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Androgens and Osteocalcin during the Menstrual Cycle¹

Department of Obstetrics and Gynecology (C.M.) and Institute of Preventive Pediatrics and Neonatology (C.D.F., D.G., F.B.), University of Siena, Siena, Italy; and European Medical Coordinator, Eli Lilly & Co. S.p.A. (D.P.A.), Sesto Fiorentino, Florence, Italy

Address all correspondence and requests for reprints to: Cosimo Massafra, M.D., Department of Obstetrics and Gynecology, Policlinico Le Scotte, 53100 Siena, Italy. E-mail: massafra{at}unisi.it

	Abstract

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The relationship between physiological variations in female sex and androgenic hormones and calciotropic hormones was investigated during the menstrual cycle. Estradiol, progesterone, total and free testosterone, androstenedione, immunoreactive PTH, calcitonin, osteocalcin (OC), and ionized calcium serum levels were determined throughout the menstrual cycle in a population of healthy eumenorrhoic women (n = 12; age range: 20–29 yr; mean: 24.2 yr). The women were studied from the first day of a menstrual phase until the first day of the following menstrual phase. Cycle length was standardized on the preovulatory estradiol peak (day 0), and values were given for the first day of a menstrual phase, and days -12, -10, -8, -6, -4, -2, 0, 2, 4, 6, 8, 10, 12, and 14 of the menstrual cycle. All subjects had a regular ovulatory cycle, as indicated by the hormonal profile. No significant cycle phase-dependent changes in calciotropic hormones were present. Significant positive correlations between total testosterone (r = 0.32, P < 0.001), free testosterone (r = 0.26, P < 0.001), androstenedione (r = 0.35, P < 0.0001), and OC were observed. The significant relations between these variables were confirmed by a time series analysis. For the first time, these findings indicate a relationship between androgens and OC serum levels during the menstrual cycle. An important regulatory role of endogenous androgens in OC secretion, bone formation, and maintenance of normal bone mineral content in the healthy eumenorrhoic woman is hence suggested.

	Introduction

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THE EFFECTS of physiological variations in female sex hormones on calciotropic hormones throughout the menstrual cycle have been investigated in several studies. Some of these have shown the absence of any changes in PTH (1, 2, 3, 4), calcitonin (CT) (1, 3), osteocalcin (OC) (5), or 1,25-dihydroxy vitamin D [1,25(OH)₂D] (1, 3, 4) levels during the menstrual cycle. Conversely, other reports have indicated significantly increased levels at or around ovulation for PTH (6) or 1,25(OH)₂D (2, 7, 8), and a serum OC peak during the luteal phase (4).

However, little information is currently available on the relationships between calciotropic and androgenic hormonal changes throughout the menstrual cycle. Androgen (9) and estrogen receptors (10) have been found in bone cells, and androgens directly stimulate proliferation of bone cells in vitro (11). Patients with an excess of ovarian androgens have high bone mineral density (BMD) (12). Positive correlations between bone mass and androgen levels have also been observed in pre- and postmenopausal women (13). Accumulating evidence supports the use of androgen replacement therapy in postmenopausal women in the prevention and treatment of osteoporosis (14, 15, 16, 17).

The aim of the present study was two-fold: first, to corroborate previous evidence on the effects of female sex steroid changes on immunoreactive PTH (iPTH), CT, OC, and ionized calcium (Ca++) serum concentrations; and second, to investigate the relationships between calciotropic hormones and total testosterone (T), free-testosterone (fT), and androstenedione (A) variations during the menstrual cycle in healthy eumenorrhoic women.

	Subjects and Methods

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Subjects

Twelve nulliparous healthy volunteers (age range: 20–29 yr; mean: 24.2 yr), with a history of regular menstrual cycles lasting from 28–30 days, entered the study. Medical history, physical examination, and laboratory data excluded the presence of any pathological condition. BMD, measured by DEXA (Hologic QDR 1000, Waltham, MA), showed results in the normal range in all subjects. Body weight was normal, and all subjects were on a typical Mediterranean diet. None of the subjects had smoking habits, made use of alcohol, or had a history of intensive exercise. None of the women received any type of medication during the previous 6 months or during the study period. Informed consent was obtained from all the participants before participation in the study.

Study design

All subjects participated in the study during the same period. Subjects were studied from the first day of a menstrual phase (M) until the first day of the following menstrual phase. Serum levels of LH, FSH, estradiol (E₂), progesterone (P), T, fT, and A were evaluated, every other day, from M to the first day of the following menstrual phase. Serum Ca++ concentrations were also determined on the same days. Because sampling on alternate days cannot accurately define the peak of the midcycle LH surge, cycle length was standardized on the preovulatory E₂ peak (day 0), and values were given for days M, -12, -10, -8, -6, -4, -2, 0, 2, 4, 6, 8, 10, 12, and 14 of the menstrual cycle, respectively. Follicular and luteal phases were defined on the basis of the hormone pattern levels and divided into six different groups: early- (days from M to -10), mid- (days from -8 to -4), and late- (days from -2 to 0) follicular phase and early- (days from 2 to 4), mid- (days from 6 to 10), and late- (days from 12 to 14) luteal phase.

Laboratory measurements

Blood samples (12 mL) were taken from the antecubital vein between 0800 and 0900 h, after overnight fasting. Blood was immediately centrifuged, and aliquots of the serum samples were stored at -30 C until hormonal assay. Hormone assessments were performed in duplicate in a single matrix at the end of the study period. LH, FSH (RADIM, Pomezia, Roma, Italy), and iPTH (Diagnostic Systems Laboratories, Inc., Webster, TX) were determined by immunoradiometric assay. E₂, P, T (Sorin Biomedica, Saluggia, Vercelli, Italy), fT (Diagnostic Systems Laboratories, Inc.), A (RADIM, Pomezia), CT and OC (INCSTAR Corp., Stillwater, MN) were determined by RIA. Sensitivity and precision data of the hormonal assays are reported in Table 1. Accuracy and specificity data of the assay are reported in the kit instructions by the manufacturers. Ca++ serum levels were determined by a calcium-sensitive electrode (Emogas ABL, Radiometer, Copenhagen, Denmark).

Data were expressed as the mean (M) ± SEM, unless otherwise stated. Cycle phase-related changes were evaluated by one-factor ANOVA for repeated measures, with post hoc pairwise comparison of means, using the Scheffé test. Correlations between variables were tested using univariate regression analysis and Pearson’s coefficients. An analysis of covariance was performed to determine whether the relationship between androgens and OC was dependent on the menstrual phase. To identify the temporal relationships between androgens and OC, the variables were analyzed by time series analysis (18); and, after theoretical elimination of intervals between peaks, cross-correlation coefficients at concurrent points were calculated using the mean values from all subjects. A two-tailed P < 0.05 was considered of statistical significance. All data were analyzed with a STATISTICA, release 4.0, software package for Windows (StatSoft, Inc. 1993, Tulsa, OK).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The mean duration of the menstrual cycle was 29 ± 1.0 days (M ± SD) (28–30 days), with menses duration of 4.0 ± 1.0 days (M ± SD). All the women examined had a regular ovulatory cycle (Table 2). Figure 1 reports the cycle-dependent variations for T, fT, and A. No significant cycle-dependent variations in iPTH, CT, or OC concentrations were evidenced (Fig. 2), and no significant correlation was found between calciotropic hormones and E₂ and P cyclical variations. No covariates to the menstrual cycle-phase were found to be significant at P < 0.05. Using individual values, significant positive correlations T/OC (r = 0.32, P < 0.001), fT/OC (r = 0.26, P < 0.001), and A/OC (r = 0.35, P < 0.0001) were obtained (Fig. 3); whereas no other significant relations between calciotropic hormones and androgens were present. A time series analysis, after theoretical elimination of intervals between peaks (mean values from all subjects), confirmed significant positive correlations between androgens and OC: T/OC (r = 0.702, P = 0.0036), fT/OC (r = 0.568, P = 0.02), and A/OC (r = 0.654, P = 0.0082) (Table 3). No significant changes in serum Ca++ levels throughout the cycle, or significant relationships between Ca++ and hormonal changes were demonstrated. No significant autocorrelations were found for the variables examined.

View larger version (32K): [in this window] [in a new window]   Figure 1. Changes in testosterone, fT, and A serum levels (M ± SEM) in eumenorrhoic women during follicular and luteal menstrual phases. The indicated P values are related to the overall variation throughout the menstrual cycle (one-factor ANOVA for repeated measures).

View larger version (37K): [in this window] [in a new window]   Figure 2. Changes in iPTH, CT, and OC serum levels (M ± SEM) in eumenorrhoic women during follicular and luteal menstrual phases. The indicated P values are related to the overall variation throughout the menstrual cycle (one-factor ANOVA for repeated measures).

View larger version (24K): [in this window] [in a new window]   Figure 3. Positive relationships between T/OC, fT/OC, and A/OC on days M, -12, -10, -8, -6, -4, -2, 0 (preovulatory peak of E2), 2, 4, 6, 8, 10, 12, and 14 of the menstrual cycle in eumenorrhoic women (linear regression analysis using individual values, n = 180).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The evidence of a similar intracellular density for androgen (9) and estrogen (11) receptors in the osteoblasts of male and female donors and direct stimulation of bone cell proliferation in vitro by androgens have been demonstrated (11). Clinical evidence suggests the stimulating role of androgens on OC secretion. In 10- to 14-yr-old boys and 9- to 12-yr-old girls, serum OC is significantly correlated with T levels (19); and in healthy boys, an age-related increase of OC levels and T plasma concentration has also been demonstrated (20).

In women, indirect evidence also shows the role of androgens in osteoblastic activity. In postmenopausal women, it has been shown that T concentrations are associated with rates of bone loss at the distal radius (13). Bone loss has been significantly associated with low androgen concentrations in premenopausal women, and low estrogens and androgens in late-, peri-, and postmenopausal women (21). These findings are consistent with the previously reported positive relationship between BMD and androgen levels in young (22), as well as pre- and perimenopausal women (23), and with the association between declining adrenal androgen levels and bone loss in aging women (24). The additional observation, that hyperandrogenic women have a positive correlation between T and fT and BMD, further supports these findings (13). Circulating A is also significantly reduced in postmenopausal women with osteoporosis, compared with postmenopausal women without osteoporosis (25). Conversely, vertebral BMD is significantly raised in young women with high endogenous A concentrations (22).

The relationship between T, fT, and A and OC serum levels, observed during the menstrual cycle, confirms previous reports that androgens may play an important role in OC secretion (16, 19, 20). This is further supported by positive results of androgenic therapies on bone mass. In fact, estrogen-plus-T replacement in postmenopausal women has shown more beneficial effects on BMD compared with either oral or parenteral estrogen alone (15, 16, 17). Women treated with oral estrogen plus methyltestosterone reportedly show not only a reduction in biochemical parameters of bone resorption (as seen with estrogen alone) but also an increase in markers of bone formation (16). The observed relationship between androgens and OC, as well as the possible effects of the androgen-mediated changes during the menstrual cycle, may be further elucidated by the contemporary determination of biochemical indicators of bone remodeling (in particular, by determining osteoclastic activity markers) (26).

	Footnotes

 
¹ This work was financed with "60%" funds for scientific research from the Italian Ministry of Education and "Fondi di Qualità" from Azienda Ospedaliera Senese, Siena, Italy.

Received June 5, 1998.

Revised November 9, 1998.

Accepted November 16, 1998.

	References

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