Erythropoiesis in women during 11 days at 4,300 m is not affected by menstrual cycle phase
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1 University of Colorado Health Sciences Center, Denver 80262;
2 University of Colorado at Denver, Denver, Colorado 80217;
3 Veterans Affairs Medical Center, Palo Alto, California 94304
4 Saint Louis University School of Medicine, St. Louis, Missouri 63110;
5 United States Army Research Institute of Environmental Medicine, Natick, Massachusetts 01760
ABSTRACT
Because the ovarian steroid hormones, progesterone and estrogen, have higher blood levels in the luteal (L) than in the follicular (F) phase of the menstrual cycle, and because of their known effects on ventilation and hematopoiesis, we hypothesized that less hypoxemia and less erythropoiesis would occur in the L than the F phase of the cycle after arrival at altitude. We examined erythropoiesis with menstrual cycle phase in 16 women (age 22.6 ± 0.6 yr). At sea level, 11 of 16 women were studied during both menstrual cycle phases, and, where comparison within women was available, cycle phase did not alter erythropoietin (n = 5), reticulocyte count (n = 10), and red cell volume (n = 9). When all 16 women were taken for 11 days to 4,300-m altitude (barometric pressure = 462 mmHg), paired comparisons within women showed no differences in ovarian hormone concentrations at sea level vs. altitude on menstrual cycle day 3 or 10 for either the F (n = 11) or the L (n = 5) phase groups. Arterial oxygen saturation did not differ between the F and L groups at altitude. There were no differences by cycle phase on day 11 at 4,300 m for erythropoietin [22.9 ± 4.7 (L) vs. 18.8 ± 3.4 mU/ml (F)], percent reticulocytes [1.9 ± 0.1 (L) vs. 2.1 ± 0.3% (F)], hemoglobin [13.5 ± 0.3 (L) vs. 13.7 ± 0.3 g/100 ml (F)], percent hematocrit [40.6 ± 1.4 (L) vs. 40.7 ± 1.0% (F)], red cell volume [31.1 ± 3.6 (L) vs. 33.0 ± 1.6 ml/kg (F)], and blood ferritin [8.9 ± 1.7 (L) vs. 10.2 ± 0.9 μg/l (F)]. Blood level of erythropoietin was related (r = 0.77) to arterial oxygen saturation but not to the levels of progesterone or estradiol. We conclude that erythropoiesis was not altered by menstrual cycle phase during the first days at 4,300-m altitude.
keywords:erythropoietin; ferritin; reticulocytes; red cell volume
INTRODUCTION
RED BLOOD CELL FORMATION, or erythropoiesis, increases in both men and women at high altitude and is considered to be an important component of altitude acclimatization (13, 16, 18, 21, 22, 32, 33). In men, after 10 days at 4,300 m compared with sea level, measurements of red cell volume, although tending to increase, are quite variable, but there is a clear doubling of the reticulocyte count and the blood level of erythropoietin (EPO) (16). Although the hematopoietic response to altitude has been extensively studied in men, there is relatively little information in women and none with regard to menstrual cycle phase, despite the known effects on erythropoiesis of ovarian steroid hormones. Andean women residing at high altitude have higher arterial oxygen saturation (SaO2) and lower hemoglobin concentration before than after menopause, which suggests hormonal influences on breathing and erythropoiesis (21, 22). Administration of estrogen and progesterone to rats at high altitude decreases polycythemia and EPO, suggesting control by ovarian steroid hormones of erythropoiesis (12, 28). Other studies in laboratory animals and cell systems support the concept that ovarian hormones modulate the erythropoietic response to hypoxia (14, 15, 29, 31, 35). Whereas progesterone, through its stimulation of ventilation, acts to decrease EPO production, estrogen decreases EPO effects (3, 8, 27, 30). Progesterone is a powerful stimulant to human breathing (34), and, in menstruating women living at altitude, progesterone is much higher in the luteal than the follicular phase of the cycle (11), as occurs at sea level. And, in women who are long-term residents at high altitude, estrogen has been reported to be higher in the luteal than in the follicular phase of the menstrual cycle (11). We expected, therefore, that women taken to altitude in the luteal compared with the follicular phase would breathe more (23) and would have higher SaO2, lower blood levels of EPO, lower reticulocyte counts, smaller red cell volumes, and, possibly, lower levels of hemoglobin and hematocrit, compared with women in the follicular phase. We conducted the present study because we are not aware that the effect of cycle phase on EPO or on erythropoiesis has been examined in women during their first days at high altitude.
METHODS
Sixteen healthy nonsmoking, eumenorrheic women (age 22.2 ± 0.6 yr, height 1.67 ± 1.0 m, weight 63.6 ± 1.9 kg, and body mass index 22.8+0.7 kg/m2) who were sea-level residents without altitude exposure in the previous year volunteered to participate in this study, as previously described (5, 6, 25). Peak oxygen uptake was 43 ± 2 ml · kg1 · min1 with a range of 33-53 ml · kg1 · min1. Informed consent as approved by the participating institutions was obtained (5, 6, 25). At sea level (barometric pressure = 752 mmHg), subjects were studied at the Aging Studies Unit of the Palo Alto, CA, Veterans Affairs Health Care System. Subsequently, the same subjects were studied at high altitude on the summit of Pikes Peak, CO (4,300 m; barometric pressure = 462 mmHg) at the US Army Maher Pikes Peak Laboratory Facility. Both at sea level and at altitude, energy intake and the level of physical activity were regulated to prevent altitude-related weight loss (5-7) and detraining. Because the initial blood screening found in some women ferritin levels that were less than the adult normal lower level of 12.0 μg/l (24), and because of the known propensity for iron stores to be depleted at altitude (32), oral supplemental iron was begun at the onset of sea-level studies and continued throughout the altitude phase. Normal ferritin levels were present during the sea-level measurements reported here, but, as noted below, ferritin levels fell during the altitude stay.
At sea level, 11 of the 16 women were studied in the follicular as well as the luteal phase of the menstrual cycle. On the basis of 3 mo of documentation by diary or history, a subject began daily testing for her luteinizing hormone (LH) surge by using an ovulation predictor kit (OvuQuick, Becton-Dickinson, Rutherford, NJ) at least 4 days before the estimated time of the surge. Subjects kept a menstrual cycle diary that noted the date and duration of menses, the date of the hormone surge, and the duration of menstrual cycles. At sea level and at high altitude, day 1 of a study cycle was the day after menses began for the follicular phase or the day after a LH surge for the luteal phase. Blood levels of ovarian steroid hormones were measured on days 3, 10, and 12 at sea level and on days 3, 6, 9, 10, and 11 at high altitude. Women were considered to be in the follicular phase when, in conjunction with cycle data, measured hormone concentrations showed estradiol was >20 pg/ml but progesterone was <2.5 ng/ml. The luteal phase was present when, in conjunction with cycle data, progesterone levels were >2.5 ng/ml. Cycles were considered abnormal where levels of progesterone failed to increase after a documented LH surge detected by ovulation predictor kits. Women whose cycles were abnormal were classified as being in the follicular phase for the purpose of analyses because they had low estradiol and progesterone concentrations, as are characteristic of the early follicular phase. Estradiol and progesterone were measured in duplicate using a chemoluminescence enzyme immunoassay (Immunolite kits, Diagnostic Products, Los Angeles, CA).
Altitude exposure followed completion of the sea-level studies by at least 1 mo. On day 1 of a preassigned cycle phase, subjects were flown to Colorado Springs and then were taken to the summit of Pikes Peak, an automobile trip of ~90 min. Because time constraints prevented measurements being made during altitude acclimatization during both phases of the menstrual cycle in each woman, subjects were initially assigned to arrive on Pikes Peak at the beginning of either their follicular (n = 8) or luteal (n = 8) phase. However, two of the women assigned to the luteal phase did not have an LH surge at sea level, but they came for study at the expected date to altitude where the absence of menses and low progesterone levels indicated they were in the follicular phase. A third woman had an LH surge late (day 21) in her cycle while at sea level but began to menstruate soon after her arrival on Pikes Peak. Her low blood progesterone levels at all measurement times at 4,300 m indicated she was in the follicular phase of the cycle. Thus, for the 11-day altitude exposure, hormonal data in the follicular phase were analyzed for 11 women in whom paired comparison of altitude with sea level could be made. Hormonal data in the luteal phase were available for five women both at sea level and at altitude.
Blood for analysis of ovarian steroid hormones was obtained by venapuncture, allowed to clot for 30 min, and then centrifuged at 3,000 rpm for 10 min. The serum was immediately frozen. Within 1 mo of the completion of the altitude studies, serum aliquots were assayed for progesterone and estradiol (Diagnostic Products Coat-A-Count radioimunnoassay) by the General Clinical Research Center Laboratory at the University of Colorado Health Sciences Center.
At sea level, resting ventilation was measured before breakfast and more than 2 h after a meal on days 1 or 2 and 7 or 8 of the 12-day sea-level test period. Measurements in a cycle phase were averaged for presentation. At high altitude, resting ventilation was measured at least 2 h after a meal the afternoon of day 1 (2-3 h after arrival) and in the mornings of days 2, 3, 5, 7, and 12. Seated, resting subjects breathed through a low-resistance respiratory valve and breathing circuit connected to a computer-controlled, breath-by-breath metabolic measurement system (Vmax229, SensorMedics, Yorba Linda, CA) for ~20 min. End-tidal PCO2 measured breath-by-breath and averaged over the last 8-10 min of the test is reported in RESULTS. SaO2, as measured simultaneously by finger pulse oximetry (Nellcor N-200), is also reported.
The measurement of red cell volume by the carbon monoxide method has previously been described (36, 37). Briefly, a subject was allowed to rest semirecumbent for at least 10 min and then to rebreathe into a system that was ~5 liters in volume and that contained 100% oxygen and a carbon dioxide absorber. After the subject rebreathed for 5 min, a 3-ml blood sample was drawn from the venous cannula in an arm vein to provide the baseline value, after which 50 ml at sea level and 80 ml at 4,300 m (under conditions of ambient temperature and pressure) of 100% carbon monoxide were injected into the rebreathing system. With the subject continuing to rebreathe, blood samples were drawn at 5, 10, and 15 min. Measurement in duplicate of hematocrit (microhematocrit technique) and hemoglobin concentration (model OSM3, Radiometer, Copenhagen, Denmark) was performed for each blood sample, and the remainder of the sample was stored at 4°C for measurement in triplicate of carboxyhemoglobin concentration by gas chromatography as previously described (10). Total blood volume was calculated as previously described (36, 37). Red cell volume was the product of blood volume times the mean hematocrit of the 0-, 5-, 10-, and 15-min samples.
Reticulocyte (erythrocytes containing at least 2 blue granules) counts were obtained from smears of venous blood stained with 0.5% methylene blue plus 1.6% potassium oxalate and were examined by oil-immersion light microscopy at a magnification of ×1,000. Measurements were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and 10 or 11 at 4,300 m. Duplicate counts (±20% from each of 2 observers) of ~1,000 erythrocytes were averaged and reticulocytes were expressed as a percentage of erythrocytes.
For EPO, because of potential diurnal variation (20), venous blood samples for analysis were drawn either midmorning or midafternoon. Measurements were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and 10 or 11 at 4,300 m. After centrifugation, serum samples were frozen until analysis by radioimunnoassay (Diagnostic Systems Laboratories, Webster, TX). For 45 samples analyzed in duplicate, the difference averaged 1.8 ± 1.0 mU/ml. For duplicate analyses, the mean is reported.
Blood ferritin was measured in the Aging Studies Unit of the Palo Alto Veterans Affairs Medical Center by chemoluminescence (Bayer ACS 180). Blood ferritin concentration was monitored periodically to confirm that iron supplementation was successful in raising blood ferritin to values within the normal range of >12 μg/l (24). In addition, blood samples for ferritin were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and either 10 or 11 at 4,300 m.
As noted in the tables, some measurements were not obtained on specified days in the event of loss of sample or equipment malfunction. Data are expressed as means ± SE. Statistical analysis utilized a one-way analysis of variance for the changes in the same subjects over time. Unpaired t-tests were employed to compare values for subjects in the follicular vs. those in the luteal phase of the menstrual cycle. Linear least squares equations were used to describe relationships of one variable on another. The null hypothesis was rejected when P < 0.05.
RESULTS
Measurements by Cycle Phase at Sea Level
Before examining whether erythropoiesis varied with cycle phase at altitude, we examined by paired analysis whether differences existed within women between cycle phases at sea level. The comparison was made on day 11 or 12 of the cycle phase, because on these days we had measurements of female steroid hormones and measurements relating to erythropoiesis (Table 1). In the 11 of the 16 subjects whose ovarian hormone levels were measured in both cycle phases, progesterone levels were higher in the luteal than the follicular phase, but estradiol levels were not different between the two phases. Of variables relating to erythropoiesis and available for paired analysis on these days, hematocrit values (n = 10) were lower in the luteal than the follicular phase, but there were no significant cycle phase-related differences in EPO (n = 5), reticulocyte count (n = 10), blood ferritin levels (n = 9), hemoglobin concentration (n = 10), or red cell volume (n = 9) (Table 1).
Measurements by Cycle Phase at 4,300 m
Progesterone and estradiol. In the comparison of altitude with sea level, on the basis of hormone levels and menstrual diaries, 5 of the 16 subjects had measurements made in the luteal phase both at sea level and at 4,300 m. For paired luteal-phase measurements, there was no difference in hormone levels when altitude values were compared with those at sea level either on day 3 or day 10 or 11 (Table 2). Paired measurements of ovarian hormone levels were obtained in the other 11 subjects in the follicular phase both at sea level and at 4,300 m (Table 2). For paired follicular-phase measurements, there was no difference in hormone levels when altitude values were compared with those at sea level either on day 3 or day 10 or 11 (Table 2).
In the comparison of hormone measurements between the groups for the 5 women in the luteal phase vs. the 11 women in the follicular phase of the menstrual cycle, progesterone blood levels were higher both at sea level and at altitude in the luteal-phase group than in the follicular-phase group (Fig. 1, A and B). Women in the luteal phase had higher estradiol levels than those in the follicular phase on day 3 of the cycle at sea level (Fig. 1C). At 4,300 m, although estradiol values tended to be higher early in the luteal than the follicular phase of the cycle, the differences were not statistically significant (Fig. 1D).
Ventilation and SaO2. End-tidal PCO2 fell progressively from sea-level values over the 12 days of observation at 4,300 m. SaO2 fell on arrival at 4,300 m and then increased as ventilatory acclimatization progressed. There was no difference at sea level or at 4,300 m between the 5 women in the luteal phase vs. the 11 women in the follicular phase with regard to the end-tidal PCO2 (Fig. 2A) or SaO2 (Fig. 2B).
Measurements of erythropoiesis. At 4,300 m, for the 5 women in the luteal phase and the 11 women in the follicular phase, levels of EPO were increased above the sea-level values, but there was no difference between the groups (Fig. 3A). Reticulocyte counts (Fig. 3B) were higher than at sea level after 6 and 11 days at 4,300 m, but here also there was no difference between the groups in the luteal vs. the follicular phase. Hemoglobin (Fig. 3C) was higher and ferritin (Fig. 3D) was lower than sea-level values, but there were no cycle phase-related differences for these variables. Red cell volumes increased from 28.0 ± 2.6 ml/kg at sea level to 31.1 ± 3.6 ml/kg after 11 days at 4,300 m in the luteal-phase group and from 30.6 ± 1.0 to 33.0 ± 1.6 ml/kg in the follicular-phase group, but neither the red cell volume nor the increase at altitude differed between the groups.
Because, in women given supplementary progestational agents, ventilation increased during the first hour of altitude exposure (23), and because, in experimental animals (12), administration of progesterone and estrogen inhibited erythropoiesis, we examined the relationship of hormone levels to erythropoiesis in our subjects. We found no relationship at high altitude between red cell volume on day 11 or erythrocyte count on days 6 and 11 with estradiol [P = not significant (NS)] or progesterone (P = NS) levels. We also found no relationship between EPO and either estradiol (P = NS) or progesterone (P = NS). However, in all subjects within our cohort, EPO levels related well (r = 0.77, R2 = 0.6, P < 0.01, n = 63) to SaO2, without regard to the duration of stay at altitude. This suggested that 60% of the variation in EPO could be accounted for by variation in SaO2. To better demonstrate possible hormonal effects, we compared the relationship between EPO and SaO2 for arbitrarily chosen extremes of hormonal levels at altitude. In these analyses, the relationship between EPO and SaO2 remained unaffected by levels of progesterone (Fig. 4A) or estrogen (Fig. 4B)
All Subjects Regardless of Cycle Phase
Because of the absence of cycle phase effects on erythropoiesis, and because of the dearth of information in women going to altitude, we report the values for the entire cohort at sea level and at altitude (Table 3). EPO levels were higher than sea level on day 3 and subsequently fell toward, but not to, sea-level values. Reticulocyte counts had not increased on day 3 at 4,300 m but rose by days 6 and 11. Red cell volume was not increased on day 3 but was increased on day 11. Hemoglobin and hematocrit values increased at altitude. Blood ferritin level fell with increasing reticulocytosis (r = 0.36, P < 0.01, n = 52).
DISCUSSION
The main finding of the present study in women was that menstrual cycle phase did not affect measurements relating to erythropoiesis, either at sea level or at 4,300 m. Menstrual cycle phase during experimental measurements was confirmed by history and by blood levels of progesterone and estradiol. Our findings of no cycle-related change in EPO confirmed those of Cotes and Canning (9) in six healthy women at sea level. In the women of their study, blood hemoglobin was slightly lower in the luteal than the follicular phase, similar to our findings for hematocrit, which may reflect expansion of plasma volume in the luteal phase. Our findings of no cycle phase-related differences in reticulocyte count and red cell volume further supported the concept of little or no change in erythropoiesis during the menstrual cycle at sea level.
At high altitude our hypothesis of diminished erythropoiesis in the luteal phase of the menstrual cycle was not substantiated. Several potential factors could have complicated this interpretation. Perhaps most important was the small number of subjects in the luteal phase of the menstrual cycle at altitude. Although we had hoped to have equal numbers in the cycle phases, we ended up with groups containing 11 in the follicular, and only 5 in the luteal, phase. Fortunately, at altitude, the phase that was present was associated with hormonal values that were within the normal sea-level range for the given phase and were similar to values obtained in the same subjects at sea level. We are not aware of previous studies that have reported sequential hormonal changes in women for the first days after arrival at altitude.
Also potentially complicating the interpretation of no difference in erythropoiesis with menstrual cycle phase was the fall in ferritin to levels that were low enough (<10 μg/l) to affect EPO and red cell formation (26). A decrease in ferritin occurred in conjunction with new red cell formation at altitude, suggesting that, despite oral iron supplementation, augmented erythropoiesis drew down the body's iron stores. In the study by Richalet et al. (32), in volunteers going above 3,600 m for 39 days (the final 21 days being at 6,542 m), ferritin levels decreased from 17.8 to 6.0 μg/l in the four women studied. In two of the four women, ferritin levels fell to 2 μg/l, but blood hemoglobin levels at altitude did not increase, suggesting that iron deficiency may have impaired erythropoiesis. We cannot exclude the possibility that iron deficiency inhibited erythropoiesis in our subjects. However, in findings against this possibility, our subject with the lowest serum ferritin (5.4 μg/l) doubled her reticulocyte count (from 0.9 to 1.9%) at 4,300 m and increased her total red cell volume by 15%, compatible with an adequate hematopoietic response. In any event, the possible influence of iron deficiency appeared to be the same in our two groups, because the levels of ferritin, reticulocytes, hemoglobin, hematocrit, and red cell volume did not differ between the follicular vs. the luteal phase at 4,300 m. Taken together, our findings supported the concept that erythropoiesis was not affected by menstrual cycle phase during the days at altitude.
Another potential difficulty in interpreting erythropoiesis in our study lay in the variable hypoxic stimulus present during the acclimatization process. SaO2 changes during altitude acclimatization. We expected that high levels of progesterone in the luteal phase would raise effective ventilation and diminish hypoxemia compared with the follicular phase, but this did not occur. Although SaO2 fell from 98% at sea level to ~80% on arrival at 4,300 m and then rose to 88% after 11 days residence, there was no cycle phase-related difference in these temporal changes between the groups.
The effect of menstrual cycle phase on ventilation during short-term (<12 h) altitude exposure has been studied at rest and during exercise. Loeppky et al. (23) studied 19 women in both phases of the menstrual cycle at low altitude (barometric pressure = 636 mmHg), and after 1 and 12 h at simulated high altitude (barometric pressure = 426 mmHg). When women were in the luteal phase, their alveolar and arterial PCO2 values at low altitude and after 1 h at high altitude were lower than in the follicular phase, but there were no statistically significant cycle phase-related differences in PCO2 after 12 h at high altitude. Furthermore, arterial PO2 values at either 1 or 12 h at altitude were not significantly different between the follicular and the luteal phases of the cycle. Beidleman et al. (2), in an exercise study of eight women during 3 h of altitude chamber exposure to a barometric pressure of 464 mmHg, found no difference with cycle phase in PCO2 or ventilation at submaximal and peak exercise. In both studies, the cycle phase comparison at low and high altitude was conducted within and not among subjects. Yet, even with these optimal experimental designs, less hypoxemia in the luteal, compared with the follicular, phase could not be shown after 12 h of resting high-altitude exposure and after 3 h of exposure to high altitude during exercise. Perhaps in these (2, 23) and the present study, the large changes in acid-base balance and in arterial oxygenation during ventilatory acclimatization to altitude may have acted, at least in part, to obscure any effect of progesterone on ventilation. If so, a longer altitude exposure might show greater ventilation and less hypoxemia in the luteal than the follicular phase, as has been found in women at high altitude who resided permanently at 4,300 m (22). However, for our study during the first days at 4,300 m, because the altitude-related hypoxic stimulus did not differ between the groups and because altitude exposure per se did not cause abnormal hormone levels, perhaps it is not surprising that erythropoiesis was not different between the luteal- and follicular-phase groups.
Also to be considered in interpreting the present results was the variation in EPO levels within individuals. Diurnal variation has been reported for EPO levels both at sea level and at altitude (20). We attempted to minimize the effect of diurnal variation by drawing blood samples at midmorning or midafternoon when variation is least (20). Furthermore, the diurnal variation is small compared with the effect of acclimatization-related variations (1, 20). The large acclimatization effect at 4,300 m on EPO levels was shown in five men by bioassay, and in nine men by ELISA (Fig. 5). Values were reported to rise to a peak ~1-2 days after arrival and then to return toward, but not to, sea-level values (1, 20), a pattern also found (by radioimmunoassay) at lesser altitudes (17, 18). At 4,300-m altitude, EPO levels in women followed a generally similar time course to that reported for men (Fig. 5). The changing EPO levels at altitude reflect, in part, the greater hypoxemic stimulus on arrival and the partial relief of that stimulus with ventilatory acclimatization (20). In any event, EPO shows remarkable change during the first few hours and days at high altitude.
To our knowledge, the only prior report showing EPO levels for women at both sea level and high altitude is that of Richalet et al. (32). Levels in women in that study showed a rather good relationship to SaO2, even without the taking of account of the duration of altitude stay (Fig. 6). When data in women from the present study are added to those in women obtained by Richalet et al., they lie along the line of best fit for the data of Richalet et al. (Fig. 6). The pronounced rise in EPO levels at lower SaO2 in the study of Richalet et al. might have been due in part to iron deficiency in some of their subjects. When ferritin concentration falls below 10-20 μg/l, EPO levels may rise (I. Asmus, unpublished observations). Although EPO depends on many factors (4, 26), hypoxia remains the primary stimulus. The results in our cohort suggest that, for the first 11 days at 4,300 m, ~60% of the variation in EPO levels reflects variation in SaO2 and that the remaining variation apparently had little to do with hormonal fluctuations during the menstrual cycle.
From the above findings, one is impressed by the number and magnitude of confounding variables that could impact hematopoiesis in healthy young women during the first days at altitude. Among these are the 1) changing levels of progesterone (and probably estrogen) during the normal menstrual cycle, 2) changing SaO2 and acid-base balance with acclimatization, 3) fluctuating iron stores as erythropoiesis proceeds, 4) diurnal variation in EPO, and 5) changes in EPO with acclimatization itself. Given all these factors, perhaps it is not surprising that, in a small cohort of women, we could not identify differences in erythropoiesis between the luteal and the follicular phases during an 11-day study at high altitude. During this interval, we did not find even the expected relative hyperventilation in the luteal, as opposed to the follicular, phase of the cycle. It was as though the known effects of progesterone on ventilation were small compared with the effects of acclimatization itself. However, published reports suggest that, once acclimatization is complete, as in altitude residents, the effects of the ovarian hormones become apparent. Women who are of child-bearing age have greater ventilation, lower hemoglobin, and less erythropoiesis than do older women who are postmenopausal (21, 22). Whatever is the impact of confounding variables on erythropoiesis in young women during the first 11 days at high altitude, such impact is likely to be temporary. Further research is needed to establish the role of menstrual cycle phase on breathing and hematopoiesis in women residing for longer periods at high altitude.
ACKNOWLEDGEMENTS
We thank the test volunteers for their participation and cooperation in this study. Also, the nurses, staff, and faculty at the Aging Studies Unit of the Palo Alto Veterans Affairs Medical Center provided essential assistance, without which this study could not have been performed.
FOOTNOTES
Deceased 28 December 1999.
This research was supported in part by US Army Contract DAMD-17-95C-5110; the General Clinical Research Center of the University of Colorado Health Sciences Center (National Institutes of Health Division of Research Resources 5-01 RR-00051); and National Heart, Lung, and Blood Institute Grant HL-14985 and Specialized Center of Research Grant HL-46481.
The views, opinions, and/or findings in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other official documentation. Citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations.
Address for reprint requests and other correspondence: J. T. Reeves MDB-131, Univ. of Colorado Health Sciences Center, Denver, CO 80262 (E-mail: john.reeves@uchsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 March 2001; accepted in final form 8 August 2001.
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2 University of Colorado at Denver, Denver, Colorado 80217;
3 Veterans Affairs Medical Center, Palo Alto, California 94304
4 Saint Louis University School of Medicine, St. Louis, Missouri 63110;
5 United States Army Research Institute of Environmental Medicine, Natick, Massachusetts 01760
ABSTRACT
Because the ovarian steroid hormones, progesterone and estrogen, have higher blood levels in the luteal (L) than in the follicular (F) phase of the menstrual cycle, and because of their known effects on ventilation and hematopoiesis, we hypothesized that less hypoxemia and less erythropoiesis would occur in the L than the F phase of the cycle after arrival at altitude. We examined erythropoiesis with menstrual cycle phase in 16 women (age 22.6 ± 0.6 yr). At sea level, 11 of 16 women were studied during both menstrual cycle phases, and, where comparison within women was available, cycle phase did not alter erythropoietin (n = 5), reticulocyte count (n = 10), and red cell volume (n = 9). When all 16 women were taken for 11 days to 4,300-m altitude (barometric pressure = 462 mmHg), paired comparisons within women showed no differences in ovarian hormone concentrations at sea level vs. altitude on menstrual cycle day 3 or 10 for either the F (n = 11) or the L (n = 5) phase groups. Arterial oxygen saturation did not differ between the F and L groups at altitude. There were no differences by cycle phase on day 11 at 4,300 m for erythropoietin [22.9 ± 4.7 (L) vs. 18.8 ± 3.4 mU/ml (F)], percent reticulocytes [1.9 ± 0.1 (L) vs. 2.1 ± 0.3% (F)], hemoglobin [13.5 ± 0.3 (L) vs. 13.7 ± 0.3 g/100 ml (F)], percent hematocrit [40.6 ± 1.4 (L) vs. 40.7 ± 1.0% (F)], red cell volume [31.1 ± 3.6 (L) vs. 33.0 ± 1.6 ml/kg (F)], and blood ferritin [8.9 ± 1.7 (L) vs. 10.2 ± 0.9 μg/l (F)]. Blood level of erythropoietin was related (r = 0.77) to arterial oxygen saturation but not to the levels of progesterone or estradiol. We conclude that erythropoiesis was not altered by menstrual cycle phase during the first days at 4,300-m altitude.
keywords:erythropoietin; ferritin; reticulocytes; red cell volume
INTRODUCTION
RED BLOOD CELL FORMATION, or erythropoiesis, increases in both men and women at high altitude and is considered to be an important component of altitude acclimatization (13, 16, 18, 21, 22, 32, 33). In men, after 10 days at 4,300 m compared with sea level, measurements of red cell volume, although tending to increase, are quite variable, but there is a clear doubling of the reticulocyte count and the blood level of erythropoietin (EPO) (16). Although the hematopoietic response to altitude has been extensively studied in men, there is relatively little information in women and none with regard to menstrual cycle phase, despite the known effects on erythropoiesis of ovarian steroid hormones. Andean women residing at high altitude have higher arterial oxygen saturation (SaO2) and lower hemoglobin concentration before than after menopause, which suggests hormonal influences on breathing and erythropoiesis (21, 22). Administration of estrogen and progesterone to rats at high altitude decreases polycythemia and EPO, suggesting control by ovarian steroid hormones of erythropoiesis (12, 28). Other studies in laboratory animals and cell systems support the concept that ovarian hormones modulate the erythropoietic response to hypoxia (14, 15, 29, 31, 35). Whereas progesterone, through its stimulation of ventilation, acts to decrease EPO production, estrogen decreases EPO effects (3, 8, 27, 30). Progesterone is a powerful stimulant to human breathing (34), and, in menstruating women living at altitude, progesterone is much higher in the luteal than the follicular phase of the cycle (11), as occurs at sea level. And, in women who are long-term residents at high altitude, estrogen has been reported to be higher in the luteal than in the follicular phase of the menstrual cycle (11). We expected, therefore, that women taken to altitude in the luteal compared with the follicular phase would breathe more (23) and would have higher SaO2, lower blood levels of EPO, lower reticulocyte counts, smaller red cell volumes, and, possibly, lower levels of hemoglobin and hematocrit, compared with women in the follicular phase. We conducted the present study because we are not aware that the effect of cycle phase on EPO or on erythropoiesis has been examined in women during their first days at high altitude.
METHODS
Sixteen healthy nonsmoking, eumenorrheic women (age 22.2 ± 0.6 yr, height 1.67 ± 1.0 m, weight 63.6 ± 1.9 kg, and body mass index 22.8+0.7 kg/m2) who were sea-level residents without altitude exposure in the previous year volunteered to participate in this study, as previously described (5, 6, 25). Peak oxygen uptake was 43 ± 2 ml · kg1 · min1 with a range of 33-53 ml · kg1 · min1. Informed consent as approved by the participating institutions was obtained (5, 6, 25). At sea level (barometric pressure = 752 mmHg), subjects were studied at the Aging Studies Unit of the Palo Alto, CA, Veterans Affairs Health Care System. Subsequently, the same subjects were studied at high altitude on the summit of Pikes Peak, CO (4,300 m; barometric pressure = 462 mmHg) at the US Army Maher Pikes Peak Laboratory Facility. Both at sea level and at altitude, energy intake and the level of physical activity were regulated to prevent altitude-related weight loss (5-7) and detraining. Because the initial blood screening found in some women ferritin levels that were less than the adult normal lower level of 12.0 μg/l (24), and because of the known propensity for iron stores to be depleted at altitude (32), oral supplemental iron was begun at the onset of sea-level studies and continued throughout the altitude phase. Normal ferritin levels were present during the sea-level measurements reported here, but, as noted below, ferritin levels fell during the altitude stay.
At sea level, 11 of the 16 women were studied in the follicular as well as the luteal phase of the menstrual cycle. On the basis of 3 mo of documentation by diary or history, a subject began daily testing for her luteinizing hormone (LH) surge by using an ovulation predictor kit (OvuQuick, Becton-Dickinson, Rutherford, NJ) at least 4 days before the estimated time of the surge. Subjects kept a menstrual cycle diary that noted the date and duration of menses, the date of the hormone surge, and the duration of menstrual cycles. At sea level and at high altitude, day 1 of a study cycle was the day after menses began for the follicular phase or the day after a LH surge for the luteal phase. Blood levels of ovarian steroid hormones were measured on days 3, 10, and 12 at sea level and on days 3, 6, 9, 10, and 11 at high altitude. Women were considered to be in the follicular phase when, in conjunction with cycle data, measured hormone concentrations showed estradiol was >20 pg/ml but progesterone was <2.5 ng/ml. The luteal phase was present when, in conjunction with cycle data, progesterone levels were >2.5 ng/ml. Cycles were considered abnormal where levels of progesterone failed to increase after a documented LH surge detected by ovulation predictor kits. Women whose cycles were abnormal were classified as being in the follicular phase for the purpose of analyses because they had low estradiol and progesterone concentrations, as are characteristic of the early follicular phase. Estradiol and progesterone were measured in duplicate using a chemoluminescence enzyme immunoassay (Immunolite kits, Diagnostic Products, Los Angeles, CA).
Altitude exposure followed completion of the sea-level studies by at least 1 mo. On day 1 of a preassigned cycle phase, subjects were flown to Colorado Springs and then were taken to the summit of Pikes Peak, an automobile trip of ~90 min. Because time constraints prevented measurements being made during altitude acclimatization during both phases of the menstrual cycle in each woman, subjects were initially assigned to arrive on Pikes Peak at the beginning of either their follicular (n = 8) or luteal (n = 8) phase. However, two of the women assigned to the luteal phase did not have an LH surge at sea level, but they came for study at the expected date to altitude where the absence of menses and low progesterone levels indicated they were in the follicular phase. A third woman had an LH surge late (day 21) in her cycle while at sea level but began to menstruate soon after her arrival on Pikes Peak. Her low blood progesterone levels at all measurement times at 4,300 m indicated she was in the follicular phase of the cycle. Thus, for the 11-day altitude exposure, hormonal data in the follicular phase were analyzed for 11 women in whom paired comparison of altitude with sea level could be made. Hormonal data in the luteal phase were available for five women both at sea level and at altitude.
Blood for analysis of ovarian steroid hormones was obtained by venapuncture, allowed to clot for 30 min, and then centrifuged at 3,000 rpm for 10 min. The serum was immediately frozen. Within 1 mo of the completion of the altitude studies, serum aliquots were assayed for progesterone and estradiol (Diagnostic Products Coat-A-Count radioimunnoassay) by the General Clinical Research Center Laboratory at the University of Colorado Health Sciences Center.
At sea level, resting ventilation was measured before breakfast and more than 2 h after a meal on days 1 or 2 and 7 or 8 of the 12-day sea-level test period. Measurements in a cycle phase were averaged for presentation. At high altitude, resting ventilation was measured at least 2 h after a meal the afternoon of day 1 (2-3 h after arrival) and in the mornings of days 2, 3, 5, 7, and 12. Seated, resting subjects breathed through a low-resistance respiratory valve and breathing circuit connected to a computer-controlled, breath-by-breath metabolic measurement system (Vmax229, SensorMedics, Yorba Linda, CA) for ~20 min. End-tidal PCO2 measured breath-by-breath and averaged over the last 8-10 min of the test is reported in RESULTS. SaO2, as measured simultaneously by finger pulse oximetry (Nellcor N-200), is also reported.
The measurement of red cell volume by the carbon monoxide method has previously been described (36, 37). Briefly, a subject was allowed to rest semirecumbent for at least 10 min and then to rebreathe into a system that was ~5 liters in volume and that contained 100% oxygen and a carbon dioxide absorber. After the subject rebreathed for 5 min, a 3-ml blood sample was drawn from the venous cannula in an arm vein to provide the baseline value, after which 50 ml at sea level and 80 ml at 4,300 m (under conditions of ambient temperature and pressure) of 100% carbon monoxide were injected into the rebreathing system. With the subject continuing to rebreathe, blood samples were drawn at 5, 10, and 15 min. Measurement in duplicate of hematocrit (microhematocrit technique) and hemoglobin concentration (model OSM3, Radiometer, Copenhagen, Denmark) was performed for each blood sample, and the remainder of the sample was stored at 4°C for measurement in triplicate of carboxyhemoglobin concentration by gas chromatography as previously described (10). Total blood volume was calculated as previously described (36, 37). Red cell volume was the product of blood volume times the mean hematocrit of the 0-, 5-, 10-, and 15-min samples.
Reticulocyte (erythrocytes containing at least 2 blue granules) counts were obtained from smears of venous blood stained with 0.5% methylene blue plus 1.6% potassium oxalate and were examined by oil-immersion light microscopy at a magnification of ×1,000. Measurements were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and 10 or 11 at 4,300 m. Duplicate counts (±20% from each of 2 observers) of ~1,000 erythrocytes were averaged and reticulocytes were expressed as a percentage of erythrocytes.
For EPO, because of potential diurnal variation (20), venous blood samples for analysis were drawn either midmorning or midafternoon. Measurements were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and 10 or 11 at 4,300 m. After centrifugation, serum samples were frozen until analysis by radioimunnoassay (Diagnostic Systems Laboratories, Webster, TX). For 45 samples analyzed in duplicate, the difference averaged 1.8 ± 1.0 mU/ml. For duplicate analyses, the mean is reported.
Blood ferritin was measured in the Aging Studies Unit of the Palo Alto Veterans Affairs Medical Center by chemoluminescence (Bayer ACS 180). Blood ferritin concentration was monitored periodically to confirm that iron supplementation was successful in raising blood ferritin to values within the normal range of >12 μg/l (24). In addition, blood samples for ferritin were obtained on cycle day 11 or 12 at sea level and on days 3, 6, and either 10 or 11 at 4,300 m.
As noted in the tables, some measurements were not obtained on specified days in the event of loss of sample or equipment malfunction. Data are expressed as means ± SE. Statistical analysis utilized a one-way analysis of variance for the changes in the same subjects over time. Unpaired t-tests were employed to compare values for subjects in the follicular vs. those in the luteal phase of the menstrual cycle. Linear least squares equations were used to describe relationships of one variable on another. The null hypothesis was rejected when P < 0.05.
RESULTS
Measurements by Cycle Phase at Sea Level
Before examining whether erythropoiesis varied with cycle phase at altitude, we examined by paired analysis whether differences existed within women between cycle phases at sea level. The comparison was made on day 11 or 12 of the cycle phase, because on these days we had measurements of female steroid hormones and measurements relating to erythropoiesis (Table 1). In the 11 of the 16 subjects whose ovarian hormone levels were measured in both cycle phases, progesterone levels were higher in the luteal than the follicular phase, but estradiol levels were not different between the two phases. Of variables relating to erythropoiesis and available for paired analysis on these days, hematocrit values (n = 10) were lower in the luteal than the follicular phase, but there were no significant cycle phase-related differences in EPO (n = 5), reticulocyte count (n = 10), blood ferritin levels (n = 9), hemoglobin concentration (n = 10), or red cell volume (n = 9) (Table 1).
Measurements by Cycle Phase at 4,300 m
Progesterone and estradiol. In the comparison of altitude with sea level, on the basis of hormone levels and menstrual diaries, 5 of the 16 subjects had measurements made in the luteal phase both at sea level and at 4,300 m. For paired luteal-phase measurements, there was no difference in hormone levels when altitude values were compared with those at sea level either on day 3 or day 10 or 11 (Table 2). Paired measurements of ovarian hormone levels were obtained in the other 11 subjects in the follicular phase both at sea level and at 4,300 m (Table 2). For paired follicular-phase measurements, there was no difference in hormone levels when altitude values were compared with those at sea level either on day 3 or day 10 or 11 (Table 2).
In the comparison of hormone measurements between the groups for the 5 women in the luteal phase vs. the 11 women in the follicular phase of the menstrual cycle, progesterone blood levels were higher both at sea level and at altitude in the luteal-phase group than in the follicular-phase group (Fig. 1, A and B). Women in the luteal phase had higher estradiol levels than those in the follicular phase on day 3 of the cycle at sea level (Fig. 1C). At 4,300 m, although estradiol values tended to be higher early in the luteal than the follicular phase of the cycle, the differences were not statistically significant (Fig. 1D).
Ventilation and SaO2. End-tidal PCO2 fell progressively from sea-level values over the 12 days of observation at 4,300 m. SaO2 fell on arrival at 4,300 m and then increased as ventilatory acclimatization progressed. There was no difference at sea level or at 4,300 m between the 5 women in the luteal phase vs. the 11 women in the follicular phase with regard to the end-tidal PCO2 (Fig. 2A) or SaO2 (Fig. 2B).
Measurements of erythropoiesis. At 4,300 m, for the 5 women in the luteal phase and the 11 women in the follicular phase, levels of EPO were increased above the sea-level values, but there was no difference between the groups (Fig. 3A). Reticulocyte counts (Fig. 3B) were higher than at sea level after 6 and 11 days at 4,300 m, but here also there was no difference between the groups in the luteal vs. the follicular phase. Hemoglobin (Fig. 3C) was higher and ferritin (Fig. 3D) was lower than sea-level values, but there were no cycle phase-related differences for these variables. Red cell volumes increased from 28.0 ± 2.6 ml/kg at sea level to 31.1 ± 3.6 ml/kg after 11 days at 4,300 m in the luteal-phase group and from 30.6 ± 1.0 to 33.0 ± 1.6 ml/kg in the follicular-phase group, but neither the red cell volume nor the increase at altitude differed between the groups.
Because, in women given supplementary progestational agents, ventilation increased during the first hour of altitude exposure (23), and because, in experimental animals (12), administration of progesterone and estrogen inhibited erythropoiesis, we examined the relationship of hormone levels to erythropoiesis in our subjects. We found no relationship at high altitude between red cell volume on day 11 or erythrocyte count on days 6 and 11 with estradiol [P = not significant (NS)] or progesterone (P = NS) levels. We also found no relationship between EPO and either estradiol (P = NS) or progesterone (P = NS). However, in all subjects within our cohort, EPO levels related well (r = 0.77, R2 = 0.6, P < 0.01, n = 63) to SaO2, without regard to the duration of stay at altitude. This suggested that 60% of the variation in EPO could be accounted for by variation in SaO2. To better demonstrate possible hormonal effects, we compared the relationship between EPO and SaO2 for arbitrarily chosen extremes of hormonal levels at altitude. In these analyses, the relationship between EPO and SaO2 remained unaffected by levels of progesterone (Fig. 4A) or estrogen (Fig. 4B)
All Subjects Regardless of Cycle Phase
Because of the absence of cycle phase effects on erythropoiesis, and because of the dearth of information in women going to altitude, we report the values for the entire cohort at sea level and at altitude (Table 3). EPO levels were higher than sea level on day 3 and subsequently fell toward, but not to, sea-level values. Reticulocyte counts had not increased on day 3 at 4,300 m but rose by days 6 and 11. Red cell volume was not increased on day 3 but was increased on day 11. Hemoglobin and hematocrit values increased at altitude. Blood ferritin level fell with increasing reticulocytosis (r = 0.36, P < 0.01, n = 52).
DISCUSSION
The main finding of the present study in women was that menstrual cycle phase did not affect measurements relating to erythropoiesis, either at sea level or at 4,300 m. Menstrual cycle phase during experimental measurements was confirmed by history and by blood levels of progesterone and estradiol. Our findings of no cycle-related change in EPO confirmed those of Cotes and Canning (9) in six healthy women at sea level. In the women of their study, blood hemoglobin was slightly lower in the luteal than the follicular phase, similar to our findings for hematocrit, which may reflect expansion of plasma volume in the luteal phase. Our findings of no cycle phase-related differences in reticulocyte count and red cell volume further supported the concept of little or no change in erythropoiesis during the menstrual cycle at sea level.
At high altitude our hypothesis of diminished erythropoiesis in the luteal phase of the menstrual cycle was not substantiated. Several potential factors could have complicated this interpretation. Perhaps most important was the small number of subjects in the luteal phase of the menstrual cycle at altitude. Although we had hoped to have equal numbers in the cycle phases, we ended up with groups containing 11 in the follicular, and only 5 in the luteal, phase. Fortunately, at altitude, the phase that was present was associated with hormonal values that were within the normal sea-level range for the given phase and were similar to values obtained in the same subjects at sea level. We are not aware of previous studies that have reported sequential hormonal changes in women for the first days after arrival at altitude.
Also potentially complicating the interpretation of no difference in erythropoiesis with menstrual cycle phase was the fall in ferritin to levels that were low enough (<10 μg/l) to affect EPO and red cell formation (26). A decrease in ferritin occurred in conjunction with new red cell formation at altitude, suggesting that, despite oral iron supplementation, augmented erythropoiesis drew down the body's iron stores. In the study by Richalet et al. (32), in volunteers going above 3,600 m for 39 days (the final 21 days being at 6,542 m), ferritin levels decreased from 17.8 to 6.0 μg/l in the four women studied. In two of the four women, ferritin levels fell to 2 μg/l, but blood hemoglobin levels at altitude did not increase, suggesting that iron deficiency may have impaired erythropoiesis. We cannot exclude the possibility that iron deficiency inhibited erythropoiesis in our subjects. However, in findings against this possibility, our subject with the lowest serum ferritin (5.4 μg/l) doubled her reticulocyte count (from 0.9 to 1.9%) at 4,300 m and increased her total red cell volume by 15%, compatible with an adequate hematopoietic response. In any event, the possible influence of iron deficiency appeared to be the same in our two groups, because the levels of ferritin, reticulocytes, hemoglobin, hematocrit, and red cell volume did not differ between the follicular vs. the luteal phase at 4,300 m. Taken together, our findings supported the concept that erythropoiesis was not affected by menstrual cycle phase during the days at altitude.
Another potential difficulty in interpreting erythropoiesis in our study lay in the variable hypoxic stimulus present during the acclimatization process. SaO2 changes during altitude acclimatization. We expected that high levels of progesterone in the luteal phase would raise effective ventilation and diminish hypoxemia compared with the follicular phase, but this did not occur. Although SaO2 fell from 98% at sea level to ~80% on arrival at 4,300 m and then rose to 88% after 11 days residence, there was no cycle phase-related difference in these temporal changes between the groups.
The effect of menstrual cycle phase on ventilation during short-term (<12 h) altitude exposure has been studied at rest and during exercise. Loeppky et al. (23) studied 19 women in both phases of the menstrual cycle at low altitude (barometric pressure = 636 mmHg), and after 1 and 12 h at simulated high altitude (barometric pressure = 426 mmHg). When women were in the luteal phase, their alveolar and arterial PCO2 values at low altitude and after 1 h at high altitude were lower than in the follicular phase, but there were no statistically significant cycle phase-related differences in PCO2 after 12 h at high altitude. Furthermore, arterial PO2 values at either 1 or 12 h at altitude were not significantly different between the follicular and the luteal phases of the cycle. Beidleman et al. (2), in an exercise study of eight women during 3 h of altitude chamber exposure to a barometric pressure of 464 mmHg, found no difference with cycle phase in PCO2 or ventilation at submaximal and peak exercise. In both studies, the cycle phase comparison at low and high altitude was conducted within and not among subjects. Yet, even with these optimal experimental designs, less hypoxemia in the luteal, compared with the follicular, phase could not be shown after 12 h of resting high-altitude exposure and after 3 h of exposure to high altitude during exercise. Perhaps in these (2, 23) and the present study, the large changes in acid-base balance and in arterial oxygenation during ventilatory acclimatization to altitude may have acted, at least in part, to obscure any effect of progesterone on ventilation. If so, a longer altitude exposure might show greater ventilation and less hypoxemia in the luteal than the follicular phase, as has been found in women at high altitude who resided permanently at 4,300 m (22). However, for our study during the first days at 4,300 m, because the altitude-related hypoxic stimulus did not differ between the groups and because altitude exposure per se did not cause abnormal hormone levels, perhaps it is not surprising that erythropoiesis was not different between the luteal- and follicular-phase groups.
Also to be considered in interpreting the present results was the variation in EPO levels within individuals. Diurnal variation has been reported for EPO levels both at sea level and at altitude (20). We attempted to minimize the effect of diurnal variation by drawing blood samples at midmorning or midafternoon when variation is least (20). Furthermore, the diurnal variation is small compared with the effect of acclimatization-related variations (1, 20). The large acclimatization effect at 4,300 m on EPO levels was shown in five men by bioassay, and in nine men by ELISA (Fig. 5). Values were reported to rise to a peak ~1-2 days after arrival and then to return toward, but not to, sea-level values (1, 20), a pattern also found (by radioimmunoassay) at lesser altitudes (17, 18). At 4,300-m altitude, EPO levels in women followed a generally similar time course to that reported for men (Fig. 5). The changing EPO levels at altitude reflect, in part, the greater hypoxemic stimulus on arrival and the partial relief of that stimulus with ventilatory acclimatization (20). In any event, EPO shows remarkable change during the first few hours and days at high altitude.
To our knowledge, the only prior report showing EPO levels for women at both sea level and high altitude is that of Richalet et al. (32). Levels in women in that study showed a rather good relationship to SaO2, even without the taking of account of the duration of altitude stay (Fig. 6). When data in women from the present study are added to those in women obtained by Richalet et al., they lie along the line of best fit for the data of Richalet et al. (Fig. 6). The pronounced rise in EPO levels at lower SaO2 in the study of Richalet et al. might have been due in part to iron deficiency in some of their subjects. When ferritin concentration falls below 10-20 μg/l, EPO levels may rise (I. Asmus, unpublished observations). Although EPO depends on many factors (4, 26), hypoxia remains the primary stimulus. The results in our cohort suggest that, for the first 11 days at 4,300 m, ~60% of the variation in EPO levels reflects variation in SaO2 and that the remaining variation apparently had little to do with hormonal fluctuations during the menstrual cycle.
From the above findings, one is impressed by the number and magnitude of confounding variables that could impact hematopoiesis in healthy young women during the first days at altitude. Among these are the 1) changing levels of progesterone (and probably estrogen) during the normal menstrual cycle, 2) changing SaO2 and acid-base balance with acclimatization, 3) fluctuating iron stores as erythropoiesis proceeds, 4) diurnal variation in EPO, and 5) changes in EPO with acclimatization itself. Given all these factors, perhaps it is not surprising that, in a small cohort of women, we could not identify differences in erythropoiesis between the luteal and the follicular phases during an 11-day study at high altitude. During this interval, we did not find even the expected relative hyperventilation in the luteal, as opposed to the follicular, phase of the cycle. It was as though the known effects of progesterone on ventilation were small compared with the effects of acclimatization itself. However, published reports suggest that, once acclimatization is complete, as in altitude residents, the effects of the ovarian hormones become apparent. Women who are of child-bearing age have greater ventilation, lower hemoglobin, and less erythropoiesis than do older women who are postmenopausal (21, 22). Whatever is the impact of confounding variables on erythropoiesis in young women during the first 11 days at high altitude, such impact is likely to be temporary. Further research is needed to establish the role of menstrual cycle phase on breathing and hematopoiesis in women residing for longer periods at high altitude.
ACKNOWLEDGEMENTS
We thank the test volunteers for their participation and cooperation in this study. Also, the nurses, staff, and faculty at the Aging Studies Unit of the Palo Alto Veterans Affairs Medical Center provided essential assistance, without which this study could not have been performed.
FOOTNOTES
Deceased 28 December 1999.
This research was supported in part by US Army Contract DAMD-17-95C-5110; the General Clinical Research Center of the University of Colorado Health Sciences Center (National Institutes of Health Division of Research Resources 5-01 RR-00051); and National Heart, Lung, and Blood Institute Grant HL-14985 and Specialized Center of Research Grant HL-46481.
The views, opinions, and/or findings in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other official documentation. Citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations.
Address for reprint requests and other correspondence: J. T. Reeves MDB-131, Univ. of Colorado Health Sciences Center, Denver, CO 80262 (E-mail: john.reeves@uchsc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 March 2001; accepted in final form 8 August 2001.
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