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Endocrinology Vol. 142, No. 6 2381-2389
Copyright © 2001 by The Endocrine Society

ARTICLES

Longitudinal Changes in Reproductive Hormones and Menstrual Cyclicity in Cynomolgus Monkeys during Strenuous Exercise Training: Abrupt Transition to Exercise-Induced Amenorrhea1

Nancy I. Williams2, Anne L. Caston-Balderrama3, Dana L. Helmreich4, David B. Parfitt, Connie Nosbisch5 and Judy L. Cameron

Departments of Psychiatry (N.I.W., A.L.C.-B., C.N., J.L.C.), Cell Biology & Physiology (N.I.W., A.L.C.-B., J.L.C.), and Neuroscience (D.L.H., D.B.P., J.L.C.), University of Pittsburgh, Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Judy L. Cameron, Ph.D., Department of Psychiatry, University of Pittsburgh, 3811 O’Hara Street, Pittsburgh, Pennsylvania 15213. E-mail: cameronj{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cross-sectional studies of exercise-induced reproductive dysfunction have documented a high proportion of menstrual cycle disturbances in women involved in strenuous exercise training. However, longitudinal studies have been needed to examine individual susceptibility to exercise-induced reproductive dysfunction and to elucidate the progression of changes in reproductive function that occur with strenuous exercise training. Using the female cynomolgus monkey (Macaca fascicularis), we documented changes in menstrual cyclicity and patterns of LH, FSH, estradiol, and progesterone secretion as the animals developed exercise-induced amenorrhea. As monkeys gradually increased running to 12.3 ± 0.9 km/day, body weight did not change significantly although food intake remained constant. The time spent training until amenorrhea developed varied widely among animals (7–24 months; mean = 14.3 ± 2.2 months) and was not correlated with initial body weight, training distance, or food intake. Consistent changes in function of the reproductive axis occurred abruptly, one to two menstrual cycles before the development of amenorrhea. These included significant declines in plasma reproductive hormone concentrations, an increase in follicular phase length, and a decrease in luteal phase progesterone secretion. These data document a high level of interindividual variability in the development of exercise-induced reproductive dysfunction, delineate the progression of changes in reproductive hormone secretion that occur with exercise training, and illustrate an abrupt transition from normal cyclicity to an amenorrheic state in exercising individuals, that is not necessarily associated with weight loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC STRENUOUS exercise training stimulates many favorable physiological adaptations; however, heavy training can also be accompanied by a range of perturbations in normal reproductive function in women. Estimates of the prevalence of secondary amenorrhea in female athletes range from 1–44%, exceeding estimates of 2–5% reported in sedentary women (1, 2, 3, 4, 5). These abnormalities have been documented in athletes participating in a wide variety of sports, but are most often noted in long-distance runners, gymnasts, and ballet dancers. In addition, the prevalence of milder reproductive abnormalities, such as luteal phase disturbances and anovulation, has recently been estimated to be as high as 42% and 16%, respectively, in women who exercise moderately (6). Although not as severe as amenorrhea, these abnormalities may compromise fertility. It is possible that shortening of the luteal phase and reduction of circulating levels of progesterone have detrimental effects on implantation and early maintenance of a conceptus, in that decreases in the duration or amount of progesterone secreted during the luteal phase have been correlated with failure to reproduce (7, 8, 9, 10). Furthermore, an exercise-induced reduction of circulating estrogens may lead to a loss of bone density (11, 12, 13), an increased risk of skeletal fractures (14), and clinically undesirable changes in lipoprotein profiles associated with increased cardiovascular risk (15).

The deficit in the functioning of the hypothalamic-pituitary-ovarian axis occurring with strenuous exercise training appears to be at the level of the hypothalamus. Several studies have found a reduction in LH pulse frequency in amenorrheic athletes and in exercising women with anovulation and luteal phase deficiency (16, 17). Moreover, ovulation has been successfully induced in amenorrheic athletes using clomiphene citrate, a stimulator of gonadotropin release (18). Most of these data have been collected in cross-sectional studies comparing groups of athletes with reproductive dysfunction to sedentary controls or with athletes maintaining normal menstrual cyclicity (16, 17, 18). While this approach accurately documents physiological conditions at the endpoint of exercise-induced reproductive dysfunction, such studies do not address issues related to the progression of reproductive changes that occur with exercise training. These include the varied susceptibility of individuals to exercise-induced disturbances in reproductive function, and the amount of training necessary for these alterations to occur. Additionally, interpretation of cross-sectional data must take into account the potential for self-selection of subjects participating in athletics, who may be highly susceptible to menstrual disturbances or may have preexisting alterations in neuroendocrine function.

To avoid the limitations of cross-sectional studies in humans, we undertook a longitudinal study of exercise-induced amenorrhea in a nonhuman primate species, the cynomolgus monkey (Macaca fascicularis). Female cynomolgus monkeys have 28–32 day menstrual cycles, like women, and offer the advantage of allowing control over exercise, diet and exposure to other environmental and social factors that may alter reproductive function throughout the training period. Unlike female rhesus monkeys (Macaca mulatta), they do not display seasonal changes in reproductive function (19 ; Cameron, unpublished data). This experimental model allowed us to clearly identify progressive changes in patterns of reproductive hormone secretion and menstrual cyclicity that occur with exercise training.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sixteen adult female cynomolgus monkeys, weighing 2.10 to 4. kg, were used for this experiment. These animals were born in the wild and imported as young adults and were maintained for several years before this study at the University of Pittsburgh Primate Research Laboratory in individual cages in rooms holding 40–80 monkeys. Animals were maintained on a controlled lighting schedule, with lights on from 0700–1900 h. Room temperature was 24 ± 2 C. Animals were fed the standard diet used at the Primate Research Laboratory, consisting of a single daily meal of approximately 300 Cal (15 pellets) of Purina high protein monkey chow (no. 5045, Ralston Purina Co., St. Louis, MO), supplemented with one quarter piece of fresh fruit (approximately 25 Cal). Monkeys were fed at 1600 h each day. The animals also received novel items such as toys or noncaloric foods, as part of a psychological enrichment program in accordance with the USDA guidelines. Water was available ad libitum. Food intake was recorded daily after removing any remaining food pellets from the previous day’s meal. Body weight was monitored every other day using a scale accurate to the nearest 0.1 kg (Acme Scale and Supply, Pittsburgh, PA). Monkeys had their vaginal area swabbed daily with a cotton-tipped applicator to detect menses. All experiments were performed in compliance with the regulations of the Animal Care and Use Committee of the University of Pittsburgh.

Blood sampling procedures
Blood samples for the measurement of serum LH, FSH, estradiol (E2), and progesterone (P4) were collected from unanesthetized animals every other day throughout the study. All animals were acclimatized to the sampling procedures over several menstrual cycles before the study, and therefore the blood sampling per se did not result in alterations in menstrual cyclicity. Samples were obtained before feeding and before the animals exercised. For collection of blood samples, each monkey was trained to jump from its cage into a transport box, allowing it to be carried to a nearby sampling room. Once in the sampling room, animals entered a specially designed cage to allow brief immobilization the monkey’s leg. Blood samples were obtained by femoral venipuncture. Samples were allowed to clot at room temperature for 1 h, were then refrigerated for 2 h, and subsequently were centrifuged at 2500 rpm for 10 min at 4 C. Serum was collected and stored at -20 C in glass vials until assays were performed. Every 6 weeks, hematocrit was measured and animals were given 50 mg im iron. Hematocrits were maintained within the normal range in all monkeys throughout the study.

Monitoring of reproductive function
Before the study, all animals were habituated to swabbing of the vaginal area and blood sampling procedures. The occurrence of at least three successive, normal menstrual cycles was documented in each monkey before the initiation of the study protocol. The first day of menses was designated as the first day of a menstrual cycle. A cycle was considered normal if it was ovulatory, 24–38 days in length, and exhibited typical cyclic changes in reproductive hormones, including a rise in estradiol (E2) in the late follicular phase, mid-cycle surges of LH and FSH, and a rise in progesterone (P4) during the luteal phase. The determination of normal cycle length was based on a statistical analysis with a large sample of monkeys from the colony which revealed that this is the range in which ovulatory cycles are most consistently observed. A monkey was considered to be amenorrheic if she did not have menses for a period equivalent to three of her average cycle lengths (90–110 days), and she exhibited low, noncyclic levels of P4 and E2, with no evidence of ovulation.

Exercise training
Animals were trained to run on standard human size treadmills (Model 910e; Precor, Inc., Bothell, WA). Each treadmill was covered by a plexiglass box, which had numerous air holes in the front and back panels to allow adequate ventilation (Fig. 1Go). Monkeys were slowly adapted to the treadmill, by first being allowed to sit on the treadmill and explore it for several days, and then being allowed to walk slowly. Speed and duration were then increased in an individualized manner, so that monkeys were not forced to run beyond a speed and duration at which they could run comfortably (Fig. 2Go). Monkeys were trained until they were running approximately 12 km per day. When fully trained, monkeys ran 7 days a week for a total of 2 h a day, with a 3-min break after each 30-min running period.



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  		Figure 1. Schematic diagram of a monkey running on a human sized treadmill covered by a ventilated plexiglass box.

 


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  		Figure 2. Patterns of daily training in two of the eight monkeys in this study. "AM1" indicates the beginning of the amenorrheic period in each monkey. Each monkey was trained in an individualized manner so that the monkey was not forced to run when it showed signs of fatigue. If a monkey became ill or received an injury during the study, her daily training was temporarily reduced or stopped (see brief period at day 100 for Monkey #2243, top panel).

 
Experimental protocol
Two groups of monkeys were studied, a group which exercised daily after an initial sedentary cycle (n = 8), and a control group which remained sedentary throughout the study (n = 8). For each monkey assigned to the exercise group a weight-matched control monkey was chosen from a larger group of monkeys within the colony (mean weight: exercise group = 2.81 ± 0.14 kg, control group = 3.12 ± 0.47 kg). The experimental protocol for the control animals was similar to that for the exercising animals, except that control animals did not participate in exercise training. Previous studies in the lab showed that there are no changes in menstrual cyclicity associated with daily sitting on a treadmill (20); thus for the current study control monkeys remained in their cages and did not sit on the treadmill daily. To determine the longitudinal pattern of changes in menstrual cyclicity and reproductive hormones during the course of the study the following parameters were monitored throughout the study in both groups of animals: the occurrence of menses, plasma concentrations of reproductive hormones, body weight, food intake, and daily mileage. For each exercising animal the study was continued until the animal had become amenorrheic for 90–100 days (3 menstrual cycle equivalents). Each control animal was studied for an equivalent number of cycles as that animal’s exercising counterpart.

Hormone assays
Serum LH and FSH were measured by RIA by the RIA Core Laboratory of the Center for Research in Reproductive Physiology at the University of Pittsburgh, using previously described methods (21, 22). The sensitivities of the LH assays ranged from 7.8–13.6 ng/ml. The intraassay and interassay coefficients of variation for the LH assays used in these studies were 7.3% and 9.4%, respectively. The sensitivities of the FSH assays ranged from 1.4–3.4 ng/ml. The intraassay and interassay coefficients of variation for the FSH assays used in these studies were 6.3% and 7.9%, respectively.

Plasma E2 concentrations were measured using the following procedure, which has not been previously published. A Diagnostic Products Estradiol RIA Kit protocol (KE2D1; Diagnostic Products Co., Los Angeles, CA), was modified by the RIA Core Laboratory of the University of Pittsburgh Center for Research in Reproductive Physiology. The following modifications were instituted: 1) standard curve tubes received 1, 2, 4, 10, 30, and 100 pg synthetic estradiol (Sigma, St. Louis, MO) in 100 µl steroid-stripped, ovariectomized (OVX) rhesus monkey serum; 2) total binding and nonspecific binding tubes received 100 µl steroid stripped OVX rhesus monkey serum instead of standard; 3) total binding tubes, standard curve tubes and sample tubes received 50 µl first antibody; 4) all tubes received 50 µl trace; 5) at the end of the incubation all tubes received 0.5 ml cold precipitating solution, followed 15 min later by 0.5 ml cold PBS. The sensitivities of the assay ranged from 2.12 to 4.54 pg/ml. The intraassay and interassay coefficients of variation for the estradiol assays used in these studies were 5.4% and 8.2%, respectively.

Plasma P4 concentrations were measured using a RIA developed by the RIA Core Laboratory of the University of Pittsburgh Center for Research in Reproductive Physiology, based on an earlier assay developed by Goodman (23). This previously unpublished protocol involved: 1) addition of 2 ml petroleum ether (Mallinckrodt, Inc. Specialty Chemicals Co, Paris, KY) to standard curve tubes, dried down using a stream of air in a water bath at 37 C, followed by addition and drying down of 5, 10, 20, 50, 100, 200, and 300 pg synthetic progesterone (a gift from Dr. Julane Hotchkiss) in ethyl alcohol; 2) samples of 50–200 µl were extracted using 2 ml petroleum ether with 5 min of vortexing on a Buchler vortex evaporator set at 6–7, followed by freezing of the aqueous phase by immersion in an ethyl alcohol dry ice bath and decanting of the ether phase into fresh tubes, followed by drying of ether under a stream of air in a water bath at 37 C; 3) fractional recovery tubes had 50 and 100 pg standard plus steroid-stripped OVX rhesus monkey serum in a volume equivalent to the sample volume; 4) all tubes, except nonspecific binding tubes (which received gel-PBS), received 100 µl anti-progesterone antibody (P11–192; Endocrine Sciences, Inc., Tarzana, CA) diluted 1:200 in gel-PBS; 5) all tubes received 100 µl 1,2,6,7-3H-progesterone (0.125 µCi/ml; NEN Life Science Products 381; Boston, MA) in gel-PBS; 6) tubes were incubated for 2 h at room temperature, followed by 30 min at 4 C; 7) tubes received 1 ml cold dextran-charcoal, were vortexed and incubated for 15 min at 4 C, centrifuged at 2,600 x G for 30 min at 4 C, and the supernatants decanted into scintillation vials with 3 ml Ultima Gold Counting Cocktail (Packard Instruments Co., Downers Grove, IL), and radioactive counts were quantified in a ß counter. The sensitivities of the assay ranged from 0.03 to 0.11 ng/ml. The intraassay and interassay coefficients of variation for the estradiol assays used in these studies were 4.4% and 7.6%, respectively.

Data analysis
All measurements reported were made throughout the study, including the sedentary period and the entire period of training in exercising animals and an equivalent period in controls. Consistent and significant changes in reproductive hormones were not noted until the last two menstrual cycles before the development of amenorrhea in exercising monkeys. Thus, in the current report, we first describe changes in menstrual cyclicity during early training and compare cyclicity to that observed during an equivalent number of menstrual cycles in sedentary control animals. We then focus our primary data analyses on reproductive hormone levels during the following six time periods during the study: when the exercising animals were sedentary and exhibiting normal menstrual cycles (Sed), when they were training but were not yet amenorrheic (Cycle-2 and Cycle-1, the last two cycles before amenorrhea developed), and when they had developed amenorrhea (AM1, AM2, and AM3; Fig. 3Go). AM1 was designated as beginning with the first day of the last menses. The time points AM1, AM2, and AM3 were each defined to extend for a duration equivalent to an individual monkey’s average cycle length. For example, if an animal normally had a menstrual cycle that was 30 days long then AM1, AM2, and AM3 would each encompass 30 days, so that the total period of amenorrhea included in the study would be from the first day of the last menses through the subsequent 90-day period.



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  		Figure 3. Schematic diagram of the experimental design. Data for reproductive hormones and kilometers run per day were analyzed during menstrual cycles when monkeys were sedentary (Sed), training hard but still eumenorrheic (Cycle-2, Cycle-1; the last two cycles before the development of amenorrhea), and then at three time points after monkeys had become amenorrheic, each equivalent to an average cycle length (AM1, AM2, and AM3).

 
The effects of exercise training on gonadotropin secretion were characterized by measuring LH and FSH in each sample obtained every other day over the course of the entire study. To characterize the effects of exercise training on circulating sex steroids, E2 was measured in samples obtained during the follicular phase of each cycle (i.e. the period from Day 1 of the cycle until the day of the LH surge). P4 was measured during the luteal phase in ovulatory cycles (i.e. in samples between the day of the LH surge and the day before menses). During the periods when monkeys were amenorrheic, E2 and P4 were measured in samples obtained every fourth day.

Across the six time periods specified above, the following parameters were calculated: 1) early follicular phase levels of LH and FSH (i.e. averages of LH and FSH concentrations measured in the first three blood samples collected during the menstrual cycle); 2) the magnitude of the preovulatory E2 peak (i.e. the highest E2 value immediately preceding the LH surge in ovulatory cycles); 3) average follicular phase E2 (i.e. the average of all the follicular phase estradiol concentrations measured); 4) peak P4 (i.e. the highest P4 level measured during the luteal phase); and 5) average P4 (i.e. the average of all the luteal phase P4 concentrations measured). During amenorrheic phases (AM1, AM2, and AM3), peak E2 and peak P4 represent the highest values measured during the amenorrheic period, and the average E2 and average P4 represent an average of all of the values during the period of amenorrhea. An additional calculation of the late luteal phase rise in FSH (i.e. the average of the last two measurements of FSH in the luteal phase) was made during Sed, Cycle-1, and Cycle-2. All FSH, E2 and P4 measures made were above detectability for their respective assays. LH values measured for some data points were at or below the level of assay detectability. To determine the effect of these data points on the LH data reported, a two-step analysis was performed. Data for LH measures were first analyzed without adjustments for varying assay sensitivities. Then, the data were reanalyzed after substituting the highest minimum detectable dose (13.6 ng/ml) obtained from all LH assays for all values below the minimum detectable dose. This adjustment for varying assay sensitivities did not change any of the statistical results; therefore, the data that are reported are not adjusted.

To detect significant effects of exercise on the parameters measured in this study two-way ANOVA (for the exercise and control groups) for repeated measures were performed. When a significant main effect was detected, Student’s t tests were performed to detect where differences occurred. All posthoc analyses were made using a Bonferoni correction to correct for multiple comparisons. A P value of less than or equal to 0.05 was considered significant. To test whether there was a correlation between training and dietary parameters and the number of months to induce amenorrhea, correlation coefficients were calculated using simple regression analyses. The significance of "r" was determined using an F test (SuperAnova, Abacus Concepts, Berkeley, CA). To compare the proportion of ovulatory cycles to total cycles, and the proportion of cycles that were of normal length (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) days between exercising and control animals we performed {chi} square tests. All data are reported as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Training and dietary parameters
Over the course of training, all exercising monkeys in the study became amenorrheic, while none of the control animals developed amenorrhea. From the onset of exercise training, the number of months to the onset of amenorrhea varied from 7 to 24, with a mean of 14.3 ± 2.2 months. Time to the onset of amenorrhea was not significantly correlated with initial body weight, average daily training distance, or average daily Cal/kg of food intake (Table 1Go). Body weight did not change significantly over the course of the study, even though food intake was held relatively constant while the monkeys progressed from being sedentary to running approximately 12 km/day (mean = 12.3 ± 0.9 km during AM3).


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  		Table 1. Training and dietary parameters in exercising animals

 
Effects of exercise on ovulation and menstrual cycle characteristics
Early training cycles. A total of 65 menstrual cycles were observed in exercising monkeys from the start of training up to, but not including Cycle-2. Fifty-three of the 65 cycles, or 81.5%, were normal length ovulatory cycles, while 4 cycles (6.2%) were anovulatory, 6 cycles (9.2%) were long ovulatory cycles and 2 cycles (3.1%) were short ovulatory cycles. In the sedentary monkeys, we observed that 61 of 65 cycles (93.9%) were normal length ovulatory cycles, with one anovulatory cycle (1.5%), 3 (4.6%) long ovulatory cycles, and no short ovulatory cycles. A {chi} square analysis of the distribution of cycles across the various menstrual cycle categories, i.e. normal length ovulatory, long ovulatory, short ovulatory, and anovulatory, revealed that there was no significant difference between exercising monkeys and their sedentary counterparts (Pearson {chi}-Square = 6.96; P = 0.073), although there was a trend of more abnormal cycles in exercising monkeys. Abnormal cycles in the exercisers occurred throughout the training period but did not appear with increasing frequency as training progressed, until Cycle-2. At any one time, before Cycle-2, there were no significant differences in menstrual cycle parameters between the exercising group and the control sedentary group.

Cycles preceding amenorrhea. When the menstrual cycles immediately before amenorrhea were considered, exercise training caused a significant increase (P < 0.05) in the total length of the menstrual cycle. The cycles for the exercising animals were 31 ± 2, 33 ± 2, and 37 ± 3 days in length, respectively, for Sed, Cycle-2, and Cycle-1. When cycle length was expressed as a percent of the length of each monkey’s Sed cycle, Cycle-2 was 106 ± 6% of Sed (P > 0.05 vs. Sed) and Cycle-1 was 122 ± 10% of Sed (P < 0.02 vs. Sed). Although all eight exercising monkeys experienced menses during Sed, Cycle-2 and Cycle-1, only five of eight exercising monkeys ovulated during Cycle-1 (i.e. three were anovulatory based on hormonal criteria but still experienced menstrual bleeding at the end of the Cycle-1 time period). In the five monkeys that ovulated during Cycle-1, the increase in cycle length from Sed to Cycle-1 was due to a lengthening of the follicular phase (14 ± 1 to 16 ± 2 to 23 ± 2 days for Sed, Cycle-2, and Cycle-1, respectively; P < 0.05 Cycle-1 vs. Sed), with no significant change in the length of the luteal phase (16 ± 1, 16 ± 1, and 15 ± 2 days for Sed, Cycle-2, and Cycle-1, respectively; Fig. 4Go). The number of days that menstrual bleeding was observed during Sed, Cycle-2, and Cycle-1 did not change significantly with training (3.3 ± 0.3, 3.2 ± 0.3, and 2.5 ± 0.4 days, respectively).



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  		Figure 4. Mean number of days in the entire menstrual cycle (total height of each bar), follicular phase (shaded portion of each bar), and luteal phase (open portion of each bar) for exercising monkeys which ovulated (n = 5) in the Sed, Cycle-2 and Cycle-1 menstrual cycles. Changes in total cycle length were a result of a progressive and significant increase in follicular phase length, with no change in luteal phase length. Asterisks represent a significant difference (P < 0.05) from values in the Sed cycles.

 
When equivalent time periods were analyzed for the monkeys in the control group, there were no significant changes in cycle length, length of the follicular phase, length of the luteal phase, or number of days of menstrual bleeding. Cycle lengths for control animals were 32 ± 4, 30 ± 2, and 31 ± 4 for the cycles equivalent to Sed, Cycle-2, and Cycle-1. Follicular phase lengths for control animals were 17 ± 4, 15 ± 2, and 15 ±3 for the cycles equivalent to Sed, Cycle-2, and Cycle-1.

Changes in reproductive hormones with exercise training
Mean data showing the changes in plasma concentrations of reproductive hormones during the sedentary cycle, the cycles immediately preceding the development of amenorrhea, and the period of amenorrhea for exercising monkeys are shown in Fig. 5Go. Similar data in the control monkeys are shown in Fig. 6Go. Individual data for a representative exercising monkey are shown in Fig. 7Go. A two-way ANOVA revealed a significant difference over time from Sed through AM3 for all measured reproductive hormones, with significant declines in each hormone occurring in the exercising animals and no significant changes over time occurring in the control animals. For the exercising group, early follicular phase levels of LH were significantly lower (P < 0.05) during Cycle-1, compared with Sed levels, and were further reduced (P < 0.05) during the amenorrheic period (AM1, AM2, and AM3) when compared with Cycle-2. Early follicular phase FSH levels were significantly lower (P < 0.05) during AM1 and AM2 compared with Sed levels in the exercising group. Although mean plasma FSH concentrations for all eight exercising monkeys declined as amenorrhea developed, 3 of the 8 monkeys actually showed an increase in mean circulating FSH concentrations during the amenorrheic period. Both average and peak follicular phase E2 levels were significantly decreased (P < 0.05) during AM1, AM2, and AM3, compared with Sed and Cycle-2 levels (P < 0.05) in the exercising group. And, during Cycle-1, AM1, AM2, and AM3, average and peak P4 concentrations were significantly lower than Sed levels (P < 0.05) in the exercising animals.



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  		Figure 5. Effects of training on plasma levels of reproductive hormones in the group of exercising monkeys (n = 8). a, Significant difference, P < 0.05, compared with Sed values in the exercising group; b, significant difference, P < 0.05, compared with the corresponding time point in the control group. Significance levels are corrected for multiple comparisons using a Bonferroni correction.

 


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  		Figure 6. Plasma levels of reproductive hormones in the group of control monkeys (n = 8) during the same time periods illustrated for the exercising group in Fig. 5Go. There were no significant changes in any parameter over time.

 


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  		Figure 7. Hormone, menses, body weight, running distance and calorie intake data from one monkey who became amenorrheic after 14 months of daily exercise training. During Sed, Cycle-2, and Cycle-1 this animal exhibited a normal hormonal profile, i.e. an LH and FSH surge preceded by a preovulatory estradiol peak, and then followed by a postovulatory increase in P4. AM1-AM3 marks the time period of amenorrhea, with low LH and FSH and noncyclic levels of E2 and P4. Data for actual caloric intake for this monkey during Sed are not available for some days.

 
Mean data for plasma levels of reproductive hormones of the five exercising monkeys that ovulated each cycle during Sed, Cycle-2, and Cycle-1 are illustrated in Table 2Go. For these animals, no significant differences over time (i.e. Sed, Cycle-2, Cycle-1) in follicular phase LH, FSH, or E2 were observed. Average luteal phase P4 concentrations declined significantly with time (P < 0.05; Cycle-1 vs. Sed), and peak P4 showed a nonsignificant trend toward decline. The late luteal phase rise in FSH declined progressively with training (34.7 ± 8.9 ng/ml, 15.6 ± 6.5 ng/ml, 8.9 ± 2.5 ng/ml in Sed, Cycle-2, and Cycle-1, respectively; P < 0.05).


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  		Table 2. Effects of training on plasma levels of reproductive hormones during ovulatory cycles only (n=5)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study is the first to prospectively document in a longitudinal study the progressive changes in menstrual cyclicity and reproductive hormone secretion that occur during the development of exercise-induced reproductive dysfunction resulting from a gradual training regimen. One of the most surprising findings of this study was that the transition into a state of exercise-induced amenorrhea was very abrupt, occurring within the span of a couple of menstrual cycles. The earliest significant changes in the activity of the reproductive axis in the exercising animals occurred during Cycle-2, and was a significant decrease in late luteal phase FSH levels, which are important for the recruitment of developing follicles at the beginning of each menstrual cycle (24, 25). The finding of an initial change in gonadotropin support to the reproductive axis supports the hypothesis that a suppression in the central drive to the reproductive axis leads to the other reproductive abnormalities that occur with strenuous exercise training, in that the other abnormalities that we detected did not occur until Cycle-1. In Cycle-1, the exercising monkeys showed an increase in menstrual cycle length, due entirely to an increase in follicular phase length, with no change in luteal phase length. This suggests that the process of follicular development leading to ovulation is impaired by exercise training. There was also a significant decrease in plasma LH concentrations in the early follicular phase in Cycle-1. In addition, average and peak luteal phase plasma concentrations of progesterone were reduced with exercise training during the cycle immediately preceding the development of amenorrhea (i.e. Cycle-1). Lastly, during the amenorrheic period there were decreases in all of the reproductive hormones we measured. None of the progressive changes noted in the exercise group were detected in the control group, which experienced the same experimental protocol, except they did not experience daily exercise (Fig. 6Go). Thus, the development of exercise-induced reproductive dysfunction appears to be initiated by a suppression of gonadotropin secretion, followed secondarily by both a lengthening of the time needed for development of a dominant follicle and the impairment of corpus luteum secretory function, and ending in complete cessation of menstrual cycles characterized by low circulating levels of gonadotropins and gonadal steroid hormones.

Our data are in agreement with data from previous cross-sectional studies showing that gonadotropin secretion is suppressed in exercising individuals (16, 17, 26, 27, 28). However, these cross-sectional studies were not able to discern the order in which reproductive changes occur during training. Moreover, most of the previous longitudinal studies have involved either very abrupt training where many changes in reproductive function have occurred simultaneously (29), or have involved mild training regimens that have often led to little or no impairment of reproductive function (30, 31, 32, 33, 34, 35). Our data support the findings of a longitudinal study by Boyden (34), who tested 19 moderately trained women at the beginning and end of 14–15 months of marathon training and showed there was a slight reduction in plasma LH concentrations. However, because subjects in the Boyden study were only tested twice, the occurrence of suppressed gonadotropin secretion in relation to other reproductive changes that occurred could not be assessed.

Two secondary changes observed just before the development of exercise-induced amenorrhea were a lengthening of the follicular phase of the menstrual cycle and a decrease in progesterone secretion by the corpus luteum. Changes in these reproductive functions became apparent in Cycle-1, the cycle before amenorrhea. Three of the eight monkeys in the exercise group failed to ovulate in this cycle. In two previous studies examining follicular phase ovarian function with exercise training, decreased estradiol secretion in trained women were reported (35, 36). However, we did not find a significant decrease in either mean or peak estradiol levels in exercising monkeys until the amenorrheic period. Failure to ovulate was reported by Bullen et al. (29) in women who were abruptly trained at a high level of activity for a 2-month period, with subjects showing 28% and 60% anovulation in the first and second months of the training protocol, respectively. Our finding that 38% of monkeys were anovulatory just before the development of amenorrhea is in line with the findings of these investigators. However, monkeys exercised 6–23 months before anovulation occurred, which was much later in exercise training than in the Bullen study. This was likely due to a more gradual training regimen, with exercise intensity and duration tailored to each monkey’s ability.

Of the secondary changes in reproductive function that we observed, the most widely reported in other studies has been a change in luteal function with exercise training. We observed a 34% reduction in mean progesterone levels in the luteal phase of Cycle-1, just before the development of amenorrhea. Similar reports of luteal phase inadequacy have been documented in exercising women in several cross-sectional and prospective studies (17, 28, 37, 38, 39, 40), and was recently estimated to occur with a 79% incidence in moderately exercising, regularly menstruating women (6). Presumably, a reduction in circulating levels of progesterone would have detrimental effects on the implantation and early maintenance of a conceptus, in that decreases in the amount of progesterone secreted during the luteal phase have been correlated with a failure to reproduce (41, 42). However, the level of progesterone that is sufficient for optimal functioning of the endometrium has yet to be elucidated. Therefore, the impact of exercise-induced suppression of luteal progesterone secretion on fertility remains speculative at this time.

Another relatively common finding in exercising women has been shortening of the luteal phase (37, 43, 44), a condition also associated with infertility (8, 9). In a case study following one runner for 18 months, Shangold (43) found an inverse relationship between the length of the luteal phase and the amount of training. Prior et al. (44) reported luteal phase shortening, with no changes in menstrual cycle length, in 14 women training for a marathon, and Rogol (33) found a slight decrease in luteal phase length in a prospective study with women running at their lactate threshold. Despite these findings in women, none of the monkeys in our study showed any indication of luteal phase shortening, even though they all progressed to amenorrhea during the course of training. It may be that there is an increased vulnerability of the human corpus luteum to early demise, when compared with the monkey corpus luteum. Clearly, larger studies in monkeys are needed before such a conclusion could be reached.

The final stage of reproductive dysfunction that occurred in all eight monkeys was the loss of menstrual cyclicity and amenorrhea. This was accompanied by a decrease in mean circulating levels of LH, FSH, E2, and P4. Amenorrhea has been reported in a number of cross-sectional studies of exercising women (16, 17, 28), but has generally not been found in the prospective studies that have been performed to date. This may well be a function of the type of training that has been employed in the prospective studies, which has been gradual, and in many cases less vigorous than the type of training that was used in this study (30, 31, 32, 33, 34, 35).

Interestingly, in 3 of 8 monkeys we found an increase in circulating levels of FSH during the amenorrheic period compared with the preceding menstrual cycles. A simultaneous decrease in circulating LH levels, accompanied by increased FSH concentrations has been shown to occur when the GnRH pulse generator is operating at slow frequencies (45). Thus, it is possible that the development of amenorrhea, accompanied by increased FSH levels, is due to an intermediate suppression of the central drive to the reproductive axis, where GnRH pulses are still secreted frequently enough to support FSH secretion but not LH secretion.

The timing of development of amenorrhea was quite variable in our study, ranging from 7 to 24 months of exercise training. The duration of training before amenorrhea was not significantly related to initial body weight, the change in body weight during training, or the average amount of daily food intake per kilogram body weight. This finding suggests a high degree of individual variability with respect to the susceptibility of the reproductive axis to an exercise stress. The question of what characteristics make an individual susceptible to exercise-induced reproductive dysfunction has received relatively little attention. We do know, however, that there is a strong link between energy availability and exercise-induced changes in reproductive hormone secretion. In short-term prospective experiments, Loucks et al. (46) and Williams et al. (47) have shown that reductions in LH pulsatility caused by exercise are due to an energy deficit created by an imbalance of energy intake and energy expenditure. In addition, in other cross-sectional studies Loucks et al. (17) and Laughlin et al. (28) have demonstrated the existence of chronic neuroendocrine and metabolic adaptations that resemble those occurring with chronic energy restriction. And in the longitudinal study by Bullen et al. (29), more severe exercise-induced reproductive disturbances occurred in the treatment group that lost body weight during their training. Despite these links to energy availability, in the current study we had no indication that overt indices of energy availability, such as initial body weight, change in body weight, or daily food intake accounted for susceptibility to developing rapid reproductive dysfunction. It is possible, however, that some aspect of energy balance, such as basal metabolic rate or changes in thyroid hormone levels, may underlie the variation in susceptibility to exercise-induced reproductive dysfunction (48). Further studies are needed to address this important question regarding identification of factors that convey sensitivity of the reproductive axis to suppression, or protection of the reproductive axis to impairment in other individuals.

In summary, this is the first report describing progressive changes in the development of exercise-induced amenorrhea resulting from gradual exercise training; these changes begin with a suppression of gonadotropin secretion and are followed by lengthening of the follicular phase and a decline in progesterone production by the corpus luteum, which eventually leads to amenorrhea. The monkey model that was used for this work shows many similarities to the pattern of exercise-induced reproductive dysfunction that has been surmised from cross-sectional and prospective studies in women. In contrast to human studies, however, use of this monkey model allowed performance of longitudinal studies with control over a variety of parameters that appear to be of strong significance to the induction of exercise-induced reproductive abnormalities. These include the level of food intake, the performance of other activities in addition to the planned exercise, and exposure to other life stresses. We believe that this monkey model of exercise-induced reproductive dysfunction will thus provide significant advantages in future studies aimed at elucidating the etiology of exercise-induced reproductive dysfunction, including the factors that cause increased susceptibility of some females to exercise-induced reproductive disturbances, and the long-term consequences of exercise-induced reproductive impairment on clinical outcomes such as bone integrity, lipid profiles, and cardiovascular risk factors.


   Acknowledgments
 
The authors are grateful for the assistance of the technical staff, whose dedication was necessary to train monkeys to run for many hours each day, including Karen Church, Dawn Murphy, Cindy Heilman, and Lisa Mattern. The resources and assistance of the staff of the Primate and RIA Core Laboratories of the Center for Research in Reproductive Physiology at the University of Pittsburgh were also greatly appreciated.


   Footnotes
 
1 This work was supported by grants from NIH, including HD-20789, HD-25929, and HD-08610. Back

2 Current address: The Noll Physiological Research Laboratory, Department of Kinesiology, Pennsylvania State University, University Park, Pennsylvania 16803. Back

3 Current address: Department of Obstetrics and Gynecology, Harbor-UCLA Medical Center, 1124 West Carson Street, RB-1, Torrance, California 90502. Back

4 Current address: Department of Biology, Middlebury College, Middlebury, Vermont 05753. Back

5 Current address: Department of Medicine, University of Washington, Seattle, Washington 98195. Back

Received October 26, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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