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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 8 2476-2482
Copyright © 1997 by The Endocrine Society

Clinical Studies

Biochemical Bone Markers and Bone Mineral Density during Postmenopausal Hormone Replacement Therapy with and without Vitamin D3: A Prospective, Controlled, Randomized Study1

Anna-Mari Heikkinen, Markku Parviainen, Leo Niskanen, Marja Komulainen, Marjo T. Tuppurainen, Heikki Kröger and Seppo Saarikoski

Departments of Obstetrics and Gynecology (A.-M.H., M.K., M.T.T., S.S.), Internal Medicine (L.N.), and Surgery (H.K.), University Hospital of Kuopio, FIN-70211 Kuopio; the Department of Clinical Chemistry, University Hospital of Helsinki (M.P.), FIN-02900 Helsinki, Finland

Address all correspondence and requests for reprints to: Dr. A.-M. Heikkinen, Department of Obstetrics and Gynecology, University Hospital of Kuopio, P.O. Box 1777, 70211 Kuopio, Finland. E-mail: anna-mari.heikkinen{at}pp.inet.fi


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The effects of postmenopausal hormone replacement therapy (HRT) and vitamin D on the serum concentrations of three bone biochemical markers and their associations with bone mineral density (BMD) were studied in a population-based 1-yr follow-up study. A total of 72 healthy postmenopausal women were randomized into 4 treatment groups: HRT group (sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate), D group (vitamin D3, 300 IU/day), HRT+D group (both of the above), and placebo group (calcium lactate, 500 mg/day). Serum concentrations of osteocalcin (OC) and bone-specific alkaline phosphatase (BAP) were measured as biochemical markers of bone formation, and serum type I collagen carboxy-terminal telopeptide was measured as a marker of bone resorption at baseline and after 6 and 12 months of treatment. To investigate the associations of these markers with BMD, lumbar (L2–L4) and femoral neck BMDs were determined by dual x-ray absorptiometry at baseline and after 2.5 yr of treatment.

In both hormone groups, the serum concentrations of the three bone metabolic markers had decreased after 12 months. Those of OC decreased by 29.2% (P = 0.017) in the HRT group and by 37.3% (P = 0.004) in the HRT+D group, and BAP concentrations decreased by 34.4% (P < 0.001) in the HRT group and by 36.2% (P < 0.001) in the HRT+D group. Serum type I collagen carboxy-terminal telopeptide concentrations had decreased by 21.6% (P = 0.012) in HRT group and by 14.1% (P = 0.011) in the HRT+D group. In the D group, the serum concentrations of BAP had decreased by 11.7% (P = 0.040) after 12 months, but the other two markers showed no change. The only change seen in the placebo group was a 19.2% increase in OC concentrations (P = 0.041) after 6 months, but at 12 months, the mean OC level was similar to that at baseline. After 2.5 yr, both lumbar and femoral BMD had decreased in the D group [2.1% (P = 0.022) and 3.6% (P = 0.019), respectively] and in the placebo group [3.3% (P = 0.009) and 2.7% (P = 0.010), respectively], whereas no significant changes occurred in the hormone groups. There were significant inverse correlations between the changes in lumbar and femoral BMDs and changes in all three biochemical markers (r = -0.240 through -0.336; P = 0.005–0.064).

Our results suggest that HRT counteracts the biochemical changes caused by increased bone turnover associated with menopause. Importantly, the changes in bone markers correlate with long term changes in BMDs of lumbar spine and femoral neck. Low dose vitamin D treatment, however, seems to have only marginal effects on bone metabolism in early postmenopausal healthy women.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
MENOPAUSE is associated with adverse changes in bone turnover. Both bone resorption and bone formation increase. When bone resorption exceeds formation, bone loss and osteoporosis will occur. As changes in bone mineral density (BMD) are late and relatively irreversible, it would be important to have a means of identifying high risk individuals and to monitor their treatment before fracture occurs.

Bone biochemical markers have been suggested to reflect postmenopausal high bone turnover. These markers could be useful in separating women into fast and slow bone losers (1). Additionally, they could be used to select patients for prevention of osteoporosis, to select therapy, and to follow treatment responses.

Osteocalcin (OC) is a noncollagenous protein synthesized by osteoblasts and released into the circulation in proportion to the rate of bone formation (2). It has been considered to be a marker of bone formation, but it may also reflect bone resorption (3). Serum OC concentrations decline markedly in both sexes up to the age of 50 yr and thereafter show a transient increase in women, reflecting high postmenopausal osteoblastic activity, but with further aging OC levels decline in both elderly men and women (4). According to the results of previous studies, hormone replacement therapy (HRT) decreases the concentrations of serum OC in postmenopausal women (5, 6, 7, 8, 9). OC has been suggested to be the best single biochemical marker for estimating the rate of bone loss in untreated postmenopausal women (9, 10), but its value as a follow-up marker during preventive medication of osteoporosis is not clear.

Bone-specific alkaline phosphatase (BAP) is a biochemical marker of bone formation, and it also reflects osteoblastic activity (11). The expression of this protein starts after cessation of cell proliferation of osteoblasts and reaches a maximum during matrix maturation, but declines during matrix mineralization (12). Circulating BAP concentrations increase with advancing age (13) and decrease during HRT (2, 14).

Type I collagen carboxy-terminal telopeptide (ICTP) is excreted from bone through collagen degradation and has, therefore, been considered as a marker of bone resorption (15). Circulating concentrations of ICTP do not correlate with age or menopausal status as clearly as do those of other bone metabolic markers, but they may reflect bone resorption in osteoporotic patients (13, 16). However, it has been suggested that ICTP is not a useful marker of bone resorption during HRT (16).

We examined the serum concentrations of OC, BAP, and ICTP at the baseline and after 6 and 12 months of sequential HRT and/or vitamin D treatment in a population-based, randomized group of 72 early postmenopausal women with normal BMD. The aim of this prospective study was to assess changes in the circulating concentrations of these biochemical bone markers and their correlations to long term BMD changes during four different treatment schedules.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

The subjects in this study comprise a subgroup of the Kuopio Osteoporosis Risk Factor and Prevention Study population. A baseline postal inquiry was sent to all 14,200 47- to 56-yr-old women in Kuopio Province, Eastern Finland, to investigate osteoporosis risk factors among perimenopausal women (17). The 464 postmenopausal women who were willing to undergo a 5-yr clinical trial, who had had their last menstrual period within 6–24 months before the study, and who had no contraindications for HRT, were included in the study. Written informed consent was obtained from the participants. The study design was approved by the ethics committee of Kuopio University Hospital. The participants were thereafter randomized by computer program into the following four treatment groups: HRT group, estradiol valerate (2 mg) on cycle days 1–21, cyproterone acetate (1 mg) on cycle days 12–21 and a treatment-free interval on cycle days 22–28 (Climen, Schering, Berlin, Germany); D group, cholecalciferol (300 IU/day) and calcium lactate (500 mg/day equivalent to 93 mg Ca2+/day; D-Calsor, Orion Corp., Espoo, Finland), with no intake during June through August; HRT+D group, Climen and D-Calsor; and placebo group, calcium lactate (500 mg/day equivalent to 93 mg Ca2+/day; Calcium Lactate, Rohto Ltd., Tampere, Finland). The study design has been described previously in detail by Tuppurainen and colleagues (18).

This analysis is a substudy based on 18 randomly selected women from each treatment group. A random sample of women who came to the baseline examination within a 2-month period during the spring formed this study population.

A total number of 69 women completed the 1-yr study. Two women receiving HRT and 1 woman receiving vitamin D stopped taking the study medication during the follow-up and were excluded from analyses. None of the participants had any disease or took medications known to cause secondary osteoporosis. Lumbar and femoral neck BMDs were analyzed in 63 women at baseline and after 2.5 yr of treatment.

Methods

Gynecological examination was performed at the beginning of the study and after 6 and 12 months. Briefly, in the baseline postal inquiry, the consumption of milk products was investigated. The daily calcium intake was calculated as the sum of calcium intake from milk, sour milk, yogurt (120 mg/dL), and cheese (87 mg/slice). The weekly duration of physical activity was noted. If the weekly duration of exercise was at least 3 h, the subject was considered to be physically active. Smoking and drinking habits were also recorded through use of a standardized questionnaire. The duration of regular smoking and the number of cigarettes consumed currently per day were registered. The lifetime amount of smoking was calculated in pack-years by dividing the lifetime number of cigarettes by 20 x 365. The weekly consumption of alcohol was expressed as absolute ethanol intake. Body mass index (BMI) was calculated as weight (kilograms) ÷ height (meters)2.

BMDs of the lumbar spine (L1–L4) and left proximal femur (femoral neck) were measured using dual x-ray absorptiometry (Lunar, Madison, WI) at Kuopio University Hospital before and after 2.5 yr of treatment, by trained personnel. The short term reproducibilities [coefficient of variation (CV)] of the spine and femoral neck measurements in our laboratory are 0.9% and 1.5%, respectively. The long term reproducibility (CV) of our dual x-ray absorptiometry instrument based on weekly repeated phantom measurements is 0.6%. To ensure that bone edges and intervertebral markers in the spine and regions of interests in the proximal femur were set consistently, all scans were reviewed by one investigator (H.K.). The BMD of vertebrae L2–L4 were used for the analyses. One lumbar measurement was excluded because of marked arthritic change.

Laboratory analyses

Serum concentrations of OC, BAP, and ICTP were determined at baseline and after 6 and 12 months of treatment, and those of vitamin D metabolites (25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D]), estradiol, FSH, calcium, and phosphate were determined at baseline. All blood samples were taken in the morning after an overnight fast.

Serum concentrations of OC were measured using a human-specific immunoassay for intact or almost intact OC (19). The interassay CV of this immunoassay was less than 11.6% during the present study. Serum concentrations of BAP were measured using a modified agarose gel electrophoretic method from Beckman (Brea, CA) as described previously more in detail (19, 20). The CV was less than 9.8% for BAP and less than 5.0% for the liver-specific isoform of the alkaline phosphatase (AP). Serum ICTP concentrations were measured using a RIA developed by Risteli et al. (21) and made available by Orion Diagnostica (Espoo, Finland). This assay had a CV less than 10.0% in our laboratory.

The concentrations of 25OHD and 1,25-(OH)2D were measured using a modification of the method described by Parviainen and colleagues (22). The interassay coefficient of variation was less than 8% in the serum 25OHD analysis and less than 14% in the 1,25-(OH)2D assay. Serum FSH concentrations were measured using a luminescence immunoassay (Byk-Sangtec, Dietzenbach, Germany; interassay CV, <5.6%). Serum calcium concentrations were measured using flame photometry (Efox, Merck Eppendorf, Darmstadt, Germany; interassay CV, <1.3%). Serum phosphate was measured using the phosphomolybdate method (Kone Specific, Espoo, Finland; interassay CV, <2.1%).

All assays were carried out at Kuopio University Hospital, Department of Clinical Chemistry.

Statistical methods

The analyses were carried out on a treatment basis. Statistical analyses of the longitudinal data for serum biochemical markers and BMDs were performed using multivariate ANOVA for repeated measures (MANOVA). Student’s t test for paired data was used to test the significance of a difference within a group if there was a time-related change within the group by MANOVA. One-way ANOVA with the Newman-Keuls post-hoc test was used to test the significance of differences between the baseline levels of serum biochemical markers and BMDs. Log transformation of ICTP data was used, as the ICTP values were not normally distributed.

Because BMIs and FSH levels were not normally distributed after log transformation, one-way ANOVA with the Kruskall-Wallis test was used to test for differences between groups. The Kruskall-Wallis test was also used to test differences between groups regarding other baseline parameters. Spearman correlations between the relative changes (follow-up - baseline value ÷ baseline level, as a percentage) in serum concentrations of bone biochemical markers and BMDs were examined.

P < 0.05 was considered statistically significant. The results are reported as the mean ± SEM. The data were analyzed using SPSS 228 for UNIX 228 (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
At baseline, there were no statistically significant differences between the groups with regard to age, BMI, duration of the menopause, previous HRT use, or baseline serum laboratory data. The reported consumption of milk products, use of alcohol and cigarettes, and amount of physical exercise were equal among the four groups (Table 1Go).


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  		Table 1. Baseline characteristics of 69 postmenopausal women according to treatment groups

 
The measurements of bone biochemical markers are summarized in Tables 2-4 and Fig. 1Go.



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  		Figure 1. The study group (n = 69) was divided into tertiles according to the baseline and 1 yr bone marker levels. This figure shows the numbers of subjects in the third tertile according to the four different treatment groups at baseline and after 1 yr. See Materials and Methods for descriptions of the groups.

 
After 6 months, serum concentrations of OC had increased by 19.2% (P = 0.041) in the placebo group, but they had not changed significantly in the other three groups. By 12 months, OC had decreased by 29.2% (P = 0.017) in the HRT group and by 37.3% (P = 0.004) in the HRT+D group, but they were similar to baseline values in the two nonhormone groups (Table 2Go). These time-related changes were also statistically significant between the groups (P = 0.002, by MANOVA).


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  		Table 2. The levels of serum OC (nanograms per mL) before and during the 6 and 12 months of therapy in four treatment groups

 
Serum concentrations of BAP decreased in subjects using HRT and/or vitamin D3, but did not change in the placebo group (Table 3Go). In the HRT group, the decrease was 26.3% (P = 0.000) after 6 months and 34.4% (P < 0.001) after 12 months. In the HRT+D group, BAP concentrations had decreased by 25.0% (P = 0.003) after 6 months and by 36.2% (P < 0.001) after 12 months. In the D group, the concentrations of BAP had not changed at 6 months, but had decreased by 11.7% (P = 0.040) after 12 months. At 1 yr, the differences between the groups were statistically significant (P < 0.001, by MANOVA).


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  		Table 3. The levels of serum BAP (units per L) before and during the 6 and 12 months of therapy in four treatment groups

 
Serum concentrations of ICTP did not change in the placebo or D groups, but they decreased in both hormone groups. This reduction was more obvious in the HRT group [22.1% after 6 months (P = 0.004) and 21.6% after 12 months (P = 0.012)] than in the HRT+D group [11.2% after 6 months (P = 0.039) and 14.1% after 12 months (P = 0.011); Table 4Go]. At the 1 yr examination, the differences in ICTP levels were statistically significant between the groups (P = 0.007, by MANOVA).


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  		Table 4. The levels of serum ICTP (micrograms per L) before and during the 6 and 12 months of therapy in four treatment groups

 
Table 5Go shows the lumbar and femoral neck BMD values at baseline and after 2.5 yr of treatment. There were no significant differences in baseline BMD values. After 2.5 yr, both lumbar and femoral BMDs had decreased in the D group [2.1% (P = 0.022) and 3.6% (P = 0.019), respectively] and in the placebo group [3.3% (P = 0.009) and 2.7% (P = 0.010), respectively]. In the HRT and HRT+D groups, both lumbar and femoral neck BMDs remained about the same. The 1 yr relative changes in concentrations of bone markers correlated inversely with the 2.5 yr changes in BMDs (Table 6Go).


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  		Table 5. BMD (grams per cm2) of the lumbar spine and femoral neck in the four treatment groups at the baseline and during the 2.5 y of therapy

 

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  		Table 6. Spearman’s correlation coefficients (r) between relative changes ({Delta}) in bone biochemical markers in 1 yr and relative changes in BMD in 2.5 yr

 
For a better demonstration of the observed changes, all the subjects were divided into tertiles according to the baseline and 1 yr bone marker levels. At baseline, the limits of the third (highest) tertile were 4.37 ng/mL for OC, 95.0 U/L for BAP, and 3.77 µg/L for ICTP. There were 23–24 subjects in the third tertile of each marker; 12 subjects had both OC and BAP, 11 subjects both OC and ICTP, and 10 subjects both BAP and ICTP concentrations in the highest tertile. All 3 baseline bone markers were in the third tertile in 5 subjects. These 5 subjects also tended to have low baseline BMDs (lumbar BMD, 1.070 ± 0.036 g/cm2; femoral neck BMD, 0.871 ± 0.025 g/cm2) compared with the other subjects (lumbar BMD, 1.144 ± 0.022 g/cm2; femoral neck BMD, 0.938 ± 0.016 g/cm2), but, possibly due to small number of subjects, the difference was not statistically significant.

Additionally, we were interested in tracking these markers of bone metabolism in the treatment groups. This is demonstrated in Fig. 1Go, which shows the numbers of subjects in the highest tertile according to treatment, at baseline and after 1 yr. The third tertile limits at 1 yr were 4.30 ng/mL, 77.0 U/L, and 3.20 µg/L for OC, BAP, and ICTP, respectively. In both hormone groups, the number of subjects with elevated serum marker levels decreased markedly, whereas in the placebo and D groups, high levels were relatively persistent during follow-up. As expected, more new subjects appeared in the highest tertile in the nonhormone groups at 1 yr.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our population-based, successfully randomized, study group gives a representative sample of the postmenopausal women in the study area. The study was started 6–24 months after natural menopause, when the absolute rates of bone turnover and bone loss show an increase, but changes in BMD are not yet seen. The present results show a clear independent effect of HRT on biochemical markers of bone formation and resorption. The serum concentrations of all three bone metabolic markers decreased significantly in both HRT groups, indicating that HRT suppresses bone turnover. This observation was confirmed by the BMD measurements. HRT inhibited the decreases in lumbar and femoral BMDs seen in the nonhormone groups. Changes in all three bone markers showed statistically significant inverse and consistent correlations with the BMD changes. On the other hand, vitamin D appeared to have only a slight effect on bone marker concentrations and no effect on BMDs.

Hansen et al. (1) suggested that fast bone losers could be identified by assay of bone biochemical markers. At advanced ages and in cases of certain bone-affecting diseases, bone resorption is high and formation low. Hence, it is likely that the serum concentrations of bone resorption markers increase, whereas those of formation markers decrease (2, 23). The levels of bone markers reflect the net balance of bone metabolism (24). However, the synthesis and release of bone formation markers reflect different developmental phases of osteoblasts. The expression of AP is highest during maturation of the osteoblast matrix, whereas OC is released during matrix mineralization, which is the final developmental phase of osteoblasts (12).

Bone resorption decreases during HRT (25). It has been suggested that the effects of estrogen on bone are mediated through calcitropic hormones, calcitriol [1,25-(OH)2D], calcitonin, and PTH (25). Estrogen may also have direct effects on osteoblasts (26). It has been noted that estrogen as well as anabolic steroids influence the proliferation of human bone-derived cells (27, 28). In a recent study, Raisz et al. (29) reported a decrease in both bone formation and resorption markers during short term estrogen therapy, but the concentrations of formation markers increased when methyltestosterone was added to the estrogen therapy. Although estrogen therapy evidently decreases the circulating concentrations of markers of bone formation, the net effect of estrogen on bone is positive. Owing to a close coupling between osteoblasts and osteoclasts, the lowering of bone formation markers in serum during HRT is most likely a consequence of decreased bone resorption.

Several previous studies have shown the efficacy of biochemical markers in assessing the short term (2, 29) and long term (5, 6, 7, 9, 14) effects of HRT on bone resorption and formation markers, and their correlations with BMD changes (23, 30, 31). Some contradiction exists, and it has been suggested that the concentrations of serum markers do not change during HRT as clearly as those of urine markers (23). Hence, the use of different serum and urinary markers is inconsistent. Urinary hydroxyproline has been used recently as a sole biochemical marker of bone resorption, although its sensitivity and specificity are limited (32). Both OC and BAP have been widely used as bone formation markers (19, 27, 33). Parviainen et al. (19) showed that the concentrations of these two markers correlate well in healthy subjects, whereas Diaz-Diego and colleagues (33) found no relationship in this respect. There are many different methods of measuring multiple forms of OC; thus, discrepancies prevail in the clinical use of OC assays (19, 34). Pyridinoline- and deoxypyridinoline-containing cross-linked peptides are relatively specific for type I collagen (21, 32), and they have been introduced as new markers of bone collagen degradation. In addition to serum ICTP, collagen degradation products that contain pyridinoline or deoxypyridinoline can be measured in urine (6). In this study we measured bone formation and resorption markers in serum only. Our electrophoretic method for BAP measurement provides a sensitive and specific means of quantitating low BAP levels even in the presence of other AP isoforms and isoenzymes (20), and this method gives results comparable to those of high performance liquid chromatography (35). Furthermore, this method allows for simultaneous quantitation of liver hepatocyte-derived AP and even other forms of serum AP.

HRT and HRT+D induced similar decreases in the serum concentrations of all three bone metabolic markers. Our results are in accordance with those of most previous studies regarding OC and BAP (5, 6, 7, 8, 9, 14, 29). In previous studies, ICTP has not been a sensitive marker of bone resorption in nonosteoporotic patients (13, 16) or during HRT (16). However, it has been suggested that ICTP is a good marker when examining bone turnover in osteoporotic subjects (13), and in metabolic bone diseases it correlates with the bone resorption rate assessed by either histomorphometry (15) or calcium kinetics (36). Our results indicate that ICTP levels are relatively persistent in nonosteoporotic early postmenopausal subjects, as shown by the lack of changes in the placebo group; therefore, changes observed in the hormone groups reflect the true effects of HRT.

The combination of serum markers gives extra information, as evidenced by the fact that about half of the subjects in the third tertile showed two of the measured markers there. By using three markers simultaneously, we could recognize a potential risk group of five (7.2%) subjects with all markers in the highest tertile and low BMD in both lumbar spine and femoral neck. Furthermore, we observed clear tracking in this risk group. Hence, it is likely that clinically rational use of bone markers requires a combination of them rather than reliance on a single marker. Unfortunately, the size of our study group was too small to show the changes in BMDs in four different treatment groups when two or three markers were combined.

Low dose vitamin D induced only minor changes in bone metabolic marker concentrations compared with placebo. Initial calcium and vitamin D metabolite levels were in the normal range, excluding the possibility of vitamin D or calcium deficiencies as confounding factors. The effect of vitamin D on bone metabolic markers in early postmenopausal subjects has not been reported in previous follow-up studies. Our results suggest that vitamin D supplementation is not necessary in healthy postmenopausal women with adequate dietary calcium and vitamin D intake. On the other hand, BAP decreased with low dose vitamin D supplementation; therefore, even this low dose may have an effect on the early maturation phase of osteoblast formation.

In the placebo group, the only statistically significant change was an increase in OC concentrations at 6 months. There were no other changes, showing that bone metabolic marker levels are relatively constant in healthy, nonosteoporotic, early postmenopausal women. The time since menopause in our study was 1.2–1.5 yr. Although OC concentrations had increased after 6 months, they had returned to baseline levels by 1 yr. This result is in accordance with that reported by Hashimoto et al. (37), showing that OC reaches a peak level during the second year after menopause. In a recent study by Reeve et al. (38), circulating OC concentrations increased slowly until a plateau was reached 5 yr postmenopause.

It has been estimated that BMD increases 1–3%/yr during HRT (39). In our study, no statistically significant increases could be found in hormone groups, whereas both femoral and lumbar BMDs decreased after 2.5 yr in both nonhormone groups. Our results regarding bone markers are strongly corroborated by their significant and consistent correlations with the changes in lumbar and femoral neck BMDs. In this regard, previous studies have been contradictory (24, 30, 31) as the major shortcomings of bone biochemical markers are their great intra- and interindividual variations (40). Therefore, most previous studies have tried to show the effect of treatment, which could be adequately reflected by changes in bone biochemical markers (14, 31, 40).

In conclusion, our population-based study shows that serum markers of bone formation and resorption are persistent, and their changes reflect those in BMD. Thus, they may be used to monitor the effect of HRT in healthy, early postmenopausal women. The measured bone markers may also be useful in finding women with a high rate of bone turnover after menopause. However, the association between biochemical changes and fracture rates remains to be proven.


   Acknowledgments
 
We thank Mrs. Sirkka Harle, Mrs. Irma Janhunen, and Ms. Seija Oinonen for technical help, Mrs. Pirjo Halonen for advice concerning statistical methods, and Dr. Nick Bolton for checking the language of the manuscript.


   Footnotes
 
1 This work was supported by Leiras Ltd., Schering Ltd., the Finnish Gynecological Society and the Pharmacal Foundation. Back

Received January 16, 1997.

Revised May 2, 1997.

Accepted May 14, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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