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Original Studies |
Departments of Obstetrics and Gynecology (I.G., Y.T., O.C., R.K., M.N., B.C.Y., S.Y., H.M.) and Pediatrics (S.Y.), Yokohama City University School of Medicine, Yokohama 236-8567, Japan
Address all correspondence and requests for reprints to: Itsuo Gorai, Department of Obstetrics and Gynecology, Yokohama City University School of Medicine, 3–9 Fukuura, Kanazawa-ku, Yokohama 236, Japan.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recent studies on the role of estrogen in bone metabolism have shown the involvement of cytokines, such as interleukin (IL)-1 and IL-6. Pacifici et al. (2, 3) reported that estrogen withdrawal allows peripheral blood monocytes to secrete more IL-1 in postmenopausal women, and that estrogen replacement inhibits IL-1 secretion by monocytes in those women. Manolagas and his co-workers reported (4) that IL-6 is involved in the stimulation of bone resorption induced by estrogen deficiency. Tamura et al. (5) showed that IL-1 greatly stimulates the differentiation of osteoclast precursors into mature osteoclasts in the presence of soluble IL-6 receptors (sIL-6R) in cocultures of mouse bone marrow and primary osteoblastic cells. Because estrogen shows significant cyclic variation during the menstrual period, these findings have prompted us to investigate variations in the levels of cytokines during the menstrual period.
Bone loss because of suppression of ovarian estrogen production may occur in women administered GnRH agonists (6, 7). A simple means of identifying women with increased bone resorption associated with accelerated bone loss is clinically important in the management of those requiring GnRH agonists. Markers of bone turnover are now being studied to identify women who will lose bone before bone density measurements can detect such losses. The percent change in bone mineral density at L1-L4 after 6 months of GnRH agonist treatment correlated inversely with the percent change of cross-linked N-telopeptides of type I collagen from baseline to month 4 (8). Evaluating the baseline values of biochemical markers is crucial for detecting a percent change from baseline in those women who start GnRH agonists therapy.
When bone turnover in pre- and perimenopausal women is examined, the specific days of the menstrual cycle on which blood and/or urine specimens are collected are of clinical importance. The determinations made of sex steroids in the early follicular phase may not adequately represent sex steroid concentrations for premenopausal women throughout the menstrual cycle. Multiple blood or urine samples (or both) may be necessary to adequately characterize the associations between estrogens and skeletal health in premenopausal women (9). It is therefore worthwhile to assess alterations in metabolic markers of bone turnover during the menstrual cycle in healthy women with normal cycles.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Ten healthy Japanese women ranging in age from 22–43 yr (mean, 33 ± 7 yr) with no history of menstrual cycle irregularity were investigated. None of the volunteers had had any chronic diseases or were taking any medication known to affect bone metabolism (e.g. renal disease; hyperparathyroidism; hyperthyroidism; diabetes mellitus; or corticosteroid, anticonvulsant, and oral contraceptive therapy). All subjects were ambulatory and engaged in their usual activities. All subjects gave informed consent before entering the study.
Blood samples were drawn on the first day of menstruation, followed by sampling three times a week (Monday, Wednesday, and Friday) until 4 days after the subsequent menstruation. All blood samples were collected in the fasting state between 0800–0900 h and were allowed to clot at room temperature for approximately 1 h. Thereafter, the serum was separated off by centrifugation and stored at -20 C. Urine samples were collected as second void at the same time and were also stored at -20 C.
Analytical methods for serum cytokines
Serum IL-6 levels (Amersham International, Buckinghamshire, UK) and serum sIL-6R levels (Quantikine, human sIL-6R immunoassay; R & D Systems, Minneapolis, MN) were measured using a sandwich enzyme-linked immunosorbent assay (ELISA).
Serum IL-1ß values were determined using a solid-phase ELISA, which uses an antibody for IL-1ß bound to a microtiter plate together with a biotinylated antibody to IL-1ß and Amdex amplification reagent (Amdex N.S., Buckinghamshire, UK).
The minimum detectable doses of serum IL-6, sIL-6R, and IL-1ß are 0.70 pg/mL, 140 pg/mL, and 0.1 pg/mL, respectively. The intra- and interassay coefficients of variation (CV) were 2.1–4.3% and 3.8–6.3% for serum IL-6, 2.3–8.6% and 4.2–6.4% for serum sIL-6R, and <10% and <10% for serum IL-1ß, respectively.
Makers of bone turnover
Bone formation. A serum bone-specific alkaline phosphatase (ALP) assay (Alkphase-B; Metra Biosystems, Mountain View, CA) was performed using a microplate coated with an anti-bone-specific ALP monoclonal antibody. The bound ALP activity was measured by using p-nitrophenyl phosphate as a substrate (10). The minimum detectable level of this assay is 2 U/L, and the intra- and interassay CVs were 2.2–3.8% and 2.1–3.8%, respectively.
Serum concentrations of carboxy-terminal propeptide of type I procollagen (PICP) were measured using an equilibrium RIA (11) whose lowest detectable concentration is 25 ng/mL. The intra- and interassay CVs were 1.7–6.7% and 1.3–5.1%, respectively.
Serum intact osteocalcin (OC) was measured using a sandwich enzyme immunoassay (EIA) that employs polyclonal antibodies against N-terminal 20 residues (amino acids 1–20) and against C-terminal 7 residues (amino acids 43–49) (12). The sensitivity of the EIA is 1.0 ng/mL and the intra- and interassay CVs were 2.5–5.4% and 4.7–5.7%, respectively.
Serum N-mid OC levels were measured based on a two-site immunoradiometric assay (IRMA) for human OC using human OC as a standard and two monoclonal antibodies raised against human OC, a solid-phase anti-25–37 region, and a tracer anti-5–13 sequence of the molecule (13). The minimum detectable concentration of IRMA is 0.4 ng/mL, and the intra- and interassay CVs were 1.7–5.2% and 2.3–6.8%, respectively.
Bone resorption. Urinary type I collagen C-telopeptide
breakdown products (CTx) were measured by an ELISA (Cross Laps ELISA,
Osteometer Biotech A/S, Herlev, Denmark) based on an immobilized
synthetic peptide with an amino acid sequence specific for a part of
the C-telopeptide of the 
1-chain of type I collagen (14, 15).
The sensitivity of the method is 50 µg/L, and the intra- and
interassay CVs were 1.0–3.7% and 2.6–4.5%, respectively.
Urinary free deoxypyridinoline (D-Pyr) was measured by an ELISA that uses a monoclonal antibody having less than 1% cross-reactivity with free Pyr (Pyrilinks-D, Metra Biosystems) and no significant interaction with cross-linked peptides (16). The urinary excretion of bone resorption markers was corrected using the urinary creatinine concentration measured by a standard colorimetric method. This technique’s lowest detectable concentration is 3 nM, and the intra- and interassay CVs were 3.6–9.5% and 6.3–10.3%, respectively.
Serum concentrations of pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) were measured with an equilibrium RIA (17). The sensitivity is 1.0 ng/mL, and the intra- and interassay CVs were 4.8–6.9% and 3.9–7.7%, respectively.
Other biochemical indices
Serum ALP (U/L) was measured spectrophotometrically using p-nitrophenyl phosphate as the substrate, according to the method recommended by the German Society of Clinical Chemistry. Serum PTH (ng/mL) was determined by RIA using intact PTH as the standard (18). Serum PTH-related protein (PTHrP) was measured using a two-site IRMA (Nichols Institute Diagnostics, San Juan Capistrano, CA) (19). The minimum detectable levels of serum PTH and PTHrP were 100 ng/mL and 0.2 pmol/L, respectively. The intra- and interassay CVs were 3.7–7.2% and 2.6–9.3% for PTH and 3.5–9.5% and 2.7–4.5% for PTHrP, respectively. Serum FSH, LH, estradiol (E2), and progesterone (P4), and the serum levels of calcium and inorganic phosphorus were analyzed according to standard laboratory methods. Serum calcium value was corrected for serum albumin using the following equation: corrected calcium = calcium + [40-albumin (g/L)] 0.02 mmol/L.
All samples from an individual woman were analyzed in the same assay.
Statistical analysis
Each cycle was divided into a follicular period (FP) and a luteal period (LP) by the serum LH peak. FP and LP were normalized by lengths to eliminate interindividual variations in cycle and phase lengths as described in the literature (20). FP was further divided into early (days -14 to -12), mid (days -10 to -6), and late (days -4 to -2), and LP into early (days 2–4), mid (days 6–10), and late (days 12–14), respectively. Values from each subject were expressed as the percent changes from values at the LH peak. The significance of the differences among cytokines and indices of bone metabolism for different menstrual periods (early FP vs. early LP, etc.) was assessed using Student’s t test for paired data by means of Statistical Package (SAS Institute, Cary, NC). Regression analysis was used in determining the relationship of ovarian steroids, sIL-6R, and bone metabolic markers during the menstrual cycle. Except where explicitly indicated, mean values are expressed as mean ± SD.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The mean cycle length was 28.5 ± 1.9 days (range, 26–31 days).
The FP consisted of 15.4 ± 1.9 days (range, 13–18 days), and the LP
of 13.1 ± 0.6 days (range, 12–14 days). All investigated cycles were
ovulatory, as judged from the rise in serum P4 values
during LP. Values at the LH peak are given in Table 1
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Gonadotropins and ovarian sex steroids. Serum LH showed its
well-known midcyclic peak (P < 0.0001), and serum FSH
varied significantly (P = 0.0329 
P
< 0.0001) showing a midcyclic rise (P < 0.0001).
Serum E2 showed an increase during the mid FP to peak
values at ovulation (P = 0.0053 P
< 0.0001), a rapid decrease after ovulation (P =
0.0002), and a rise during the mid LP. Serum P4 showed an
increase after ovulation (P < 0.0001) with a midluteal
maximum (P < 0.0001).
Serum cytokines. Serum sIL-6R showed a rise in the early FP,
followed by a fall in the mid FP and an increase in the late FP.
Following a fall in the early LP, serum sIL-6R rose, showing an overall
increase during the LP (Fig. 1A). There
were significant differences in serum sIL-6R between both the early FP
(P = 0.0423) and the mid FP (P =
0.0334) and the early LP. Serum concentrations of IL-6 and IL-1ß were
below detectable levels.
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). The differences in serum
bone-specific ALP values were significant between the early and late
FPs (P = 0.0265), between the early FP and the early LP
(P = 0.0272), and between both the late FP
(P = 0.0364) and the early LP (P =
0.0373) and the late LP.
Serum PICP increased in the early FP, fluctuated during mid and late
FPs, and decreased during the LP (Fig. 1C). There was a significant
difference in PICP levels between the early FP and the mid LP
(P = 0.0161).
Serum intact OC showed a rise during the late FP and a fall during the mid and late LPs. Serum N-mid OC showed a rise in the mid FP and a fall in the late FP and mid LP. The variations in serum intact OC and N-mid OC, however, did not show any significant differences (data not shown).
Bone resorption markers. Urinary CTx showed a fall during the
early and mid FPs, a rise in the late FP, and a fall throughout the LP
(Fig. 2A). The differences in urinary CTx
were significant between the early and late FPs (P =
0.0086), and between the late FP and early LP (P =
0.0170), mid LP (P = 0.0008) or late LP
(P = 0.0143).
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). There was a significant difference between the
early FP and the early LP (P = 0.0347).
Serum ICTP decreased in the late FP and increased in the early and mid
LPs (Fig. 2C). The difference in serum ICTP was significant between the
late FP and the early LP (P = 0.0440).
Other biochemical indices
Serum PTH showed an increase during the FP and a decrease during
the LP (Fig. 3A). There was a significant
difference in serum PTH between the late FP and mid LP
(P = 0.0128). Serum PTHrP showed a fall in the early
and mid FPs and in the late LP, fluctuation in the late FP and the mid
LP, and a rise in the early LP (Fig. 3B). The difference in serum PTHrP
was significant between the mid FP and the early LP (P
= 0.0375).
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Relation between variables
The correlations between steroid hormones, sIL-6R, and bone
metabolic markers during the menstrual cycle are summarized in Table 2. Peaks of serum sIL-6R and bone
metabolic marker levels are summarized in Table 3. We then calculated the correlations in
the percent changes from values at the LH peak between E2
and serum sIL-6R or the other biochemical indices when the intervals
between the peaks of each of the markers and that of E2
were eliminated theoretically. Serum E2 significantly
correlated with serum PTH (r = 0.252, P = 0.0164)
when PTH preceded 8 days before E2 peak (Table 3).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Variations in serum and urinary levels of biochemical indices across
the menstrual cycle have been investigated in several studies (20, 24, 25, 26, 27, 28, 29, 30, 31). Recently, specific receptors for estrogen have been
demonstrated in osteoblasts (32, 33) and osteoclasts (34), and
P4 receptors have been found in bone cells (35), indicating
the possibility that sex steroids may act directly on bone cells. In
this study, serum E2 significantly correlated with intact
OC and serum ALP, whereas serum P4 correlated with serum
bone-specific ALP and intact OC and inversely with urinary CTx and
serum calcium (Table 2).
To the best of our knowledge, there has been only one report that dealt
with OC levels during the menstrual cycle. Nielsen et al.
(20) showed a variation in serum OC with significantly higher levels
during the LP. The authors measured serum OC by RIA modified from that
of Price and Nishimoto (36). In the present study, we measured serum
intact OC with sandwich EIA (12), and serum N-mid OC with two-site IRMA
(13). However, we could not find any significant variations in serum
intact OC and N-mid OC levels during the menstrual period. The most
likely explanation for these discrepancies is the difference in the
epitope specificity of the antibodies between their study and our
study. Nielsen et al. (20) further showed that serum OC and
serum E2 correlated best when the OC peak occurred
approximately 7 days after E2 (r = 0.69;
P < 0.05), suggesting a stimulatory effect of estrogen
on osteoblastic activity with a time lag of 7 days. The authors
ascribed the time lag to stimulation of osteoblastic proliferation
rather than to stimulation of the activity of the existing osteoblasts.
The peak in intact OC preceded the E2 peak by 2.5 ± 2.1
days in this study. There was no significant correlation between intact
OC and E2 when intact OC preceded before E2 by
2 days. However, serum intact OC significantly correlated with serum
E2 when there was no time lag (Table 2).
PICP is a globular trimer that is cleaved extracellularly from the carboxy-terminus of procollagen. As PICP, which is not incorporated into bone matrix, is not cleared by the kidney, circulating PICP levels correlate with the bone collagen synthesis rate and osteoblastic activity (37, 38, 39). Serum PICP showed a variation dissimilar to that of serum bone-specific ALP, and behaved somewhat differently from serum OC and bone-specific ALP in that it was not significantly higher in women in either early or late postmenopause than in premenopausal women (40, 41).
Based on these results, a divergence among the changes in bone-specific ALP, intact OC, and PICP could reflect a complex process of bone formation, a stimulatory effect of estrogen on osteoblastic activity involving stimulation of osteoblastic differentiation and proliferation and of the activity of existing osteoblasts (42), and differences in the behavior of bone remodeling units at different sites in the skeleton.
In addition to the above direct effects of estrogen on bone cells, an indirect mechanism could explain variations in markers of bone metabolism during the menstrual cycle. Serum PTH was significantly correlated with serum E2 with a time lag of 8 days. Our results support the findings and the explanations of Pitkin et al. (25) and El-Hajj Fuleihan et al. (31), but are not in agreement with reports in which no midcyclic elevations were found in serum PTH (26, 29, 30). Recently, Thys-Jacob and Alvir (43) observed a significant decline in total and ionized calcium at midcycle, with an increase of E2 in both the asymptomatic and the premenstrual syndrome groups. Only in the premenstrual syndrome group was the peak midcycle intact PTH significantly elevated compared with early follicular levels. However, we could not find any explanations for these discrepancies. Concerning this mechanism, Riggs et al. (44, 45, 46) found a 40–50% increase in serum immunoreactive PTH and a concomitant decrease in serum calcium after 10–26 weeks of conjugated estrogen treatment in postmenopausal osteoporotic women. An in vitro study (47) suggested that estrogen might directly enhance PTH secretion.
A variation in urinary CTx during the menstrual cycle confirmed our previous finding of a significant variation in the urinary levels of cross-linked N-telopeptides of type I collagen (48). The same profile was observed for urinary free D-Pyr. The serum ICTP, however, showed a different pattern from urinary CTx or free D-Pyr. This is consistent with another study that showed the nadir of serum ICTP during the FP and its peaks during the LP (49). ICTP is reported to be cleaved during the degradation of type I collagen and to correlate with bone resorption assessed by either histomorphometry or calcium kinetic studies (50) and is mainly cleared by the kidney (17). Twenty four-hour endogenous creatinine clearance has been reported to be higher during the LP than during the FP and to correlate with the production of ovarian hormones (51, 52), indicating that the decrease in urinary CTx and free D-Pyr during the LP was a result of decreased production rather than decreased renal clearance.
In conclusion, our observations of significant cyclic variations in biochemical markers of bone metabolism suggest that cyclic variations in markers of bone formation and resorption and a bone-resorbing cytokine might be modulated by cyclic changes in sex steroids during the menstrual period. In addition, the specific days of biochemical events in the menstrual cycle are crucial for evaluating osteoblastic and osteoclastic activities in pre- and perimenopausal women, or in women who start GnRH agonist therapy. The physiological significance of the variation in osteoclastic and osteoblastic activities during the menstrual cycle remains to be elucidated further in relation to skeletal health in premenopausal women.
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Acknowledgments |
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Received August 13, 1997.
Revised October 30, 1997.
Accepted November 5, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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ller-Petersen J, Larsen CE, Raffn K. 1987 Endogenous overnight creatinine clearance. Serum
beta2-microglobulin and serum water during the menstrual
cycle. Acta Med Scand. 221:191–197.[Medline]
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