators Ltd., St. Albans, England) at four standard sites (biceps, triceps, subscapularis, and suprailiac). The percentage of body
234
Hohtari et al.
Stress response in athletic amenorrhea
Table 1 Clinical and Endocrine Characteristics of the Subjects Studied (Mean ± SE)
Variable
Amenorrheic athletes N = 12
Eumenorrheic athletes N=9
Age (yr) Height (em) Weight (kg) Age at menarche (yr) Body fat(%) vo2 max (mllkg/min) FSH (IU/L) LH (IU/L) E 2 (nmol/1)
18.5 ± 0.6 167 ± 1.9 52.3 ± 1.9 13.8 ± 0.4 18.8 ± 1.4 58.3 ± 1.7 5.2 ± 0.7 4.5 ± 0.9 0.1 ± 0.0
16.2 ± 0.5" 168 ± 1.9 58.6 ± 2.1" 12.9 ± 0.2 23.4 ± 1.3" 54.6 ± 2.0 5.4 ± 0.7 4.7 ± 0.3 0.1 ± 0.0
• P < 0.05 compared to amenorrheic athletes.
fat was calculated from the sum of the four skinfold measurements, according to Durnin and Rahaman. 12 Exercise Tests
For determination of V0 2 max, the first exercise test was performed on a bicycle ergometer. Two graded submaximalloads and a maximal load were used without intervening pauses. The submaximal workloads lasted 6 minutes each. To reach the maximal level, the load was increased every 2 minutes by 150 kpm/min (25W) until subjective exhaustion. During the last 2 minutes, the subjects were encouraged to increase the pedaling rate. Respiratory gas analyses (oxygen consumption and carbon dioxide production) were made automatically with a gas-analyzer (Mijnhardt Oxycon-2, Odijk, Netherlands). The second acute exercise test was made at least 1 week later between 10:00 A.M. and 2:00P.M., and in eumenorrheic athletes in the early follicular phase (days 3 to 8) of the cycle. The athletes refrained from training for at least 12 hours and did not have coffee, tea, or chocolate before the test. The basal plasma levels of follicle-stimulating hormone (FSH), LH and estradiol (E 2 ) were measured in the early follicular phase. An intravenous catheter was inserted before the test. The duration of the exercise on the bicycle ergometer was 12 minutes at the level of 80% of vo2 max and about 2 minutes at the level of 100% V0 2 max. A 30-minute pre-exercise rest and a 20-minute postexercise rest period were taken in a sitting position. Blood samples were collected serially before (at 0 minutes), during (at 10 and 14 minutes), and after
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the exercise (at 24 and 34 minutes) for measurements of plasma CRH, ACTH, !3-E plus !3-lipotropin (!3-LPH), and lactate. CRH was measured in the pre-exercise rest, after 100% exercise, and in the postexercise rest, and lactate at the end of the 80% and 100% of V0 2 max exercise periods. Blood samples were collected in prechilled ethylenediamintetraacetic acid, disodium salt (EDT A) tubes for the hormone assays and in sodium fluoride tubes for the determination of lactate. After centrifugation, the plasma specimens were stored at -20°C until assayed. Laboratory Methods
For the assay of CRH, synthetic human/rat CRH and tyrosine-CRH were purchased from Cambridge Research Laboratories (Cambridge, England). Labeled 125 -I-Tyr-CRH was prepared by the chloramine-T method. A CRH antiserum, raised against synthetic human/rat CRH in rabbits, was purchased from Peninsula Laboratories Europe Ltd. (St. Helens, England). The extraction of CRH from 1-ml samples of plasma and the radioimmunoassay (RIA) were performed as described in detail earlier .13 The recovery of reference CRH added to plasma was 62%, and the intra-assay variation was 6.8%. The lowest detectable concentration of immunoreactive CRH was 1 pmol/1. Corticotropin and endorphins were extracted from 2-ml samples of plasma with Sep Pak C18 cartridges (Waters Associates Inc., Milford MA) as described by Cahill et al. 14 The RIA of plasma endorphins was carried out as described in detail previously. 15 The antiserum "K 2" used cross-reacted on a molar basis 100% with !3-E and !3-LPH but <0.01% with enkephalins and ACTH. The intra-assay variation of the determination of !3-E and !3-LPH was 11%, and therecoveries of the reference !3- E and !3- LPH added to plasma were 78% and 77%, respectively. The RIA of ACTH was carried out as described in detail by Nicholson et al./ 6 with some modifications. The reference human ACTH (1-39) was purchased from Peninsula Europe Laboratories Ltd. (St Helens, UK). The ACTH antiserum was raised in a guinea pig. The bound and unbound radioactivity was separated with an anti-guinea pig solidphase antibody-coated cellulose suspension (Wellcome Research Laboratory, Beckenham, UK). The intra-assay variability was 8.2%, and the mean recovery of ACTH added to plasma was 80%. RIAS of LH and FSH were performed with kits
Vol. 50, No.2, August 1988
purchased from Amersham International, Little Chalfont, UK. The within-assay coefficient of variation ranged from 2.4% to 5.8%. A kit for the assay of E 2 was purchased from Farmos Diagnostica (Oulunsalo, Finland). The intra-assay variation varied from 4.1% to 9.8%. Blood lactate was measured ftuorometrically in a perchloric acid extract according to Lowry and Passonneau/ 7 as modified by Harki::inen and Kormano.18 The significances of the changes of hormone levels in relation to time and the differences between the groups were tested by analysis of variance for repeated measures (Program 4V ofBMDP statistical Software Inc. Los Angeles, CA). At each time point, differences between the groups were also tested with Student's t-test and differences within the groups with paired t-test.
RESULTS Comparison of physical characteristics (Table 1) showed that the subjects in the amenorrheic group were slightly older and leaner than those with regular cycles. The age at menarche and the mean V0 2 max did not differ between the groups. Differences in the mean levels of LH, FSH, and E2 between eumenorrheic athletes in the early follicular phase and amenorrheic athletes were not significant (Table 1). The mean concentrations (±SE) of lactate in the eumenorrheic and amenorrheic groups at the end of the 80% vo2 max test were 2.8 ± 0.33 and 3.46 ± 0.29 mmol/1, respectively, and at the end of the 100% vo2 max test, 6.98 ± 0.42 and 6.61 ± 0.38 mmol/1, respectively. There were no significant differences between the groups. Paired comparison of the concentrations of CRH between samples taken at rest and at the end of the 100% exercise test did not show any significant difference, neither were there any differences in the mean CRH values at rest or during exercise between the eumenorrheic and amenorrheic groups (Table 2). In the eumenorrheic group, the concentration of {j-E and !3-LPH increased during exercise in every subject. The percentage increase varied from 29% to 380% (mean 110%). The responses were more variable in the amenorrheic group; in two amenorrheic endurance runners no response was found, and in the remaining amenorrheic women the increase varied from 8.9% to 460%. The mean pre-
Hohtari et al. Stress response in athletic amenorrhea
235
Table 2 The Mean ± SE Concentrations of CRH, Endorphins, and ACTH in Amenorrheic .
Rest 1 CRH pmol/1 P-E/P-LPH pmol/1 ACTH pmol/1
7.9 ± 1.1 9.8 ± 2.0 4.8 ± o.8" 2.9 ± 0.2 3.5 ± 0.0 2.2 + 0.4
A E A E A E
8.0 ± 1.6 4.5 ± 0.6 6.9 ± 1.8 3.6 ± 0.4
100% 8.0 ± 10.2 ± 9.2 ± 5.7 ± 7.9 ± 4.4 ±
0.8 1.7 1.8 0.7 1.7 0.9
Rest2
7.6 ± 6.4 ± 6.4 ± 4.6 ±
1.6 0.9 1.4 0.9
Rest 3 10.1 ± 10.4 ± 5.2 ± 4.8 ± 4.3 ± 3.8 ±
2.2 0.9 0.8 0.6 0.7 0.5
• p < 0.05.
exercise concentration of ,8-E and ,8-LPH was greater in the amenorrheic athletes than in the eumenorrheic athletes (P < 0.05, Fig. 1, Table 2). In the total group of 21 subjects, analysis of variance showed significant increases in the plasma levels of ACTH (P = 0.0009) and ,8-E and ,8-LPH (P = 0.0016) in relation to time but no significant differences between the amenorrheic and eumenorrheic groups. The area under the curve shown in Figure 1 was also calculated in each subject. Comparison of areas under the curves did not show any significant differences between the groups. The mean concentrations of ACTH during rest or exercise did not show any significant differences between the amenorrheic and eumenorrheic groups (Table 2). The mean levels of ACTH increased significantly during the test, both in the eumenorrheic (from 2.19 ± 0.44 to 4.4 ± 0.9 pmol/1, P < 0.05) and in the amenorrheic (from 3.5 ± 0.6 to 7.9 ± 1.7 pmol/1, P < 0.01) groups. There were highly significant correlations between the increases of ACTH and ,8-E and ,8-LPH concentrations during the exercise in the eumenorrheic (R = 0.91, P < 0.001) and amenorrheic (R = 0.92, P < 0.001) groups.
~12~--------------.
§. 10
·g."
ii :3" ~
8
6 4
c
~ 2 ~ ~
0
+-_.,...::::;:::=;:......._,.__-,.--..----!
-10
"'
10
20
Time, min
30
40
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Figure 1 The mean (± SE) concentrations of P-E + P-LPH and corticotropin before, during and after the acute exercise test in amenorrheic and eumenorrheic athletes.
236
Hohtari et al. Stress response in athletic amenorrhea
DISCUSSION
With an increase of intensity and duration of training, menstrual periods appear to become more irregular. 19 Differences in training did not, however explain amenorrhea in this study, as the pres~nt amenorrheic and eumenorrheic subjects both were trained and did not differ in physical fitness. Athletes with an initially low body weight and low body fat are widely believed to be at risk as regards menstrual disorders, 20 although no association between body fat and athletic amenorrhea has also been reported. 21 The present amenorrheic athletes were lighter and had a slightly lower percentage body fat than the control athletes, but most of their weights were within the percentages of ideal body weight as compared with a large number of Finnish women. The weights of the two leanest amenorrheic athletes differed only -11% and -12%, respectively from the ideal body weight. Thus the slight difference in body composition was not likely to be the only factor behind amenorrhea in the present series of athletes. An exercise test requiring the maximal V02 max was chosen in the present study, because it has been shown that a strenuous exercise, 80% to 100% of vo2 max, is required to elicit a response in the plasma levels of endorphins and ACTH in experienced endurance athletes. 22 Increased lactate levels in the blood showed anaerobic responses to exercise in all subjects. Concentrations of endorphins and ACTH in the plasma increased in response to exercise testing in both groups, but without significant differences between the groups. Thus, significantly abnormal endorphin or ACTH responses to exercise were not found in the amenorrheic athletes. However, varia-
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tion in the responses of plasma ACTH and endorphins to exercise was larger among the subjects in the amenorrheic group, and the two subjects showing no response were amenorrheic. Prolonged training has been reported to augment the endorphin response to exercise,9 although it has not been observed in all studies. 23 Differences in training did not affect the present endorphin responses, as the amenorrheic and eumenorrheic groups were similarly trained. Our amenorrheic group may not have been entirely homogeneous in relation to "severity" of hypothalamic amenorrhea, as some of the patients may have been in the stationary phase and some in the recovery phase of amenorrhea at the time of the study. Indeed, in three of the amenorrheic athletes, menstrual periods began 1 to 3 months after the end of the study. Stress has been found to increase the release of CRH into the rat hypophyseal portal circulation. 7 In man, insulin-induced hypogycemia is the only stress-related situation where the concentration of CRH has been reported to increase in peripheral plasma. 10 In the present study, no significant change was found in the plasma level of CRH during physical exercise, neither were there any differences between eumenorrheic and amenorrheic athletes. CRH in the circulation has been proposed to originate in part from peripheral tissues, 10 and this may mask possible changes of hypothalamic origin and explain the failure to detect any change in plasma CRH during exercise. In the present and in earlier studies/ 5 •24 increased basal concentrations of endorphins were found in the plasma of amenorrheic athletes. Earlier, increased basal cortisol secretion was reported in highly trained amenorrheic and eumenorrheic runners. 25 These findings suggest an increase in the basal activity of the pituitary-adrenal axis. Training was not found to increase basal levels of endorphins in earlier studies. 9•23 It has been reported that amenorrheic athletes experience more stress in their running than athletes with regular cycles. 20 In such persons, increased basal endorphin concentrations may represent an "abnormal" response to training. To summarize, we did not find any significantly changed responses of plasma ACTH or endorphins during acute exercise in amenorrheic athletes, but the pre-exercise basal levels of endorphins were increased in amenorrheic versus eumenorrheic athletes, suggesting a different long-term response to stress.
Vol. 50, No.2, August 1988
REFERENCES 1. Shangold MM: Exercise and amenorrhea. Seminars Reprod Endocrinol 3:35, 1985 2. Ronkainen H, Pakarinen A, Kirkinen P, Kauppila A: Physical exercise-induced changes and season-associated differences in the pituitary-ovarian function of runners and joggers. J Clin Endocrinol Metab 60:416, 1985 3. Prior JC: Luteal phase defects and anovulation: Adaptive alterations occurring with conditioning exercise. Sem Reprod Endocrinol 3:27, 1985 4. Cumming DC, Vickovic MM, Wall SR, Fluker MR: Defects of pulsatile LH release in normally menstruating runners. J Clin Endocrinol Metab 60:810, 1985 5. Cumming DC, Vickovic MM, Wall SR, Fluker MR, Belcastro AN: The effect of acute exercise on pulsatile release of luteinizing hormone in women runners. Am J Obstet Gynecol 153:482, 1985 6. Veldhuis JD, Evans WS, Demers LM, Thorner MO, Wakat D, Rogol AD: Altered neuroendocrine regulation of gonadotropin secretion in women distance runners. J Clin Endocrinol Metab 61:557, 1985 7. Plotsky P, Vale W: Haemorrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat hypophysial portal circulation and its inhibition by glucocorticoids. Endocrinology 114:164, 1984 8. Petraglia F, Rivier C, Vale W: Opioids, CRF and gonadotropin secretion: central mechanisms acting during stress. In Gynecological Endocrinology, Vol 1, Edited by AR Genazzani, A Volpe, F Facchinetti. Carnforth, United Kingdom, Parthenon Publishing Group, 1987, p 31 9. Carr DB, Bullen BA, Skrinar GS, Arnold MA, Rosenblatt M, Beitins IZ, Martin JB, McArthur JW: Physical conditioning facilitates the exercise-induced secretion of betaendorphin and beta-lipotrophin in women. N Eng! J Med 305:560, 1981 10. Suda T, Tomori N, Yajima F, Sumitomo T, Nakagami Y, Ushiyama T, Demura H, Shizume K: Immunoreactive corticotropin-releasing factor in human plasma. J Clin Invest 76:2026, 1985 11. Watabe T, Tanaka K, Kumagae M, Itoh S, Hasegawa M, Horiuchi T, Miyabe S, Ohno H, Shimizu N: Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects. Life Sci 40:1651, 1987 12. Durnin JVG, Rahaman MM: The assessment of the amount of fat in the human body from measurements of skinfold thickness. Br J Nutr 21:681, 1967 13. Laatikainen T, Virtanen T, Riiisiinen I, Salminen K: Immunoreactive corticotropin-releasing factor and corticotropin in plasma during pregnancy, labor and puerperium. Neuropeptides 10:343, 1987 14. Cahill CA, Matthews JD, Akil H: Human plasma j3-endorphin-like peptides: a rapid high recovery extraction technique and validation of radioimmunoassay. J Clin Endocrinol Metab 56:992, 1983 15. Laatikainen T, Virtanen T, Apter D: Plasma immunoreactive j3-endorphin in exercise-associated amenorrhea. Am J Obstet Gynecol 154:94, 1986 16. Nicholson WE, Davis DR, Sherell BJ, Orth DN: Rapid radioimmunoassay for corticotropin in unextracted human plasma. Clin Chern 30:259, 1984 17. Lowry OH, Passonneau JV: A collection of metabolite
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18.
19. 20.
21.
assays. In A Flexible System of Enzymatic Analysis, Edited by OH Lowry, JV Passonneau. New York, Academic Press, 1972, p 146 Hiirkonen M, Kormano M: Acute cadmium-induced changes in the energy metabolism of the rat testis. J Rep rod Fertil 21:221, 1970 Dale E, Gerlach DH, Wilhite AL: Menstrual dysfunction in distance runners. Obstet Gynecol 54:47, 1979 Schwartz B, Cumming DC, Riordan E, Selye M, Yen SSC, Rebar RW: Exercise-associated amenorrhea: a distinct entity? Am J Obstet Gynecol 141:662, 1981 Sanborn CF, Albrecht BH, Wagner WW, Jr: Athletic amenorrhea: lack of association with body fat. Med Sci Sports Exerc 19:207, 1987
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22. Rahkila P, Hakala E, Salminen K, Laatikainen T: Response of plasma endorphins to running exercises in male and female endurance athletes. Med Sci Sports Exerc 19:451, 1987 23. Howlett TA, Tomlin S, Ngahfoong L, Rees LH, Bullen BA, Skrinar GS, McArthur JW: Release of 13-endorphin and metenkephalin during exercise in normal women: response to training. Br Med J 288:1950, 1984 24. Russell JB, Mitchell DE, Musey PI, Collins DC: The role of 13-endorphins and catechol estrogens on the hypothalamicpituitary axis in female athletes. Fertil Steril 42:690, 1984 25. Villanueva AL, Schlosser C, Hopper B, Liu JH, Hoffmann DI, Rebar RW: Increased cortisol production in women runners. J Clin Endocrinol Metab 63:133, 1986
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Vol. 50, No. 2, August 1988 Printed in U.S.A.
Plasma corticotropin-releasing hormone, corticotropin, and endorphins at rest and during exercise in eumenorrheic and amenorrheic athletes*
Hannele Hohtari, M.D.t Risto Elovainio, M.D.t Katariina Salminen, B.Sc.t Timo Laatikainen, M.D.t§
The Finnish Foundation of Exercise and Sports Medicine, and Helsinki University Central Hospital, Helsinki Finland
The hypothalamic-pituitary response to exercise was studied in 12 amenorrheic and in 9 eumenorrheic athletes by comparing the concentrations of corticotropin-releasing hormone (CRH), corticotropin (ACTH), and endorphins ({3-endorphin + {3-lipotropin) in plasma at rest and during an acute exercise on a bicycle ergometer requiring 80% and 100% of the maximal oxygen uptake (V0 2 max). Plasma CRH levels did not change during the exercise, and the mean CRH values did not differ between the amenorrheic and eumenorrheic groups. In both groups, significant increases in the response to exercise were found in the concentrations of ACTH and endorphins. The only significant difference between the groups was a larger mean pre-exercise concentration of endorphins in amenorrheic than in eumenorrheic athletes (4.8 ± 0.8 standard error [SE] and 2.9 ± 0.2 pmol/1, respectively). It is concluded that in amenorrheic athletes the capacity of the anterior pituitary to secrete ACTH and endorphins in response to exercise does not significantly differ from that in eumenorrheic atheletes, although basal endorphin secretion may be increased. Fertil Steril 50:233, 1988
Received February 10, 1988; revised and accepted April 18, 1988. * Supported by a grant from the Academy of Finland. t The Finnish Foundation of Exercise and Sports Medicine. :(:Department I of Obstetrics and Gynecology, Helsinki University Central Hospital. § Reprint requests: Timo Laatikainen, M.D., Department I of Obstetrics and Gynecology, Helsinki University Central Hospital, Haartmanink 2, 00290 Helsinki, Finland.
cretion of luteinizing hormone (LH) in eumenorrheic athletes, 4 •5 as well as in amenorrheic athletes. 6 These findings suggest a hypothalamic disturbance. In the central nervous system, corticotropin-releasing hormone (CRH) and ~-endorphin (~-E) are released in response to stress. 7 CRH and ~-E play a role in the inhibitory control of LH secretion,8 and their activation has been proposed to be a mechanism through which stress inhibits LH secretion. In the peripheral plasma, the concentration of ~- E of pituitary origin increases in response to physical exercise. 9 The presence of CRH has also been reported in the peripheral plasma. 10 Demonstration of a diurnal variation in the plasma level of CRH 11 suggests that changes in central CRH activity are reflected in the peripheral plasma. No data are available on CRH in eumenorrheic and amenorrheic athletes during exercise. The aim of the
Vol. 50, No.2, August 1988
Hohtari et al.
Among female athletes, anovulatory cycles, oligomenorrhea, and amenorrhea occur more often than in the general population. 1 Hard endurance training may suppress the pituitary-ovarian axis 2 •3 and "other factors" such as psychologic stress, decrease in body fat and increase in energy output may contribute. The cause of exercise-associated amenorrhea has not been conclusively established. Acute exercise and training decrease pulsatile se-
Stress response in athletic amenorrhea
233
present study was to investigate possible differences in hypothalamic-pituitary function between eumenorrheic and amenorrheic athletes by comparing plasma concentrations of CRH, corticotropin (ACTH), and endorphins at rest and during acute exercise.
MATERIALS AND METHODS Subjects
Twenty-one healthy female athletes participated in the study. All subjects filled out a questionnaire on physical aspects, reproductive endocrine characteristics, and sports activities. Seven of the athletes were endurance runners, and there were four skiers, four rowers, three orienteers, two trackand-field athletes, and one volleyball player. Twelve of these subjects were amenorrheic, and nine had regular periods. The mean (± standard deviation [SD]) duration of amenorrhea was 13.6 (±15.1) months, the range being from 4 months to 5 years, and the range of the menstrual cycle of eumenorrheic athletes was 21 to 35 days. Other endocrine causes of amenorrhea were excluded in these women. All subjects had trained regularly for an average of 6 years. The average (mean± SD) hours used in training activities weekly at the time of the study were 12.2 ± 2.8 hours in the eumenorrheic athletes and 11.4 ± 3.6 hours in the amenorrheic subjects, and the ranges of weekly distances were 20 to 90 km and 20 to 200 km, respectively. The groups did not show any significant differences in training as compared by means of days/week training, weekly training mileage, power training schedule, or years of training. Some physical and endocrine characteristics in both groups are shown in Table 1. Gynecologic examination of the amenorrheic athletes was carried by one of us (HH). None of the subjects in the study had been on a regimen of contraceptive steroids or on any other hormonal therapy for at least half a year before the study. During the first visit the percentage of body fat and maximal oxygen uptake (V0 2 max) were determined. Body fat was assessed by measuring skinfold thicknesses with a Harpender skinfold caliper (British Indicators Ltd., St. Albans, England) at four standard sites (biceps, triceps, subscapularis, and suprailiac). The percentage of body
234
Hohtari et al.
Stress response in athletic amenorrhea
Table 1 Clinical and Endocrine Characteristics of the Subjects Studied (Mean ± SE)
Variable
Amenorrheic athletes N = 12
Eumenorrheic athletes N=9
Age (yr) Height (em) Weight (kg) Age at menarche (yr) Body fat(%) vo2 max (mllkg/min) FSH (IU/L) LH (IU/L) E 2 (nmol/1)
18.5 ± 0.6 167 ± 1.9 52.3 ± 1.9 13.8 ± 0.4 18.8 ± 1.4 58.3 ± 1.7 5.2 ± 0.7 4.5 ± 0.9 0.1 ± 0.0
16.2 ± 0.5" 168 ± 1.9 58.6 ± 2.1" 12.9 ± 0.2 23.4 ± 1.3" 54.6 ± 2.0 5.4 ± 0.7 4.7 ± 0.3 0.1 ± 0.0
• P < 0.05 compared to amenorrheic athletes.
fat was calculated from the sum of the four skinfold measurements, according to Durnin and Rahaman. 12 Exercise Tests
For determination of V0 2 max, the first exercise test was performed on a bicycle ergometer. Two graded submaximalloads and a maximal load were used without intervening pauses. The submaximal workloads lasted 6 minutes each. To reach the maximal level, the load was increased every 2 minutes by 150 kpm/min (25W) until subjective exhaustion. During the last 2 minutes, the subjects were encouraged to increase the pedaling rate. Respiratory gas analyses (oxygen consumption and carbon dioxide production) were made automatically with a gas-analyzer (Mijnhardt Oxycon-2, Odijk, Netherlands). The second acute exercise test was made at least 1 week later between 10:00 A.M. and 2:00P.M., and in eumenorrheic athletes in the early follicular phase (days 3 to 8) of the cycle. The athletes refrained from training for at least 12 hours and did not have coffee, tea, or chocolate before the test. The basal plasma levels of follicle-stimulating hormone (FSH), LH and estradiol (E 2 ) were measured in the early follicular phase. An intravenous catheter was inserted before the test. The duration of the exercise on the bicycle ergometer was 12 minutes at the level of 80% of vo2 max and about 2 minutes at the level of 100% V0 2 max. A 30-minute pre-exercise rest and a 20-minute postexercise rest period were taken in a sitting position. Blood samples were collected serially before (at 0 minutes), during (at 10 and 14 minutes), and after
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the exercise (at 24 and 34 minutes) for measurements of plasma CRH, ACTH, !3-E plus !3-lipotropin (!3-LPH), and lactate. CRH was measured in the pre-exercise rest, after 100% exercise, and in the postexercise rest, and lactate at the end of the 80% and 100% of V0 2 max exercise periods. Blood samples were collected in prechilled ethylenediamintetraacetic acid, disodium salt (EDT A) tubes for the hormone assays and in sodium fluoride tubes for the determination of lactate. After centrifugation, the plasma specimens were stored at -20°C until assayed. Laboratory Methods
For the assay of CRH, synthetic human/rat CRH and tyrosine-CRH were purchased from Cambridge Research Laboratories (Cambridge, England). Labeled 125 -I-Tyr-CRH was prepared by the chloramine-T method. A CRH antiserum, raised against synthetic human/rat CRH in rabbits, was purchased from Peninsula Laboratories Europe Ltd. (St. Helens, England). The extraction of CRH from 1-ml samples of plasma and the radioimmunoassay (RIA) were performed as described in detail earlier .13 The recovery of reference CRH added to plasma was 62%, and the intra-assay variation was 6.8%. The lowest detectable concentration of immunoreactive CRH was 1 pmol/1. Corticotropin and endorphins were extracted from 2-ml samples of plasma with Sep Pak C18 cartridges (Waters Associates Inc., Milford MA) as described by Cahill et al. 14 The RIA of plasma endorphins was carried out as described in detail previously. 15 The antiserum "K 2" used cross-reacted on a molar basis 100% with !3-E and !3-LPH but <0.01% with enkephalins and ACTH. The intra-assay variation of the determination of !3-E and !3-LPH was 11%, and therecoveries of the reference !3- E and !3- LPH added to plasma were 78% and 77%, respectively. The RIA of ACTH was carried out as described in detail by Nicholson et al./ 6 with some modifications. The reference human ACTH (1-39) was purchased from Peninsula Europe Laboratories Ltd. (St Helens, UK). The ACTH antiserum was raised in a guinea pig. The bound and unbound radioactivity was separated with an anti-guinea pig solidphase antibody-coated cellulose suspension (Wellcome Research Laboratory, Beckenham, UK). The intra-assay variability was 8.2%, and the mean recovery of ACTH added to plasma was 80%. RIAS of LH and FSH were performed with kits
Vol. 50, No.2, August 1988
purchased from Amersham International, Little Chalfont, UK. The within-assay coefficient of variation ranged from 2.4% to 5.8%. A kit for the assay of E 2 was purchased from Farmos Diagnostica (Oulunsalo, Finland). The intra-assay variation varied from 4.1% to 9.8%. Blood lactate was measured ftuorometrically in a perchloric acid extract according to Lowry and Passonneau/ 7 as modified by Harki::inen and Kormano.18 The significances of the changes of hormone levels in relation to time and the differences between the groups were tested by analysis of variance for repeated measures (Program 4V ofBMDP statistical Software Inc. Los Angeles, CA). At each time point, differences between the groups were also tested with Student's t-test and differences within the groups with paired t-test.
RESULTS Comparison of physical characteristics (Table 1) showed that the subjects in the amenorrheic group were slightly older and leaner than those with regular cycles. The age at menarche and the mean V0 2 max did not differ between the groups. Differences in the mean levels of LH, FSH, and E2 between eumenorrheic athletes in the early follicular phase and amenorrheic athletes were not significant (Table 1). The mean concentrations (±SE) of lactate in the eumenorrheic and amenorrheic groups at the end of the 80% vo2 max test were 2.8 ± 0.33 and 3.46 ± 0.29 mmol/1, respectively, and at the end of the 100% vo2 max test, 6.98 ± 0.42 and 6.61 ± 0.38 mmol/1, respectively. There were no significant differences between the groups. Paired comparison of the concentrations of CRH between samples taken at rest and at the end of the 100% exercise test did not show any significant difference, neither were there any differences in the mean CRH values at rest or during exercise between the eumenorrheic and amenorrheic groups (Table 2). In the eumenorrheic group, the concentration of {j-E and !3-LPH increased during exercise in every subject. The percentage increase varied from 29% to 380% (mean 110%). The responses were more variable in the amenorrheic group; in two amenorrheic endurance runners no response was found, and in the remaining amenorrheic women the increase varied from 8.9% to 460%. The mean pre-
Hohtari et al. Stress response in athletic amenorrhea
235
Table 2 The Mean ± SE Concentrations of CRH, Endorphins, and ACTH in Amenorrheic .
Rest 1 CRH pmol/1 P-E/P-LPH pmol/1 ACTH pmol/1
7.9 ± 1.1 9.8 ± 2.0 4.8 ± o.8" 2.9 ± 0.2 3.5 ± 0.0 2.2 + 0.4
A E A E A E
8.0 ± 1.6 4.5 ± 0.6 6.9 ± 1.8 3.6 ± 0.4
100% 8.0 ± 10.2 ± 9.2 ± 5.7 ± 7.9 ± 4.4 ±
0.8 1.7 1.8 0.7 1.7 0.9
Rest2
7.6 ± 6.4 ± 6.4 ± 4.6 ±
1.6 0.9 1.4 0.9
Rest 3 10.1 ± 10.4 ± 5.2 ± 4.8 ± 4.3 ± 3.8 ±
2.2 0.9 0.8 0.6 0.7 0.5
• p < 0.05.
exercise concentration of ,8-E and ,8-LPH was greater in the amenorrheic athletes than in the eumenorrheic athletes (P < 0.05, Fig. 1, Table 2). In the total group of 21 subjects, analysis of variance showed significant increases in the plasma levels of ACTH (P = 0.0009) and ,8-E and ,8-LPH (P = 0.0016) in relation to time but no significant differences between the amenorrheic and eumenorrheic groups. The area under the curve shown in Figure 1 was also calculated in each subject. Comparison of areas under the curves did not show any significant differences between the groups. The mean concentrations of ACTH during rest or exercise did not show any significant differences between the amenorrheic and eumenorrheic groups (Table 2). The mean levels of ACTH increased significantly during the test, both in the eumenorrheic (from 2.19 ± 0.44 to 4.4 ± 0.9 pmol/1, P < 0.05) and in the amenorrheic (from 3.5 ± 0.6 to 7.9 ± 1.7 pmol/1, P < 0.01) groups. There were highly significant correlations between the increases of ACTH and ,8-E and ,8-LPH concentrations during the exercise in the eumenorrheic (R = 0.91, P < 0.001) and amenorrheic (R = 0.92, P < 0.001) groups.
~12~--------------.
§. 10
·g."
ii :3" ~
8
6 4
c
~ 2 ~ ~
0
+-_.,...::::;:::=;:......._,.__-,.--..----!
-10
"'
10
20
Time, min
30
40
O~~~E•e~
-1 0
10 20 Time, min
30
40
Figure 1 The mean (± SE) concentrations of P-E + P-LPH and corticotropin before, during and after the acute exercise test in amenorrheic and eumenorrheic athletes.
236
Hohtari et al. Stress response in athletic amenorrhea
DISCUSSION
With an increase of intensity and duration of training, menstrual periods appear to become more irregular. 19 Differences in training did not, however explain amenorrhea in this study, as the pres~nt amenorrheic and eumenorrheic subjects both were trained and did not differ in physical fitness. Athletes with an initially low body weight and low body fat are widely believed to be at risk as regards menstrual disorders, 20 although no association between body fat and athletic amenorrhea has also been reported. 21 The present amenorrheic athletes were lighter and had a slightly lower percentage body fat than the control athletes, but most of their weights were within the percentages of ideal body weight as compared with a large number of Finnish women. The weights of the two leanest amenorrheic athletes differed only -11% and -12%, respectively from the ideal body weight. Thus the slight difference in body composition was not likely to be the only factor behind amenorrhea in the present series of athletes. An exercise test requiring the maximal V02 max was chosen in the present study, because it has been shown that a strenuous exercise, 80% to 100% of vo2 max, is required to elicit a response in the plasma levels of endorphins and ACTH in experienced endurance athletes. 22 Increased lactate levels in the blood showed anaerobic responses to exercise in all subjects. Concentrations of endorphins and ACTH in the plasma increased in response to exercise testing in both groups, but without significant differences between the groups. Thus, significantly abnormal endorphin or ACTH responses to exercise were not found in the amenorrheic athletes. However, varia-
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tion in the responses of plasma ACTH and endorphins to exercise was larger among the subjects in the amenorrheic group, and the two subjects showing no response were amenorrheic. Prolonged training has been reported to augment the endorphin response to exercise,9 although it has not been observed in all studies. 23 Differences in training did not affect the present endorphin responses, as the amenorrheic and eumenorrheic groups were similarly trained. Our amenorrheic group may not have been entirely homogeneous in relation to "severity" of hypothalamic amenorrhea, as some of the patients may have been in the stationary phase and some in the recovery phase of amenorrhea at the time of the study. Indeed, in three of the amenorrheic athletes, menstrual periods began 1 to 3 months after the end of the study. Stress has been found to increase the release of CRH into the rat hypophyseal portal circulation. 7 In man, insulin-induced hypogycemia is the only stress-related situation where the concentration of CRH has been reported to increase in peripheral plasma. 10 In the present study, no significant change was found in the plasma level of CRH during physical exercise, neither were there any differences between eumenorrheic and amenorrheic athletes. CRH in the circulation has been proposed to originate in part from peripheral tissues, 10 and this may mask possible changes of hypothalamic origin and explain the failure to detect any change in plasma CRH during exercise. In the present and in earlier studies/ 5 •24 increased basal concentrations of endorphins were found in the plasma of amenorrheic athletes. Earlier, increased basal cortisol secretion was reported in highly trained amenorrheic and eumenorrheic runners. 25 These findings suggest an increase in the basal activity of the pituitary-adrenal axis. Training was not found to increase basal levels of endorphins in earlier studies. 9•23 It has been reported that amenorrheic athletes experience more stress in their running than athletes with regular cycles. 20 In such persons, increased basal endorphin concentrations may represent an "abnormal" response to training. To summarize, we did not find any significantly changed responses of plasma ACTH or endorphins during acute exercise in amenorrheic athletes, but the pre-exercise basal levels of endorphins were increased in amenorrheic versus eumenorrheic athletes, suggesting a different long-term response to stress.
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REFERENCES 1. Shangold MM: Exercise and amenorrhea. Seminars Reprod Endocrinol 3:35, 1985 2. Ronkainen H, Pakarinen A, Kirkinen P, Kauppila A: Physical exercise-induced changes and season-associated differences in the pituitary-ovarian function of runners and joggers. J Clin Endocrinol Metab 60:416, 1985 3. Prior JC: Luteal phase defects and anovulation: Adaptive alterations occurring with conditioning exercise. Sem Reprod Endocrinol 3:27, 1985 4. Cumming DC, Vickovic MM, Wall SR, Fluker MR: Defects of pulsatile LH release in normally menstruating runners. J Clin Endocrinol Metab 60:810, 1985 5. Cumming DC, Vickovic MM, Wall SR, Fluker MR, Belcastro AN: The effect of acute exercise on pulsatile release of luteinizing hormone in women runners. Am J Obstet Gynecol 153:482, 1985 6. Veldhuis JD, Evans WS, Demers LM, Thorner MO, Wakat D, Rogol AD: Altered neuroendocrine regulation of gonadotropin secretion in women distance runners. J Clin Endocrinol Metab 61:557, 1985 7. Plotsky P, Vale W: Haemorrhage-induced secretion of corticotropin-releasing factor-like immunoreactivity into the rat hypophysial portal circulation and its inhibition by glucocorticoids. Endocrinology 114:164, 1984 8. Petraglia F, Rivier C, Vale W: Opioids, CRF and gonadotropin secretion: central mechanisms acting during stress. In Gynecological Endocrinology, Vol 1, Edited by AR Genazzani, A Volpe, F Facchinetti. Carnforth, United Kingdom, Parthenon Publishing Group, 1987, p 31 9. Carr DB, Bullen BA, Skrinar GS, Arnold MA, Rosenblatt M, Beitins IZ, Martin JB, McArthur JW: Physical conditioning facilitates the exercise-induced secretion of betaendorphin and beta-lipotrophin in women. N Eng! J Med 305:560, 1981 10. Suda T, Tomori N, Yajima F, Sumitomo T, Nakagami Y, Ushiyama T, Demura H, Shizume K: Immunoreactive corticotropin-releasing factor in human plasma. J Clin Invest 76:2026, 1985 11. Watabe T, Tanaka K, Kumagae M, Itoh S, Hasegawa M, Horiuchi T, Miyabe S, Ohno H, Shimizu N: Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects. Life Sci 40:1651, 1987 12. Durnin JVG, Rahaman MM: The assessment of the amount of fat in the human body from measurements of skinfold thickness. Br J Nutr 21:681, 1967 13. Laatikainen T, Virtanen T, Riiisiinen I, Salminen K: Immunoreactive corticotropin-releasing factor and corticotropin in plasma during pregnancy, labor and puerperium. Neuropeptides 10:343, 1987 14. Cahill CA, Matthews JD, Akil H: Human plasma j3-endorphin-like peptides: a rapid high recovery extraction technique and validation of radioimmunoassay. J Clin Endocrinol Metab 56:992, 1983 15. Laatikainen T, Virtanen T, Apter D: Plasma immunoreactive j3-endorphin in exercise-associated amenorrhea. Am J Obstet Gynecol 154:94, 1986 16. Nicholson WE, Davis DR, Sherell BJ, Orth DN: Rapid radioimmunoassay for corticotropin in unextracted human plasma. Clin Chern 30:259, 1984 17. Lowry OH, Passonneau JV: A collection of metabolite
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assays. In A Flexible System of Enzymatic Analysis, Edited by OH Lowry, JV Passonneau. New York, Academic Press, 1972, p 146 Hiirkonen M, Kormano M: Acute cadmium-induced changes in the energy metabolism of the rat testis. J Rep rod Fertil 21:221, 1970 Dale E, Gerlach DH, Wilhite AL: Menstrual dysfunction in distance runners. Obstet Gynecol 54:47, 1979 Schwartz B, Cumming DC, Riordan E, Selye M, Yen SSC, Rebar RW: Exercise-associated amenorrhea: a distinct entity? Am J Obstet Gynecol 141:662, 1981 Sanborn CF, Albrecht BH, Wagner WW, Jr: Athletic amenorrhea: lack of association with body fat. Med Sci Sports Exerc 19:207, 1987
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22. Rahkila P, Hakala E, Salminen K, Laatikainen T: Response of plasma endorphins to running exercises in male and female endurance athletes. Med Sci Sports Exerc 19:451, 1987 23. Howlett TA, Tomlin S, Ngahfoong L, Rees LH, Bullen BA, Skrinar GS, McArthur JW: Release of 13-endorphin and metenkephalin during exercise in normal women: response to training. Br Med J 288:1950, 1984 24. Russell JB, Mitchell DE, Musey PI, Collins DC: The role of 13-endorphins and catechol estrogens on the hypothalamicpituitary axis in female athletes. Fertil Steril 42:690, 1984 25. Villanueva AL, Schlosser C, Hopper B, Liu JH, Hoffmann DI, Rebar RW: Increased cortisol production in women runners. J Clin Endocrinol Metab 63:133, 1986
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