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1The John B. Pierce Laboratory, and Departments of 2Epidemiology & Public Health and 3Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut
Submitted 7 January 2005 ; accepted in final form 15 February 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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-estradiol (2 x 0.1 mg/day patches) for days 13–16 (P4-E2). On days 2 (GnRH antagonist), 9 (P4), and 16 (P4-E2), we estimated ECF and PV. To determine the rate of protein and thus water movement across the ECF, we also measured transcapillary escape rate of albumin. In P4, 
increased from 2.5 ± 1.3 to 12.0 ± 2.8 ng/ml (P < 0.05) with no change in 
(21.5 ± 9.4 to 8.6 ± 2.0 pg/ml). In P4-E2, plasma concentration of P4 remained elevated (11.3 ± 2.7 ng/ml) and plasma concentration of E2 increased to 254.1 ± 52.7 pg/ml (P < 0.05). PV increased during P4 (46.6 ± 2.5 ml/kg) and P4-E2 (48.4 ± 3.9 ml/kg) compared with GnRH antagonist (43.3 ± 3.2 ml/kg; P < 0.05), as did ECF (206 ± 19, 244 ± 25, and 239 ± 27 ml/kg for GnRH antagonist, P4, and P4-E2, respectively; P < 0.05). Transcapillary escape rate of albumin was lowest during P4-E2 (5.8 ± 1.3, 3.5 ± 1.7, and 2.2 ± 0.4%/h for GnRH antagonist, P4, and P4-E2, respectively; P < 0.05). Serum aldosterone increased during P4 and P4-E2 compared with GnRH antagonist (79 ± 17, 127 ± 13, and 171 ± 25 pg/ml for GnRH antagonist, P4, and P4-E2, respectively; P < 0.05), but plasma renin activity and plasma concentration of ANG II were only increased by P4-E2. This study is the first to isolate P4 effects on ECF; however, the mechanisms for the ECF and PV expansion have not been clearly defined. extracellular fluid regulation; gonadotropin-releasing hormone antagonist; ganirelix acetate; blood volume; estrogen
In contrast to proteins, sodium and other electrolytes diffuse freely across the capillary membrane and so are not effective osmotic gradients. Thus any increase in PV due to changes in sodium retention will cause concomitant increases in overall ECFV, which in turn would be caused by greater renal fluid and sodium retention. Progesterone effects on PV have not been as well studied as those of estrogens but are likely to be related to changes in ECFV and overall fluid retention rather than changes in the Starling forces. Progesterone competes with aldosterone for the mineralocorticoid receptor (19), which under some conditions leads to compensatory increases in aldosterone production followed by increases in renal sodium and water retention (30). Our recent study demonstrated that, whereas estradiol alone caused PV expansion independent of changes in ECFV, combined progesterone and estradiol administration caused PV expansion proportional to overall ECFV expansion, concomitant with renin-angiotensin-aldosterone system (RAAS) stimulation (33). The interpretation of the progesterone impact on these findings is complicated by the fact that estradiol can stimulate the RAAS by enhancing angiotensinogen synthesis, inhibiting angiotensin-converting enzyme activity, and augmenting plasma and tissue concentrations of renin (15, 16), so the independent effect of progesterone on PV and ECFV has not been determined. The purpose of the present study was to determine the effects of progesterone, with and without estradiol, on PV and ECFV regulation. We hypothesized that progesterone administration (P4) would have little independent effect on either PV or ECFV, but that combined progesterone-estrogen administration (P4-E2) would lead to ECFV expansion with proportional increases in PV. We further hypothesized that this increase in sodium and fluid retention would be a function of RAAS stimulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Experimental design.
To suppress reproductive function for the duration of the study, the subjects received the GnRH antagonist ganirelex acetate (250 µg/day, Antagon, Organon, West Orange, NJ) each day for 16 days (Fig. 1). Beginning on day 5 of GnRH antagonist administration, the women received 200 mg/day of oral progesterone for 11 days. Beginning on day 13 of GnRH antagonist administration, the women added estradiol for 4 days (17-estradiol, 2 transdermal patches, 0.1 mg/day each). Experimental protocols were performed after 48 h of GnRH antagonist administration, on day 9 (GnRH antagonist with P4) and again on day 16 of administration (GnRH antagonist with P4-E2). This design permitted within-subject comparisons concerning progesterone and estradiol effects on changes in ECF distribution and changes in total ECFV. This hormone suppression, add-back design eliminated other potential confounders such as GnRH and the gonadotropins (follicle-stimulating hormone, luteinizing hormone) as well as other ovarian products, thus providing a direct assessment of the estrogen and progesterone influences on ECFV and PV.
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The GnRH antagonist administration began 9–10 days after the subject's luteinizing hormone peak. This peak precedes ovulation, usually day 12–14 of a 28-day menstrual cycle (see Fig. 1), and was determined individually by the use of ovulation prediction kits (OvuQuick, Quidel, San Diego, CA). The subjects self-administered daily subcutaneous injections of the GnRH antagonist after training by qualified medical personnel. This method of GnRH antagonist administration was chosen because it is easily discontinued in the event of uncomfortable side effects, such as headaches or vasomotor symptoms (i.e., "hot flashes"). The hypothalamic-pituitary-ovarian axis suppression is reversed on cessation of drug therapy.
Adding progesterone and estradiol treatment to GnRH antagonist treatment.
Progesterone was administered orally, with 100-mg doses each morning and each evening (200 mg/day, Prometrium, Solvay Pharmaceuticals, Marietta, GA). For estradiol treatment, the subjects received 17-estradiol administered using two transdermal patches delivering 0.1 mg/day each (Vivelle; CIBA Pharmaceuticals, Summit, NJ).
Experimental protocol.
For each experiment, the subjects arrived at the laboratory at 
7:00 AM after having eaten a light (300 kcal), low-fat breakfast (bagel or toast and jelly), having drunk 10 ml/kg body wt of water at least 1 h before their arrival at the laboratory, and having refrained from alcohol and caffeinated beverages and food for the previous 12 h. The subjects were also instructed to avoid high-sodium foods the night before the study. On reporting to the laboratory, subjects voided their bladders and were weighed to the nearest 10 g on a beam balance. Subjects were then seated in a semi-recumbent position for a 60-min control period in an environmental chamber (27°C, 30% relative humidity) to ensure a steady state in extracellular volume, PV, and constituents. During this control period, a 22-gauge Teflon intravenous catheter was placed in an antecubital or forearm vein in each arm with a heparin block (20 U/ml) to maintain catheter patency. A blood pressure cuff was positioned for automatic readings by a sphygmomanometer (Colin Medical Instruments, Komaki, Japan) to monitor changes in blood pressure. A three-lead electrocardiogram (Colin Medical Instruments) was used to monitor heart rate. At the end of the control period, a baseline blood sample was taken (42 ml), immediately followed by the simultaneous injections of Evans blue dye (New World Trading, DuBarry, FL) and inulin (Cypress Pharmaceutical, Carlsbad, CA).
ECFV was determined using the inulin dilution technique (17). ECFV determination involved an injection of a volume of a 10% inulin solution containing 60-mg inulin/kg body wt in an arm vein followed by a series of blood samples. The injection was given over 4.5 min, and blood was drawn before injection and at 5, 7, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, and 180 min after the start of the injection. The subject did not change posture throughout the control period or the inulin measurement to ensure that ECFV was stable. ECFV is calculated by the rate of clearance of inulin from the plasma. This method is valid with inulin because it is distributed slowly and only throughout the extracellular space, the kidneys excrete 95–100% of the infused inulin via glomerular filtration, and the tracer concentration becomes well mixed in the plasma. ECFV was calculated using the methods described by Ladegaard-Pedersen, using a semilogarithmic plot of the clearance of the tracer from the plasma over 180 min (17).
Transcapillary escape rate of albumin (TERalb) and PV were determined using an injection of a body weight-standardized volume of Evans blue dye (0.20 ml/kg) into an arm vein and taking blood samples for determination of dilution. The dye itself attaches rapidly to plasma albumin and therefore becomes evenly mixed. TERalb was determined from Evans blue dye washout over 1 h after dye injection and is calculated from the initial slope of the exponential curve fit through the optical density of post-dye injection plasma samples at 620 nm (13). Absolute PV was measured after complete mixing of Evans blue dye (10 min). PV was determined from the product of the concentration and the volume of dye injected divided by the concentration in plasma, taking into account 1.5% lost from the circulation within the first 10 min. The Evans blue dye is prebound to plasma by premixing 1 ml of Evans blue dye (depending on body weight) with 9 ml of whole blood, creating a fixed-binding ratio of 1:1 dye to albumin molecule. The Evans blue dye injection began simultaneously with the inulin infusion from the other catheter.
Urine was collected before the infusions, at the end of the inulin infusion, and at the end of the protocol for determination of renal function. The volume of urine excreted was replaced with an equal volume of tap water by mouth after subtracting the amount of fluid infused. Blood pressure using the automated blood pressure device was recorded at the end of the control period before the infusions were begun.
All blood samples were analyzed for hematocrit (Hct), hemoglobin (Hb), and total protein. Plasma osmolality, plasma concentrations of albumin and creatinine, and serum concentrations of sodium and potassium were determined every 30 min during the infusion. Plasma samples at baseline were analyzed for plasma renin activity (PRA), and plasma concentrations of 17-estradiol (), progesterone (), sex hormone binding globulin (P[SHBG]), angiotensin II (P[ANG II]), atrial natriuretic peptide (P[ANP]), and cortisol P[CORT], and serum concentrations of aldosterone (S[Ald]). Volume, osmolality, Na+, K+, and creatinine concentrations were measured from all urine samples.
Blood/urine analysis.
Blood samples were separated into aliquots. One aliquot was immediately analyzed for Hb and Hct in triplicate by cyanomethemoglobin and microcentrifuge, respectively. Plasma protein was also determined immediately from the same aliquot by refractometry. A second aliquot was transferred to a heparinized tube to be analyzed for plasma osmolality and plasma concentrations of albumin and creatinine. A third aliquot for the determination of serum concentrations of Na+, K+, and S[Ald] was placed into tubes without anticoagulant. The remaining aliquots were placed in tubes containing EDTA for analysis of , , P[SHBG] P[ANG II], P[CORT], PRA, and P[ANP]. The tubes were centrifuged at 4°C, and the plasma was taken off. After centrifugation, the EDTA samples were frozen immediately at –70°C until analysis. Plasma and urine sodium and potassium concentrations were measured by flame photometry (Instrumentation Laboratory model 943, Lexington, MA). Plasma and urine osmolality were measured by freezing-point depression (Advanced Instruments 3DII, Needham, MA), and plasma concentrations of albumin and creatinine were determined by colorimetric assay (Diagnostic Chemicals, Oxford, CT). Serum and urine osmolality and electrolytes were determined the same day as collection and were not frozen before analysis. Plasma inulin concentration was determined with enzymatic analysis according to the methods of Sugita et al. (37) and Delanghe et al. (7).
PRA and plasma concentrations of E2, P4, sex hormone-binding globulin, ANP, ANG II, and cortisol, as well as S[Ald], were measured by radioimmunoassay. With the exception of PRA, all samples were run in the same assay. For PRA, intra- and interassay coefficients of variation for the midrange standard were (3.3 ng ANG I·ml–1·h–1) 2.9 and 3.2%, respectively (Diasorin, Stillwater, MN); for the rest of the assays, intra-assay coefficients of variation for the midrange standards were as follows: (167 pg/ml) 5.0% (Diagnostic Products, Los Angeles, CA), (2.4 ng/ml) 3.6% (Diagnostic Products), P[SHBG] (70.6 ng/ml) 2.9% (Diagnostic Systems Labs, Webster, TX), S[Ald] (147 pg/ml) 9.5% (Diagnostic Products); P[ANP] (23.4 pg/ml) 6.9% (ALPCO Diagnostics, Windham, NH); P[ANG II] (21.3 pmol/l) 6.5% (IBL, Hamburg, Germany), and P[CORT] (12.0 pg/ml) 6.5% (Diagnostics Products).
Calculations. Body water regulation was determined through the assessment of the renal clearance of free water, osmolality, and sodium. The following equations were used to calculate renal function: Glomerular filtration rate was estimated from creatinine clearance, urine Na+ excretion was calculated as the product of urine Na+ concentration and volume (ml/min); fractional excretion of Na+ (FE Na+) = [(urine volume·urine Na+ concentration)/(glomerular filtration rate·[Na+]f)]·100, where [Na+]f is the Donnan factor for cations (i.e., 0.95)·serum concentration of Na+; and fractional excretion of water = (urine volume/glomerular filtration rate)·100. Blood volume was calculated from PV with the following equation: (PV·100)/[100 – (0.847·Hct)], with corrections for trapped plasma and whole body Hct by multiplying by 0.96 and 0.91, respectively (12).
Statistics. Data are expressed as means ± SE. The variables over time (GnRH antagonist alone tests, hormone intervention tests) were analyzed by conditions (P4, P4-E2, vs. GnRH antagonist alone) using ANOVA for repeated measures. When significant differences were found, orthogonal contrasts tested differences between specific means related to the hypothesis of interest. Differences were considered statistically significant when P < 0.05 (SPSS, Chicago, IL).
Sample size calculation.
Sample size calculations were based on our two primary outcome variables of interest: 1) PV and 2) ECFV. The desired statistical test was two sided, and we assumed an alpha level of 0.01 to account for multiple comparisons (14). In similar experiments, Stachenfeld et al. (33) observed effect sizes and standard deviations for PV of 290 ± 162 ml (progesterone with estradiol) and ECFV of 65 ± 31 ml/kg (estradiol only). A sample size of eight women per group allowed us >80% statistical power (1- < 0.80) to test within-group comparison to distinguish differences from chance alone.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Neither P4 nor P4-E2 altered body weight or blood pressure (Table 1). As expected, during P4, increased over the levels observed during GnRH antagonist, with a small drop in . During P4-E2, and both increased over the levels observed during GnRH antagonist alone, and was increased relative to the P4-only trial (Table 1; P < 0.05).
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45% of the variance within the subjects (mean r2 = 0.45).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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and , and administration of estradiol and progesterone increased and to levels similar to those seen during the midluteal phase of the menstrual cycle and, as such, are physiologically relevant. These are the first data to demonstrate that progesterone increases PV independent of estradiol and show that progesterone increases in PV are proportional to increases in ECFV. The increases in PV during progesterone treatment were also associated with lower albumin transcapillary escape rates. Thus P4, with and without estradiol, increased PV through the combined mechanisms of greater protein retention in the vascular space as well as overall expansion of ECFV. Despite the greater ECFV and increase in sodium-regulating hormones, changes in renal function were not evident in this study. We suspect that water and sodium retention had already taken place during the 4–8 days of the hormone treatments and had already reached a steady state by the time the subjects were tested in the laboratory (30). Moreover, although it appears that the RAAS was stimulated during P4-E2, this does not seem to be the case during P4 because neither PRA nor ANG II was increased. Our finding that PV was increased in proportion to ECFV during P4 was consistent with our earlier study (33). PV expansion during combined hormone administration has been generally considered an estradiol effect because PV consistently expands when estradiol is administered alone in both young (3, 8, 9, 25, 29, 31, 33, 34) and postmenopausal (38) women both with and without P4. Our recent study demonstrated that E2 administered without P4 expanded PV but did so despite ECFV contraction and solely by decreasing TERalb (33).
The large PV and ECFV expansions during P4 administration are consistent with those seen during progesterone-dominated states such as pregnancy (6). Our finding that concomitant increases in progesterone and estradiol led to PV expansion conflicts with earlier findings of PV contraction (of 3–9%) during the midluteal phase of the menstrual cycle (i.e., when both endogenous estradiol and progesterone are elevated) (31, 34–36). The PV loss in these earlier studies has been generally attributed to P4-induced reduction in ECFV due to transient natriuresis related to P4 competition with aldosterone for the mineralocorticoid receptor (19, 22). The present data demonstrate that the PV loss is not solely due to P4 because when we isolated P4, PV increased, as did ECFV. We suspect that the contrasting findings in our study from those in the luteal phase are due to the sequence we used to administer the sex hormones. There is no time over the course of the cycle that progesterone is elevated without concomitant elevations in estradiol and no time when progesterone peaks before estradiol as it did in the present investigation. In addition, when we delivered the sex hormones, they were provided as steady-state treatments rather than the fluctuating and seen during the menstrual cycle. Nonetheless, our data suggest that, under steady-state conditions, progesterone is not the primary cause of the PV contraction during the menstrual cycle because when progesterone was administered alone or combined with estradiol, PV expanded rather than contracted. The cause of the midluteal phase PV contraction needs to be investigated and may be due to changes in gonadotropins, other ovarian-produced hormones, or androgens. In the present investigation, most of these hormones also would have been partially or completely suppressed during the GnRH antagonist administration along with estradiol and progesterone.
Both PRA and ANG II were unaffected by P4, indicating that the aldosterone secretion occurred independent of the RAAS. The elevation in S[Ald] is a common finding during progesterone administration (4, 5, 27, 28, 32) and, as stated earlier, is the result of competitive binding by progesterone to mineralocorticoid receptor sites in the periphery that lead to compensatory increases in aldosterone (19, 22). However, during the menstrual cycle, the increase in S[Ald] may be a result of RAAS stimulation, which appears to be closely tied to the midluteal peak (18, 27, 28) because both PRA and aldosterone secretion increase only when ovulation occurs (18), indicating that a functioning corpus luteum (and the progesterone it secretes) is a necessary component of the augmented RAAS activity. Our finding of P[ANG II] stimulation along with the greater PRA and S[Ald] provide further support for RAAS stimulation when estradiol is increased along with progesterone, a finding consistent with previous studies (26, 30). In many systems in the body, upregulation of progesterone receptors is required to induce progesterone actions (24), so this may be the case with PRA, ANG II, and aldosterone release and actions in the kidney (26, 30). Thus it is possible that, had we administered the combined treatment first, we may have seen some activation of the RAAS during P4, as we did in our earlier cross-sectional study, because the progesterone receptors would have already been primed.
Alternatively, the stimulation of these fluid-regulating hormones may be a direct estradiol effect. Estradiol independently enhances ANG synthesis (10, 15, 16), inhibiting ANG-converting enzyme activity and augmenting plasma and tissue concentrations of renin (15, 16). Thus the greater PRA may simply reflect greater ANG levels and suggests that estradiol may have increased PRA independent of the added progesterone. Still another explanation may be that the increase in PRA during P4-E2 was the result of progesterone acting as an anti-aldosterone compound, causing sodium loss and stimulating compensatory increases in PRA. This response occurred only during P4-E2, suggesting that estradiol is a necessary condition for this anti-aldosterone system, perhaps by upregulating progesterone receptors in the adrenal cortex. Clearly, more studies in humans are required to determine the individual and combined effects of estrogens and progesterone on the RAAS.
During P4 and P4-E2, the lower TERalb suggests that greater protein retention in the vasculature would have been associated with greater plasma oncotic pressure and thus in some selective fluid retention in the plasma. However, it is clear that PV increased only insofar as ECFV was increased under both hormonal conditions. TERalb is not a measure of oncotic pressure, so our assumption that TERalb led to greater oncotic pressure may be erroneous. Indeed, the within-subject relationships between TERalb and PV were only moderate and not statistically significant. Compensation in Starling forces, such as capillary filtration coefficient or permeability, may have prevented selective increases in fluid retention in the plasma during overall ECFV expansion (1, 2, 11). Studies in which both TERalb and plasma interstitial oncotic pressures are measured simultaneously during different hormonal treatments may resolve these discrepancies. Finally, unlike our previous studies, plasma osmolality was not reduced during P4-E2, which may also be an indicator that PV changed in proportion to overall ECFV. The similar plasma osmolality across hormonal treatments conflicts with all of our earlier studies and so is not easily explained other than to again speculate that the order of hormone administration has important effects on ECFV and osmoregulation.
This is the first study to demonstrate that progesterone increases PV, and does so primarily by increasing total ECF. Although TERalb was reduced during both P4 and P4-E2, TERalb did not correlate with changes in PV within individuals across conditions. This weak relationship suggests that, although changes in ECF distribution play a role in progesterone-mediated PV regulation, it is not as important as the role played by expansion of the ECFV. The increase in aldosterone secretion under both conditions supports this conclusion, as does the likely RAAS stimulation during P4-E2.
Perspectives. The impact of sex hormones on body fluid and sodium regulation has important implications for a number of syndromes for which women are at risk, including orthostatic hypotension, hypertension, premenstrual symptoms, and neurological consequences from postoperative hyponatremia. Although estrogen effects are widely studied, progesterone effects on body fluids are often ignored. Independent progesterone effects are important because of the widespread use of progesterone-only oral contraceptives and fertility treatments and because states such as early pregnancy and endometriousis are dominated by progesterone. Although many earlier studies have postulated that progesterone plays a key role in sodium regulation over the course of the menstrual cycle or during pregnancy, our study is the first to isolate progesterone effects on this system and indicate that progesterone administration in the absence of estrogens can have important, independent effects on PV and ECFV. The plasma concentration of progesterone induced in this study was similar to that found during the luteal phase. Thus it is possible that when the body is exposed to higher blood and tissue levels of progesterone (i.e., during pregnancy, oral contraception, endometriosis), greater S[Ald] or RAAS stimulation may occur and induce greater sodium and fluid retention. These effects on PV and overall ECFV, as well as a direct alteration in renal function, may play a role in the hypertension that results during these conditions in some women.
The data from our study do not definitively demonstrate whether RAAS stimulation during combined progesterone and estradiol administration was the result of independent estradiol effects or due to upregulation of progesterone receptors and subsequent progesterone effects on this system. Our data support a pivotal role for progesterone for ECFV and PV regulation.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Significantly different from progesterone treatment. Differences were considered statistically significant at P < 0.05.