Hannele Hohtari, M_D.t Katariina Salminen-Lappalainen, M.Sc.:j: Timo Laatikainen, M.D.:j:

Transcription

1 FRTILITY AND STRILITY Copyright e 1991 The American Fertility Society Vol. 55, No.2, February 1991 Printed on acid-free paper in U.S.A. Response of plasma endorphins, corticotropin, cortisol, and luteinizing hormone in the corticotropin-releasing hormone stimulation test in eumenorrheic and amenorrheic athletes* Hannele Hohtari, M_D.t Katariina Salminen-Lappalainen, M.Sc.:j: Timo Laatikainen, M.D.:j: The Finnish Foundation of xercise and Sports Medicine, Helsinki, and Departments of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland We compared pituitary response in the corticotropin-releasing hormone (CRH) test between 9 eumenorrheic and 10 amenorrheic endurance athletes. The maximal oxygen capacity (V0 2 max), determined using an exercise test on a bicycle ergometer, was larger in amenorrheic (62.7 ± 1.0 S ml/min per kg) than in eumenorrheic (54.7 ± 2.3 ml/min per kg) athletes. A 100 ILg bolus of human CRH was administered intravenously, and blood samples were collected at -15, 0, 30, 60, 90, and 120 minutes. The mean basal concentrations of endorphins, adrenocorticotropic hormone (ACTH), and cortisol did not show significant differences between the groups. The cumulative response of ACTH in the CRH test was larger in eumenorrheic (7.7 ± 1.3 pmoljl) than in amenorrheic (3.6 ± 0.6 pmoljl) athletes, but the response of endorphins and cortisol did not differ between the groups. A negative correlation was found between the V0 2max and the ACTH response during the CRH test in the total group of athletes. These findings indicated changes in the function of hypothalamic-pituitary-adrenal axis in amenorrheic athletes that can be attributed to intensive training. Fertil SteriI55:276, 1991 Hard endurance training is known to suppress the pituitary-ovarian axis. 1,2 This explains anovulatory cycles, oligomenorrhea, and amenorrhea common in female endurance athletes. 3 The mechanism by which training leads to disturbances of the menstrual cycle is still unclear. Physical stress can result in hypothalamic disturbances, shown by decreased pulsatile secretion of luteinizing hormone (LH) in eumenorrheic 4 and amenorrheic5 athletes. Received May 4, 1990; revised and accepted September 13, * Supported by a grant from the Paulo Foundation. t The Finnish Foundation of xercise and Sports Medicine. :j: Department of Obstetrics and Gynecology, University of Helsinki. Reprint requests and present address: Timo Laatikainen, M.D., Department of Obstetrics and Gynecology, University of Oulu, SF Oulu, Finland. Increased plasma and urinary cortisol values have been found in female athletes, 6 especially in those who had amenorrhea. 7 These findings suggest that disorders in the menstrual cycle in athletes may be associated with abnormalities of the hypothalamic-pituitary-adrenal (HPA) axis. Corticotropin-releasing hormone (CRH) has a central role in the regulation of HP A axis. It is released from the hypothalamus to stimulate pituitary secretion of corticotropin and endorphins.s Corticotropin -releasing hormone may suppress gonadotropin secretion in the hypothalamus directly or by stimulating the release of,b-endorphin.s,9 To elucidate the role of CRH in athletic amenorrhea, we have compared the effect of exogenous CRH on pituitary corticotropin,,b-endorphin, and LH secretion between eumenorrheic and amenorrheic female endurance athletes. 276 Hohtari et al. CRH test in athletic amenorrhea

2 Table 1 Clinical Characteristics of the Subjects Studied a Variable Age (y) Height (cm) Weight (kg) Body fat (%) V0 2 max (ml/kg/min) Blood lactate (mmol/l at V02 max) umenorrheic athletes (n = 9) 18.2 ± ± ± ± ± ± 0.65 a Values are means ± S. b P < 0.01 compared with eumenorrheic athletes. Subjects MATRIALS AND MTHODS Amenorrheic athletes (n = 10) 18.8 ± ± ± l.4 b 20.2 ± ± LOb 7.47 ± 0.95 Nineteen healthy female athletes participated in the study. Six of them were endurance runners, three orienteers, two track-and-field athletes, two skiers, two rowers, one triathlon athlete, and one football player. Ten of these subjects were amenorrheic and nine had regular periods. The mean duration (±S) of amenorrhea was 17.2 ± 2.7 months. None of the subjects had been on a regimen of contraceptive steroids or on other hormonal therapy for at least half a year before the study. Some physical characteristics in both groups are shown in Table 1. During the first visit, the percentage of body fat and the maximal oxygen uptake (V02max) were determined. Body fat was assessed with a Harpender skinfold caliper (British indicators Ltd., St. Albans, United Kingdom) by measuring skinfold thickness at four standard sites (biceps, triceps, subscapularis, and suprailiac). The percentage of body fat was calculated from the sum of the four skinfold measurements, according to Durnin and Rahaman.1o For determination of V02max, the exercise test was performed on a bicycle ergometer. Two graded submaximal loads and a maximal load were used without intervening pauses. The submaximal workloads lasted for 6 minutes each. To reach the maximal level, the load was increased every 2 minutes by 150 kpm/min (25 w) until exhaustion. During the last 2 minutes, the subjects were encouraged to increase the pedaling rate. Respiratory gas analyses (oxygen consumption imd carbon dioxide production) were made automatically with a gas analyzer (Mijnhardt Oxygon-2, Odijle, The Neth- erlands). Blood samples for hormone determinations were taken before and immediately after the exercise test. During the last visit, a CRH test was performed between 12:00 A.M. and 5:00 P.M. A 100 ILg bolus of human CRH (Bissendorf peptide GmbH; Wedemark, West Germany) was administered intravenously (IV). Blood samples were collected at -15, 0,30,60,90, and 120 minutes in ethylenediamintetraacetic acid tubes. After centrifugation, the plasma specimens were stored at -20 C until assayed. Laboratory Methods The radioimmunoassay (RIA) of plasma endorphins was performed as described in detail previouslyy The antiserum "K 2" used cross-reacted on a molar basis 100% with ~-endorphin and ~-lipotropin. Thus, ~-endorphin + ~-lipotropin was measured with the present assay. The intra-assay variability of the determinations of ~-endorphin + ~-lipotropin was 11 %. The RIA of corticotropin (ACTH) was carried out as described previously.12 The reference human ACTH (1-39) was purchased from Peninsula Laboratories Ltd. (St. Helens, United Kingdom). The intra-assay variability was 8.2%. Plasma concentration of LH was measured by a time-resolved immunofluorometric assayy Reagents were obtained from LKB-Wallac, Turku, Finland. The intra-assay coefficient of variation was 3.8%. Plasma cortisol was measured using a commercial RIA kit (Farmos Diagnostica, Turku, Finland). The intra-assay and interassay variations were 2.8% and 5%, respectively. Blood lactate was determined fluorometrically as described previously.14 The differences between the groups were tested with Student's t-test. The changes of hormone levels in relation to time and the differences between the groups were tested by ANOV A for repeated measures. A cumulative response was a sum of values obtained by subtracting the value of a hormone at time point 0 from values at 30 to 120 minutes. RSULTS Amenorrheic athletes did not differ from eumenorrheic athletes in relation to age but weighed less and had a larger V02max in the exercise test (Table 1). Blood lactate concentration did not Vol. 55, No.2, February 1991 Hohtari et al. CRH test in athletic amenorrhea 277

3 f' Table 2 Concentrations of Corticotropin, ndorphins, and Cortisol at Rest, and the Peak Concentrations After CRH Stimulation and After the xercise Test With Maximal Intensity in umenorrheic and Amenorrheic Athletes umenorrheic Amenorrheic (n = 9) (n = 10) ACTH (pmol/l) Basal 2.3 ± ± 0.91 Increase after CRH 3.3 ± ± 0.51 Increase after maximal exercise 4.2 ± ± 1.8 {:I-ndorphin + {:I-lipotropin (pmol/l) Basal 4.1 ± ± 1.1 Increase after CRH 7.3 ± ± 1.4 Increase after maximal exercise 3.9 ± ± 0.85 Cortisol (nmol/l) Basal 260 ± ± 45 Increase after CRH 300± ± 46 Values are means ± S. differ significantly between the groups at the end of the maximal exercise test. Basal concentrations of ACTH, fj-endorphin + fj-lipotropin, and cortisol before the CRH test did not show significant differences between the eumenorrhea and amenorrhea groups (Table 2). The increase after CRH administration or after the maximal exercise test was calculated by subtracting the basal value from the peak value. There were no statistically significant differences between the eumenorrhea and amenorrhea groups in the increases in the levels of these hormones, although the responses in the CRH test tended to be lower in the amenorrhea group. Similar increases in the ACTH and fj-endorphin + fj-lipotropin levels were found after the exercise test in the eumenorrhea and amenorrhea groups. Mean responses of ACTH, fj-endorphin + fj-lipotropin, and cortisol concentrations during the CRH stimulation test can be seen in Figure 1. The cumulative increase in plasma ACTH level was greater in the eumenorrhea group (7.7 ± 1.36 pmoljl [±S]) than in the amenorrhea group (3.6 ± 0.94 pmoljl, P = 0.022). The cumulative increase in the plasma cortisol (797 ± 136 nmoljl and 704 ± 148 nmoljl, respectively) or fj-endorphin + fj-lipotropin (14.6 ± 4.7 pmoljl and loa ± 2.2 pmoljl, respectively) concentrations did not show significant differences between the eumenorrhea and amenorrhea groups.. In the total group of athletes, correlations between weight, percentage body fat, or V0 2max, and the maximal or cumulative increase of the hormones during the CRH test were calculated. There was a negative correlation (R = -0.58, P = 0.024) between V0 2max value and the cumulative increase of ACTH concentration during the CRH test. The basal levels of LH in the eumenorrhea and amenorrhea groups were 3.85 ± 0.66 IU /L and 2.69 ± 0.57 IU/L, respectively. No significant change in the plasma LH level was found during the CRH test in either group. DISCUSSION The women in the present study were all endurance athletes and were training actively at the time of the study. However, the physical fitness evaluated by means of V0 2max values varied considerably between the subjects. In the age group of the ~ 10 9 B 8.a. 7 e 8. 6 ::::; 5 co e- 2 o W co 4 ~3 B2.a. g 1 8 ' r-~~--_, -_.., ::J 'is g ' 100 () 50 o o ~~---,----.., o Time (min) Figure 1. The mean (±S) responses of plasma ACTH, endorphins ({:I-endorphin + (:I-lipotropin), and cortisol concentrations in the CRH stimulation test in eumenorrheic and amenorrheic athletes. 278 Hohtari et ai. CRH test in athletic amenorrhea

4 women studied, V0 2max ;::: 63 ml/kg per minute is generally considered to signify a good physical fitness. Five of the 10 amenorrheic athletes were in this category as compared with none of the 9 athletes menstruating regularly. So the present amenorrheic athletes were more trained than the eumenorrheic athletes. Menstrual periods have been shown to become more irregular with an increase of intensity and duration of training. 15 Only in a few earlier studies, V0 2max has been determined, and no significant correlation to amenorrhea was found.7,16 No significant differences were found in the basal concentrations of endorphins, ACTH, and cortisol between the groups of amenorrheic and eumenorrheic athletes. Increased urinary cortisol secretion6 and elevated morning values of plasma cortiso16,7 have been reported in athletic women. Plasma cortisol levels and urinary cortisol secretion were increased in women with hypothalamic amenorrhea because of simple weight loss or psychological stress. 17 The plasma levels of endorphins at rest were also increased in amenorrheic athletes. ll These findings suggested that the basal activity of HP A axis is increased in hypothalamic amenorrhea related to stress. This is not primarily of adrenal origin because ACTH stimulation test did not reveal any altered sensitivity ofthe adrenal cortex to secrete cortisol in female athletes. 6 The response of hormones to stress is episodic and therefore the cumulative response may describe the changes better than the increase of the concentration during the test. In the present study, the response of plasma ACTH to an IV bolus of CRH was decreased in amenorrheic athletes, indicating a decreased sensitivity of the anterior pituitary to CRH stimulation in athletic amenorrhea. Also Loucks et al. 7 reported recently a blunted ACTH response to CRH administration in athletic women when compared with sedentary cycling women. The present amenorrheic athletes were more trained than the eumenorrheic athletes, and there was a negative correlation between the V02_ max and the ACTH response during the CRH test. Thus the blunted ACTH response to CRH stimulation can be attributed to training. The basal levels of ACTH, endorphins, and cortisol in the present amenorrheic athletes were not decreased, and even increased basal cortisol levels have earlier been reported in amenorrheic athletes.s,7 Increased release of endogenous CRH in the hypothalamus as a re- suit of intensive training may explain these findings. Corticotropin -releasing hormone has been shown to decrease gonadotropin secretion in primates18 probably by increasing the release of endogenous opioids in the hypothalamus. 19 Thus, CRH may be a factor mediating the effect of stress on the menstrual cycle. Infusion of CRH has been found to decrease plasma levels of LH and FSH in the midluteal phase of the cycle in healthy women. 7 In the present study, we were not able to demonstrate any significant effect of CRH on plasma LH levels in athletic women. Corticotropin -releasing hormone probably acts on the gonadotropin secretion in the hypothalamus and not at the level ofthe pituitary as was in the case of ACTH and endorphins. Because of the blood-brain barrier, CRH given into the peripheral circulation is transferred poorly to the central nervous system. This limits the value of the CRH test in the evaluation of the role of CRH in the regulation of gonadotropin secretion in athletic women. In the present series of women endurance athletes, the amenorrheic athletes were better trained than the eumenorrheic athletes. The blunted response of plasma ACTH level to CRH stimulation test in amenorrheic athletes suggests changes in the function of HP A axis. Our findings support the hypothesis that hard endurance training increases the endogenous release of CRH, which may be a mechanism whereby it induces disturbances of the menstrual cycle. RFRNCS 1. 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 ndocrinol Metab 60:416, Prior JC: Luteal phase defects and anovulation: adaptive alterations occurring with conditioning exercise. Sem Reprod ndocrinol 3:27, _ Shangold MM: xercise and amenorrhea. Semin Reprod ndocrinol 3:35, Cumming DC, Vickovic MM, Wall SR, Fluker MR: Defects of pulsatile LH release in normally menstruating runners. J Clin ndocrinol Metab 60:810, Veldhuis JD, vans WS, Demers LM, Thorner MO, Wakat D, Rogol AD: Altered neuroendocrine regulation of gonadotropin secretion in women distance runners. J Clin ndocrinol Metab 61:557, Villaneuva AL, Schlosser C, Hopper B, Liu JH, Hoffman DI, Rebar RW: Increased cortisol production in women runners. J Clin ndocrinol Metab 63:133, Loucks AB, Mortola JF, Girton L, Yen SSC: Alterations in Vol. 55, No_ 2, February 1991 Hohtari et al. err test in athletic amenorrhea 279

5 the hypothalamic-pituitary-ovarian and the hypothalamicpituitary-adrenal axis in athletic women. J Clin ndocrinol Metab 68:402, Rivier C, Vale W: Influence of corticotropin releasing factor on reproductive functions in the rat. ndocrinology 11:295, Barbarino A, de Marinis L, Tofani A, Della Casa S, D' Amico C, Mancini A, Corsello SM, Sciuto R, Barini A: Corticotropin-releasing hormone inhibition of gonadotropin release and the effect of opioid blockade. J Clin ndocrinol Metab 68:523, 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, Laatikainen T, Virtanen T, Apter D: Plasma immunoreactive /3-endorphin in exercise-associated amenorrhea. Am J Obstet GynecoI154:94, Hohtari H, lovainio R, Salminen K, Laatikainen T: Plasma corticotropin-releasing hormone, corticotropin and endorphins at rest and during exercise in eumenorrheic and amenorrheic athletes. Fertil Steril 50:233, Stenman U-H, Alfthan H, Koskimies A, Seppiilii M, Pettersson K, Lovgren T: Monitoring the LH surge by ultrarapid and highly sensitive immunofluorometric assay. Ann NY Acad Sci 442:544, Hiirkonen M, Kormano M: Acute cadmium-induced changes in the energy metabolism of the rat testis. J Reprod Fertil21:221, Dale, Gerlach DH, Wilhite AL: Menstrual dysfunction in distance runners. Obstet Gynecol 54:47, Loucks AB, Horvath SM: xercise-induced stress responses of amenorrheic and eumenorrheic runners. J Clin ndocrinol Metab 59:1109, Biller BMK, Federoff HJ, Koenig JI, Klibanski A: Abnormal cortisol secretion and responses to corticotropin-releasing hormone in women with hypothalamic amenorrhea. J Clin ndocrinol Metab 70:311, Olster DH, Ferin M: Corticotropin-releasing hormone inhibits gonadotropin secretion in the ovariectomized rhesus monkey. J Clin ndocrinol Metab 65:262, Gindoff PR, Ferin M: ndogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate. ndocrinology 121:837, Hohtari et al. CRH test in athletic amenorrhea