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Bone gain after calcium enriched soy milk, food supplement, and lifestyle changes in women with low bone mass: a pilot project in course form

Authors:
  • optimum nutrition, denmark
Eur J Nutr (2004) 43 :246 –257
DOI 10.1007/s00394-004-0497-8 ORIGINAL CONTRIBUTION
Eva Lydeking-Olsen
Jens-Erik Beck-Jensen
Kenneth D. R.Setchell
Trine Holm-Jensen
Soymilk or progesterone
for prevention of bone loss
A 2 year randomized, placebo-controlled trial
EJN 497
Summary Background Given
concerns over the use of hormone
replacement therapy (HRT),
women are seeking natural alterna-
tives to cope with the symptoms
and effects of menopause. The bone
sparing effects of soy protein and
its isoflavones is well established in
animal studies, while 5 previous hu-
man studies on soy and bone have
Received: 8 October 2003
Accepted: 18 February 2004
Published online: 14 April 2004
E. Lydeking-Olsen () · T. Holm-Jensen
Institute for Optimum Nutrition
Teg l g aardstraede 4, 1st.floor
1452 Copenhagen K, Denmark
Tel.: +45-33324486
Fax: +45-33324481
E-Mail: forskning@optinut.dk
J.-E. Beck-Jensen
The Osteoporosis and Metabolic Bone Unit
Dept. of Endocr inology
Copenhagen University Hospital
Hvidovre, Denmark
K. D. R. Setchell
Clinical Mass Spectrometry
Children’s Hospital and Medical Center
Cincinatti (OH), USA
yielded variable outcomes due in
part to their short duration of
study. Progesterone has been sug-
gested as a bone-trophic hormone,
but the effect of long-term, low dose
transdermal progesterone is un-
known.Aim The aim of the study
was to compare for the first time the
long-term effects of soymilk, with
or without isoflavones with natural
transdermal progesterone, or the
combination, on bone mineral den-
sity in the lumbar spine and hip.
Methods Postmenopausal,Cau-
casian women with established os-
teoporosis or at least 3 risk-factors
for osteoporosis, were randomly as-
signed,double-blind to one of four
treatment-groups: soymilk contain-
ing isoflavones (soy+, n= 23),trans-
dermal progesterone (TPD+,
n=22), or the combination of soy+
and TDP+, (n= 22) or placebo
(isoflavone-poor soymilk, soy÷ and
progesterone-free-cream TDP÷,
n=22).All subjects received com-
parable intakes of calcium, minerals
and vitamins. Bone mineral content
(BMC) and density (BMD) were
measured in lumbar spine and hip
by using dual-energy X-ray absorp-
tiometry (DEXA) at baseline and
after 2 years. Findings The percent-
age change in lumbar spine BMD
and BMC respectively,did not differ
from zero in the soy+ group
(+1.1 %,+2.0 %) and TDP+ group
(÷1.1 %,+0.4 %) but significant
bone loss occurred in the control
group (÷4.2%, ÷4.3 %) and the
combined treatment group (÷2.8 %,
÷2.4 %).No significant changes oc-
curred for femoral neck BMD or
BMC.Interpretation Daily intake of
two glasses of soymilk containing
76 mg isoflavones prevents lumbar
spine bone loss in postmenopausal
women. Transdermal progesterone
had bone-sparing effects but when
combined with soy milk a negative
interaction between the two treat-
ments occurs resulting in bone-loss
to a greater extent than either treat-
ment alone.
Key words soy – isoflavones –
genistein – daidzein – equol –
transdermal progesterone – bone
density – bone conservation –
postmenopausal women
Introduction
Current therapies for prevention and treating osteo-
porosis include estrogen and hormone replacement
therapy (ERT and HRT), the selective estrogen receptor
modulator (SERM,i.e. raloxifene),bisphosphonates and
calcitonin.
The increased risk of breast and uterine cancer with
long-term ERT and HRT [1] and unwanted side effects
account for poor compliance and loss of treatment effect
[2, 3].Recent results from the Women’s Health Intiative
E. Lydeking-Olsen et al. 247
Soy or progesterone for bone conservation
showing a lack of cardioprotective effect of HRT [4, 5]
should highten interest in natural alternatives to acute
and chronic menopausal symptomatology.
Soy protein and its constituent isoflavones have be-
come a focus of much interest for their potential bone-
sparing effects. Several lines of circumstantial evidence
warrant clinical trials of soy foods as a natural alterna-
tive to HRT. First, the non-steroidal isoflavones which
bind selectively to estrogen receptors [6, 7] have been
found in vitro to stimulate osteoblasts and inhibit os-
teoclast activity,effects that are consistent with reduced
bone turnover [8–11]. Second, animal studies using ro-
dent models of postmenopausal bone loss have consis-
tently found bone-sparing effects of soy protein or
isoflavones [12].Third, short-term human studies have
provided tantalizing evidence that isoflavones can re-
duce bone loss as measured by surrogate markers of
bone turnover and changes in bone mineral density in
postmenopausal women [13–17].And finally, epidemio-
logical studies from Japan and China have found that
postmenopausal women with the highest intake of
isoflavone-rich soy foods have the highest BMD in the
lumbar spine compared with women with low intakes.
Somekawa etal. showed that women in the two
+50mg/d groups of isoflavone-intake had a 7–9 %
higher lumbar spine BMD – both in the early and late
postmenopausal group [18],whereas Mei found a 6.4%
difference in lumbar spine BDM and 8.4% difference in
wards triangle BMD between the highest and lowest ter-
tile of isoflavone intake [19].
Based on these observations a number of dietary in-
tervention studies have been performed to determine
the effectiveness of soy protein in preventing bone loss in
postmenopausal women [13–17, 20, 21]. These have
yielded variable outcomes but all have been of a duration
of no more than 12 months,making it difficult to observe
significant changes in bone mineral density given the
slow rate of bone turnover.A potential confounder in hu-
man isoflavone research is the heterogeneity in bioavail-
ability and metabolism of the isoflavones – thus the
equol-status clearly separates subjects in two distinct
groups who are likely to have markedly different re-
sponses to soy food feeding, 30–50% being equol-pro-
ducers in various populations [37]. Equol is a non-
steroidal metabolite of one of the major isoflavones,
daidzein, produced by the gut bacterial flora [37]. It is
much less researched than the other main isoflavone
genistein, due to not being available in purified form for
in vitro and animal research. We now report the first
long-term double-blinded randomized controlled study
of 2-year duration in which soy protein with and without
isoflavones was compared for its effects on bone by mea-
surement of bone mineral density and content as the pri-
mary end-points.A second and increasingly popular al-
ternative therapy used by postmenopausal women has
been the use of natural transdermal progesterone
creams.Progesterone is considered to be a bone-trophic
agent [22–24]. An open longitudinal case-series found
that progesterone added to a conventional treatment
program of healthy lifestyle and low-dose ERT (Pre-
marin) actually increased bone mineral density (BMD)
by up to 22.4% over 3 years [25].However,the only con-
trolled trial of transdermal progesterone showed no ef-
fect on BMD after one year but it did improve vasomotor
symptoms [26].
If isoflavone-rich soy foods and/or progesterone pre-
vent bone loss then these interventions could serve as al-
ternative or adjuvant intervention during menopause
and in older age for women who are poor candidates for
or choose not to receive traditional HRT. Soy foods could
also serve as a cheap and available non-pharmacologi-
cal intervention in parts of the world where pharmaco-
logical treatment is less available due to socio-economic
or other factors.
The purpose of this study was to examine the hy-
pothesized bone-sparing effect of isoflavone-rich
soymilk, transdermal progesterone or both, on bone
mineral density (BMD) and bone mineral content
(BMC) of the lumbar spine and hip in post-menopausal
women. Biochemical markers of bone formation
[serum-type I procollagen-N-terminal-peptide (PINP)]
and bone resorption [serum type I C-terminal telopep-
tide (ICTP)] were also measured.
Subjects and methods
Subjects
Subjects were recruited through newspaper advertise-
ments and feature articles in local newspapers and mag-
azines. Potential subjects were screened initially via tele-
phone to ensure that they were at least one year
postmenopausal, with a maximum age of 75 years, had
not received any bone-active medication for at least 2
years previously and had at least 3 risk-criteria for os-
teoporosis: early menopause <45 years of age, low-en-
ergy bone-fractures with typical localization, smoking,
low body weight (BMI <19 kg/m2), low physical activity
level,low intake of calcium/vitamin D,heritage of osteo-
porosis, previous systemic steroid treatment ≥ 6 month
duration or had been diagnosed as osteoporotic by
DEXA scanning or due to fractures.
Exclusion criteria were drug or alcohol addiction,
malignant disease, immobility, current steroid treat-
ment, osteomalacia, diabetes, unstable thyroid disease,
severe osteoarthritis in lumbar spine/hip, chronic in-
flammatory diseases, active liver and kidney disease.
Of 507 responders, 290 passed the telephone screen
and 177 (61%) were assessed for eligibility. Of these 57
were not-eligible and 13 withdrew before randomiza-
tion (11 for social reasons and 2 for other health rea-
248 European Journal of Nutrition (2004) Vol. 43, Number 4
© Steinkopff Verlag 2004
sons), leaving 107 for inclusion. Before baseline testing
an information meeting was conducted at which the
subjects were assured that participation was completely
voluntary and after oral and written information, signed
a consent form.
The women came to the institute every 3 months the
first year and every 6 months (or as needed) the second
year for testing, teaching,group sessions and supply re-
plenishment.
Of 107 women entered in the study 7 withdrew within
3 months (3 for disliking the soymilk, 4 for reasons un-
related to the study), six developed intolerance to
soymilk, two developed intolerance to the skin cream
and 3 were self-reported non-compliant due to aversion
to the soy milk, leaving 89 for analysis. The protocol and
consent forms were approved by the Regional Ethical
Committee for Copenhagen and Frederiksberg, Den-
mark (J.no 02–131/1997), The Danish Medicines Agency
(J.no. 5312–240–1997) and The Danish Data Protection
Agency (J.no. 1998–1200–113).
Research design and treatment
Participants were randomly assigned double blind to
two years intervention in the following groups:
Isoflavone-rich soymilk (soy+, n = 23), transdermal
progesterone (TPD+, n =22), combined (soy+, TDP+,
n=22) or placebo (isoflavone-poor soymilk, soy÷ and
progesterone-free cream TDP÷, n= 22). Subjects were
supplied with a total of 500 ml of soymilk/d supplying
17.5 g protein/d as divided servings – one glass in the
morning and evening, respectively, with an aglycone
isoflavone content of 76.0mg/day for 500ml.The agly-
cone isoflavones are the unconjugated absorbable forms
of the isoflavones.
The soymilk was produced in Belgium from selected
isoflavone-rich soybeans (soy+) or alcohol washed soy
concentrate providing only 1.0mg isoflavones/day
(soy÷). The soymilk provided ~ 0.8 MJ (200 Kcal) and
was shelf stable for 1 year. Subjects were instructed to
incorporate the soymilk in their meals while cutting
down on cow’s milk products. Suggestions for using
soymilk in different ways were provided in written form
and 99 % of subjects participated in group meetings and
cooking classes every 3 months in the first year to
ensure long-term compliance. The soymilk was en-
riched with calcium to a level of ~ 150mg/100 ml and
all subjects were provided a commercial non-prescrip-
tion food supplement (Osforte®) containing 680 mg cal-
cium (citrate and carbonate), 300mg magnesium
(aminochelate), 20 mg silicium (sodium metasilicate),
15mg zinc (aminochelate), 6mg manganese
(aminochelate), 3 mg boron (proteinate), 2mg copper
(aminochelate), 200mg Vit. C, 40mg Pyridoxin, 200 iu
Vit. D3 and 1 mg Vit. K1.
The total calcium-intake from diet, soymilk and food
supplement was in the 1,500mg-range.
Skin cream (either containing progesterone or not)
was applied cyclically at ~ 30 g (1oz)/3 week followed by
a one week break, thus supplying 540mg micronized
progesterone/3 week cycle equivalent to 25.7mg/d. Skin
cream was applied to skin surfaces like the inner thigh,
inner arms or face,according to manufacturer’s instruc-
tion.Each batch of soymilk and skin cream was analyzed
to ensure their contents of isoflavones and progesterone,
respectively. Study flow is summarized in Fig.1.
Fig. 1 Flow diagram
E. Lydeking-Olsen et al. 249
Soy or progesterone for bone conservation
Data collection and measurements
Individual information on health,medical, reproductive
and menopausal history, physical activity and lifestyle
was obtained for all subjects by interview,aided by semi-
quantitative questionnaires.
The questionnaire included information on bone
health, family history and heritage, weight and dieting
history,environmental exposures and general health.
Estrogen exposure (in years) was calculated for each
subject by subtracting her age at menarche from her age
at last menstrual cycle. Lifetime use of cigarettes was ex-
pressed as pack-years.
Subjects underwent a normal general physical exam-
ination with weight (in underwear) and height mea-
sured using a precision scale (Seca) to the nearest 0.1 kg
and 0.5 cm respectively.Body mass index (BMI) was cal-
culated as weight in kilograms divided by height in m2
(kg/m2).
Nutrition and lifestyle history was used to elicit usual
intake patterns of milk products, grains, fish, caffeine-
containing drinks and food-items, alcohol,special diets,
fats and oils as well as vitamin and mineral supplement
use.
Seven-day weighed food records were obtained at
baseline, one and two years, after thorough instruction
and demonstration of the supplied electronic scale
(Soehnle Domino, limits of 2000 g). Weights were con-
trolled for correct measurement by weighing items of
known weight in each round of food recording. Food
recording covered all seven days of the week and sub-
jects provided food-labels and recipes of mixed dishes
to ensure correct coding of items.
Diet records are considered the best method for as-
sessing usual dietary intake [27] and were used to assess
typical mean intakes of energy,macronutrients, dietary
fiber,alcohol, caffeine,vitamins, minerals and fatty acids
at baseline and throughout the study. Food records were
analyzed by 3rd year nutrition students and B.Sc in nu-
trition staff members, using DANKOST 3000 computer-
ized nutrient database program (Danish Catering Cen-
ter, Inc, Denmark) based on the national Danish
composition of foods table (The Veterinary and Food
Administration, fourth edition,1996).
These analyses did not include vitamin and mineral
supplements at baseline or those we provided the sub-
jects with. Information on nutrient content of vitamin
and mineral supplements was obtained from manufac-
turers and/or the VFA administration database and
recorded for each subject. Total intakes reported are di-
etary plus supplemented levels.
Physical activity level was recorded using both life-
time and actual levels of work and leisure activity, ac-
cording to The Nordic Nutrition Recommendation [28]
– guidelines to a physical activity level (PAL) used for
calculating an estimate of dietary energy need based on
basal metabolic rate (BMR) calculations with the appro-
priate age, weight and PAL for each subject (NNR 1996).
Bone mineral density was measured at baseline and
at 2 years by dual x-ray absorptiometry (Norland xR 26
Mark II), and expressed as areal density (gHA/cm2) or
content (g HA).The lumbar spine,L2-L4and the femoral
neck were assessed by the same two trained research as-
sistants. Equipment was calibrated each week with a
phantom and the laboratory’s long-term within-subject
in vivo reproducibility CVs of lumbar spine BMD and
BMC are 2.0% and 1.8 %, respectively. The correspond-
ing CVs for the hip are 3.5% and 3.3 %.No attempt were
made to drop subjects after their DEXA results was
known as we also collected cross-sectional data on psy-
chological profiles, pH measurements in the stomach
and duodenum,heavy metal profiles in hair and homo-
cysteine values.
Fasting blood samples were collected at baseline and
1 and 2 years to measure safety parameters, lipids,
isoflavones and bone markers.
Safety parameters performed were routine hemato-
logical counts, serum-liver and -kidney biochemistry
(ALAT,ASAT,alkaline phosphatase, calcium, potassium,
sodium, uric acid, creatinine), thyroid stimulating hor-
mone and se-cobalamin.All analyses were performed in
a GLP-certified laboratory (Nova Medical Medi-Lab,
Copenhagen, Denmark).
Serum and plasma for bone-marker and isoflavone
analysis were stored frozen at –20°C for subsequent
analysis. Bone-markers measured were type-I C-termi-
nal telopeptide (ICTP) and type-I procollagen N-termi-
nal peptide (PINP) – both with radioimmunoassay kits
(Orion Diagnostica, Finland) according to the manufac-
turer’s instruction. The intra-assay variability was 4.8%
for ICTP and 8.6% for PINP and the corresponding in-
ter-assay variability was 5.7% and 5.1%.
Analysis of soymilk for isoflavones by HPLC
Isoflavones and their glycosidic conjugates were ex-
tracted from soymilk (5mL) by refluxing for 1 hour in
80% methanol (50 mL). After filtering the sample
through Whatman No.1 filter paper, the aqueous
methanolic phase was then accurately made up to
100 mL in a graduated volumetric.An internal standard,
equilenin (60µg), was added to three replicate 1.0mL
(1/100th) portions of this extract which was then taken
to dryness under a stream of nitrogen, and resuspended
in 10mL of 0.5M sodium acetate buffer (pH 4.5).
Isoflavone glycosides were hydrolyzed at 37 °C overnight
by addition of β-glucosidases present in Helix pomatia
digestive juice (0.1mL; Sigma Chemical Company, St.
Louis, MO). Isoflavones, now converted to their agly-
cones, were then extracted by passage of the hydrolysate
through a precharged C18-Bond Elut solid-phase car-
tridge and recovered by elution with methanol (5 mL).
250 European Journal of Nutrition (2004) Vol. 43, Number 4
© Steinkopff Verlag 2004
The metanolic extract was then taken to dryness under
a stream of nitrogen gas and resuspended in the HPLC
mobile phase for analysis by HPLC (100µL). The condi-
tions for separation of individual isoflavones have been
described in detail previously [29]. The sample size in-
jected was 10µL, flow rate 1.0 mL/min and the UV ab-
sorbance was monitored at 260nm using a Waters 2487
UV detector. Separation of the individual isoflavones
was accomplished on a 250 4.6 mm ODS (C18) reversed
phase HPLC column (Keystone Scientific, Bellefone PA)
and the isocratic mobile phase consisted of
methanol:ammonium acetate (75/25, v/v). Quantifica-
tion of total daidzein, genistein and glycitein as their re-
spective aglycones was achieved by compairing the peak
area responses given by these values against calibration
standards of each isoflavone.
Determination of isoflavones in plasma and urine
by gas chromatography-mass spectrometry
The concentration of daidzein, genistein and equol was
measured by GC-MS using stable isotopically labeled in-
ternal standards. These internal standards were added
to the plasma sample prior to its extraction and work-
up.Total and individual isoflavones were determined af-
ter extraction and enzymatic hydrolysis of the conju-
gates with a combined sulfatase and glucuronidase
enzyme preparation. The plasma (0.5mL) was equili-
brated with 50ng of the internal standards
[13C]daidzein, and [13C]genistein, and [13C]equol, di-
luted with 10 vol of 0.5M triethylamine sulfate (pH 5.0)
and heated to 64 °C before passage through a pre-wetted
solid-phase C18-Bond Elut cartridge. The solid-phase
cartridge was then washed with distilled water (10mL)
and isoflavones and their conjugates were recovered by
elution with methanol (5 mL).The methanol extract was
evaporated to dryness under nitrogen, reconstituted in
10 mL of 0.5M acetate buffer (pH 4.5), and hydrolyzed at
37 °C overnight with a solution of 10,000 Fishman Units
of a mixed β-glucuronidase/sulfatase (Helix pomatia,
Sigma Chemicals Inc.) in 0.5M sodium acetate buffer,
pH 4.5, that had been previously filtered through a car-
tridge of C18-Bond Elut to remove naturally occurring
isoflavones present in this enzyme preparation. After
hydrolysis, isoflavones were isolated by solid-phase ex-
traction on a C18-Bond Elut cartridge as described
above. The sample was taken to dryness under a stream
of nitrogen gas and isoflavones converted to their tert-
butyldimethylsilyl (tBDMS) ether derivatives for analy-
sis by GC-MS with selected ion monitoring. tBDMS
ethers were prepared by addition of acetonitrile
(100µL) and N-methyl-N-t-butyldimethylsilyltri-
flouroacetamide in 1% t-butylmethylchlorosilane
(100µL) and the sample was heated at 65°C for 2h. The
reagents were removed by evaporation in a stream of ni-
trogen and the derivates dissolved in hexane (100µL).
Isoflavone tBDMS ethers were separated and quantified
by gas chromatography-mass spectrometry with se-
lected ion monitoring.Chromatographic separation was
achieved on a DB-1 fused silica capillary column (30m
0.25mm i. d., 0.25µ film thickness; j & W Scientific
Inc., Folsom CA) using helium as the carrier gas (flow-
rate approx. 2mL/min) and with a temperature program
from 260–310°C with increments of 10°C/min. Selected
ion monitoring GC-MS of specific and characteristic
ions in the electron ionization (70eV) spectra of the
tBDMS ether derivatives of each isoflavone permitted
highly sensitive and specific quantification. The follow-
ing ions were monitored: m/z 425 (daidzein), m/z 426
([13C]daidzein), m/z 470 (equol), m/z 471 ([13C]equol),
m/z 555 (genistein), and m/z 556 ([13C]genistein). The
individual isoflavones were quantified by comparing the
peak area in the specific ion channels at the correct
retention time determined from authentic compounds,
with the peak area response for the internal standard.
This area ratio was then interpolated against calibration
curves constructed for known amounts (0–200ng) of
the individual isoflavones. Concentrations were ex-
pressed as ng/mL or µmol/L for individual plasma
isoflavones
Statistical analysis and randomization
Power calculations were based on an alpha-level of 0.05
and a beta-level (risk of type II error) of 15%, based on
an expected overall difference between groups of 5%,
equivalent to 0.5 SD with a detection limit of 1 %. The
overall power of 85 % required approx. 22 subjects in
each group and to allow for drop-outs the protocol
called for 25 in each arm.
Statistical analysis was performed with PC SAS, ver-
sion 8.2 (SAS Institute,Inc. Cary,NC, USA). Descriptive
statistics include means for normally distributed data
(i.e. age, BMI, energy intake, menopausal age, dietary
energy,percents of protein,carbohydrates and fats),me-
dians for data not normally distributed (i. e.calcium and
vit D intake, exercise,ALAT, cobalamin) and frequencies
(bone, reproductive and smoking history).
Analysis of variance (ANOVA) was used to determine
differences between groups. To determine whether
changes over the intervention period were different
from zero, paired t-tests were performed. Changes in
bone markers were analyzed using a multiple linear re-
gression model with log transformation of not-nor-
mally distributed data.An alpha-level of 5% was used in
all statistical tests. Randomization was done centrally,
generated by a table of random numbers and subjects
entered consecutively at recruitment. The randomiza-
tion code was not broken until final statistical analysis
had been performed.
E. Lydeking-Olsen et al. 251
Soy or progesterone for bone conservation
Results
Subjects
The mean age of these postmenopausal women was 58.2
y, with a mean time since menopause of 10.9 y.Based on
lumbar spine T-scores at baseline, comparing the bone
mass to that of normal, young women, 14 subjects were
classified as being osteoporotic (T-score < ÷2.5 SD,
equivalent to a bone mass ~ 25% below normal) and of
these 13 completed the study. 58 were osteopenic (T-
score between ÷ 2.49 and ÷ 1.0 SD) and of these 52 com-
pleted the study. 23 subjects had normal values (T-score
between ÷ 1.0 and +1.0 SD) and 19 completed the study.
Five had T-scores above 1.0 SD and all completed the
study. 25 subjects reported of earlier, low-energy frac-
tures with typical osteoporotic localization but only 7
were previously diagnosed by their physician as being
osteoporotic.Three had tried bisphosphonate treatment
years ago, but quit either due to side effects (n= 1) or
lack of effect (n = 2).25 had more than 2 years previously
received HRT, usually during menopausal transition,but
did not wish to continue.
Four of the women were taking medication for hy-
pothyroidism at baseline (two in soy+ group, one each
in the control and combined groups) but they were well
controlled and euthyroid. Fourteen cases of cobalamin
deficiency were found and treated at baseline (n= 12)
and at one year (n= 2),respectively. Three of these were
non-completers, four were in the TDP+, three in the
soy+, two in the combined and one in the control
groups, respectively. The treatment of cobalamin defi-
ciency did not affect the outcomes in the trial.Four were
treated for hypertension with diuretics at baseline (two
in the control and one each in the combined and TDP+
groups) and this treatment was maintained during the
study. Dietary and lifestyle assessment showed a mean
energy intake of 7.5 MJ/d (SD 1.8) with protein, fat and
carbohydrate energy percentages of 14.7 %, 31.9% and
49.6% respectively,leaving 3.8% of the energy-intake to
alcohol. Median alcohol intake was 3 servings per week
(range 0–23). None of the groups differed from these
overall values, nor did they change significantly over
time.
Daily caffeine intake was 290.0mg/d (SD 209.6).
There was a small decrease of caffeine intake over time,
to 258.4 mg/d representing a nonsignificant 11% differ-
ence over two years, with no significant group differ-
ences.
Physical activity levels were modest with median PAL
at 1.5 (range 1.4–1.8) representing a generally sedentary
lifestyle with limited recreational physical activity, al-
though a subgroup bicycled to work and 1–2 subjects in
each group had higher levels of leisure time exercise.
Physical activity levels did not change over time.
There were 48.3% past smokers and 20.2% [n = 18]
current smokers, reflecting the high prevalence of
smoking among Danish women. Three of the smokers
quit smoking for a period of less than 6 months during
the study, but began smoking again; thus, the overall
smoking status was the same in the end as at baseline.
During the study there was a non-significant weight
gain of 1.4–1.9% of baseline weight (0.9–1.2 kg) in the
soy+, combined and control groups, whereas the TDP+
group did not gain weight.
Additional baseline characteristics are shown in
Tabl e 1.
Compliance
Compliance was controlled by counting of left-over pills
(food supplement),weighing of left-over skin cream and
monthly diaries for the use of soymilk (liter/month) as
well as measurements of plasma isoflavone levels.
Overall compliance with the intervention was in the
range of 96.3–97.9% (SD 3.3–8.6) for the food supple-
ment; 95.0–98.7% (SD 6.0–12.6) for the skin cream and
83.9–98.7% (SD 9.4–13.7) for the soymilk. There were
no differences between the groups regarding compli-
ance. Poor compliers, defined as reporting 25–75%
compliance are included in the analysis and represent 2,
4, 3 and 3 subjects in the control, combined, TDP+ and
soy+ groups,respectively.
Median plasma total isoflavone levels at baseline
were 18.8ng/ml (range 9.3–388.8 ng/ml) in the control
group and 39.2ng/ml (range 9.7–411.1 ng/ml) in the
soy+ group due to the presence of 2 vs.4 habitual
soymilk users in the two groups (p= 0.03). Total
isoflavone level remained low in the control group, me-
dian 24.9ng/ml (range 15.9–225.7 ng/ml) and increased
seven to tenfold in the soy+ group to a median level of
281.9ng/ml (range 63.0–1252.7 ng/ml), p= 0.0001,
demonstrating good compliance.
Tolerance and side effects
Participants were requested to call if any undesired
events occurred and intervention was stopped for a few
days until complaints had cleared and then restarted
one-by-one to clarify if the complaint was caused by the
intervention. If so, the reaction was reproduced twice
and a decision about cessation made.For the food sup-
plement,11 % reported mild digestive upsets if taken on
an empty stomach, but all tolerated the supplement if
taken with food. One subject reduced the dosage due to
loose stools.
For the skin cream 22% experienced mild side ef-
fects, not leading to cessation: skin irritation if used in
face, but tolerated elsewhere (11 %),breast tenderness of
a few days’duration at the beginning of the study (9%),
252 European Journal of Nutrition (2004) Vol. 43, Number 4
© Steinkopff Verlag 2004
spotting of a few days’ duration or a single menstrual
bleeding (6%, evenly distributed among control and ac-
tive groups) and mild hot flushes (3%). Two subjects
stopped using the cream,one had severe local skin irri-
tation, the other an aversion to the use, but no physical
symptoms.For the soymilk, 29% experienced mild and
temporary side effects, not leading to cessation: mild di-
gestive trouble (nausea,bloating, flatulence 23%), unde-
sired weight gain (fluid retention,9%), mild throat irri-
tation (4%), mild hot flushes (2%) and temporary joint
pain (1%).
Six subjects (6%) had side effects leading to cessa-
tion:2 subjects had severe digestive trouble with nausea,
flatulence and diarrhea and 4 subjects had both diges-
tive trouble and generalized symptoms of tiredness,
fluid retention, feeling malaise, sleep disturbances,
shortness of breath and joint pain. These reactions oc-
curred 2–11 months into the study and cleared within
days after stopping the soymilk intake.They were evenly
distributed across the groups and cannot be attributed
to the isoflavone level, but demonstrate intolerance to
soy protein.
Hematological, liver and kidney parameters were
normal and remained so throughout the study as well as
TSH remained normal and unchanged with no group
differences.We could not identify a vulnerable subgroup
with respect to thyroid function, based on TSH screen-
ing, related to soy in this sample of women.
Bone measurement response to treatment
Lumbar spine
Mean percentage change in lumbar spine BMD and BMC
did not decline in the soy+ or the TDP+ group;however
significant losses occurred in the combined group and
the control group, shown in Fig. 2. Descriptive results for
BMD and BMC are shown in Table 2.
Absolute values for bone measurements at baseline
were not significantly different between the four inter-
Table 1 Baseline characteristics of postmenopausal women, n = 891
Measure/group 1. soy+ (n = 23) 2. TDP+ (n = 22) 3. Combined (n = 22) 4. Control (n = 22)
Age (y)257.8± 8.4 59.4 ± 7.0 59 ± 7.4 56.3± 6.7
Weight (kg)266.2± 10.3 63.9 ± 10.8 61.3 ± 11.1 64.8± 9.4
BMI (kg/m2)224.0± 4.2 24.4 ± 3.9 23.3 ± 4.2 23.7± 3.5
Time since menopause (y)36.0 (1–29) 9.5 (1–26) 10.5 (1–30) 6.0 (1–26)
Diet and lifestyle:
Percentage past smokers [n] 47.8 [11] 40.9 [9] 54.5 [12] 50 [11]
Percentage current smokers [n] 21.7 [5] 9.1 [2] 36 [8] 13.6 [3]
Mean pack-y, current smokers 27.3 (18–55) 18 (16–20) 13.6 (3–21.6) 20.4 (2–40)
Energy intake (MJ/d) 7.3± 1.6 6.9± 1.3 7.4± 1.6 8.3± 2.1
Total vit. D (µg)3, 5 7.27 (2.75–17.83) 7.59 (4.20–20.88) 9.84 (1.53–31.15) 6.64 (0.88–25.11)
Exercise (h/we)32 (0–11.5) 1.9 (0–9) 2 (0–10) 2 (0–14)
Serum variables:
TSH, mIU/l31.8 (0.63–7.1) 1.3 (0.38–6) 1.7 (0.33–5.8) 1.5 (0.63–12)
ALAT (U/l)323.0 (9–35) 21.5 (11–59) 27.5 (14–62) 28.0 (8–73)
ASAT (U/l)222.0± 4.4 22.8 ± 6.5 24.3 ± 6.6 23.6± 5.8
Alkaline Phosphatase3151.0 (86–238) 180.0 (132–306) 179.0 (131–260) 185.5 (89–579)
se-creatinine (µmol/l)281.0± 8.9 81.0 ± 8.7 81.3 ± 7.3 79.9± 8.3
1There were no significant differences among the groups for these characteristics
2Values are mean ± SD
3Medians (range)
4Dietary intake calculated from 7d weighed record by Dankost 2000
5From diet and supplements
Fig. 2 Long-term effect of soymilk and progesterone on bone in postmenopausal
women, n = 89, 2y, by DEXA. * P = 0.05; ** P = 0.01
E. Lydeking-Olsen et al. 253
Soy or progesterone for bone conservation
vention groups, but absolute BMD and BMC changed
significantly in the control group (p= 0.006 for BMD
and p= 0.005 for BMC) and in the combined group
(p =0.003 for BMD and p =0.03 for BMC) and remained
unchanged in the soy+ and TDP+ groups.
Results of ANOVA indicated that treatment had a sig-
nificant effect on percentage change in BMD (p=0.04)
and BMC (p= 0.03).
Paired t-test for percentage change showed a signifi-
cant loss in the control group (–4.2%, p= 0.01 for BMD
and –4.3%, p= 0.01 for BMC) and in the combined
group (–2.8%, p= 0.01 for BMD and –2.4%, p= 0.05 for
BMC) whereas the soy+ and TDP+ groups remained un-
changed at +1.1% and +2.0% in the soy+ group and
–1.1% and +0.4 % in the TDP+ group for BMD and
BMC, respectively.
The difference between the groups was non-signifi-
cant comparing control and combined groups and sig-
nificantly different comparing control and soy+
(p= 0.009 for BMD and p=0.006 for BMC). For control
vs. TDP+ there was a significant difference for BMC per-
centage change (p =0.03)
Response in relation to specific serum isoflavone lev-
els showed that equol-producer status was associated
with a better response, which did not reach statistical
significance due to low numbers.
Subgroup analysis, using a cut off level of 10ng/ml
for plasma-equol divided the soy+ group into 10 Eq+
subjects and 12 Eq– subjects (one missing value)
with mean Equol levels of 44.0 ng/ml (SD 25.20)
and 3.2ng/ml (SD 0.74) (p <0.0001) and corresponding
responses of +2.4 % and +2.8% changes for BMD and
BMC in the Eq+ group,compared to the Eq– group just
maintaining bone mass at +0.6% and +0.3% respec-
tively.
Due to the small numbers,results are not statistically
significant but the result indicates that equol could be
the major isoflavone-metabolite responsible for a clini-
cally important effect in bone.
Levels of the other isoflavones daidzein, genistein
and total isoflavones did not differ significantly between
the Eq+ and Eq– groups.
For TDP+ a subgroup of 4 women had bone gains
ranging 4.3–12.9% for BMD indicating that there might
exist a subgroup with special sensitivity to progesterone.
Hip
There were no significant differences between groups
for BMD and BMC measurements at the femoral neck,
wards triangle or trochanter at baseline and no signifi-
cant changes occurred with treatment. All four groups
had minimal changes in femoral neck BMD with per-
centage changes 0.2% and –1.3 % for control and com-
bined groups; –0.9% and –0.5% for soy+ and TDP+
groups, indicating that a factor not differing between
groups: Soy protein (regardless of isoflavone level) or
the food supplement had a stabilizing effect.
Biochemical markers of bone formation
Values for serum PINP and ICTP were not normally dis-
tributed and were thus log-transformed for the statisti-
cal analysis. Baseline and posttreatment were not signif-
icantly different between the groups for PINP, ICTP or
the PINP/ICTP ratio.
A multiple regression model was used to determine
the effect of the bone markers on the bone measurement
outcome in relation to treatment and known explana-
tory variables for bone status such as age,BMI, estrogen
exposure time and baseline bone values.
There was a non-significant percentage change in the
formation marker, PINP,in the two groups that lost bone
Table 2 Lumbar spine bone mineral density BMD and content at baseline and post-treatment in postmenopausal women1
Measure/ Baseline Post treatment
treatment group
Minimum X
±SD maximum minimum X
±SD maximum
BMD (g/cm2)
Control 0.528 0.865± 0.190 1.380 0.404 0.835±0.212a1.416
Combined 0.614 0.821 ± 0.153 1.205 0.627 0.796± 0.138b1.101
TDP+ 0.507 0.868±0.153 1.181 0.532 0.855± 0.144 1.188
Soy+ 0.478 0.925±0.260 1.475 0.500 0.933±0.265 1.504
BMC (g)
Control 20.030 39.515± 8.865 56.290 15.110 38.031± 9.549c58.060
Combined 25.960 36.369± 8.319 58.480 25.130 35.330± 7.618d53.790
TDP+ 21.780 37.657± 7.100 54.000 23.200 37.569± 6.622 52.760
Soy+ 21.760 42.546±11.202 69.820 23.410 42.794 ±10.917 69.420
1Control isoflavone free soymilk n = 22
soy+ isoflavone rich soymilk group, n = 23; TDP+ transdermal progesterone cream group, n = 22; combined group combination of soy+ and TDP+, n = 22
ap=0.006; bp=0.003; cp=0.005; dp=0.03; all compared to baseline values
254 European Journal of Nutrition (2004) Vol. 43, Number 4
© Steinkopff Verlag 2004
–6.7% (control) and –11.3% (combined), whereas the
two groups that maintained bone had changes of +3.9 %
(TDP+) and –0.2% (soy+), see Fig.3. The same trend
was seen for the PINP/ICTP ratio with ÷9.0% and
÷9.5% decreases from baseline in the control and com-
bined groups and +4.5% and +0.4 % increases in the
TDP+ and soy+ groups. Only minimal changes were
seen in ICTP (±1.8%) across the groups.
Discussion
In this study we examined the hypothesized bone-spar-
ing effect of isoflavone-rich soymilk, transdermal pro-
gesterone, or the combination on bone mineral density
(BMD) and bone mineral content (BMC) of the lumbar
spine and hip in postmenopausal women. Biochemical
markers of bone formation, serum-type I procollagen-
N-terminal-peptide (PINP), and bone resorption,serum
type I C-terminal telopeptide (ICTP), were also mea-
sured.
This is the first study designed specifically to exam-
ine bone effects of soy and progesterone in post-
menopausal women over several bone-remodelling cy-
cles, showing a positive effect of soy and its isoflavones
or progesterone on bone mass. Bone remodelling is the
process by which bone is deposited, resorbed and
formed through controlled functions of osteoblasts, os-
teoclasts and their associated activation factors and co-
factors. A full, normal cycle is approx. 180 days but the
effect of intervention in one cycle is small; thus,for test-
ing clinically relevant changes,studies of at least 2 y du-
ration are warranted. Results indicated that soymilk
with isoflavones prevented bone loss in the lumbar
spine, whereas the control group had a significant loss.
Progesterone slowed bone loss and combined treatment
had a negative interaction resulting in a greater bone
loss than either treatment alone, although not as pro-
nounced as placebo.
Isoflavones found predominantly in soy products are
structurally and functionally estrogen-like substances
similar to 17-beta estradiol. Isoflavones bind weakly to
the estrogen-receptor-alpha (ER-alpha) [6,7] dominant
in uterine and breast-tissue and both genistein and
equol, but not daidzein, exerts a strong binding to the es-
trogen-receptor-beta (ER-beta) dominant in bone [7].
Animal experiments and short-term studies indicated
that soy has a positive effect on bone,both in peri- and
postmenopausal women; findings that are in agreement
with recent epidemiological studies. Animal studies
have shown that soymilk-based diets increased calcium
absorption in rats [30], and isoflavones in soy protein
isolate were shown to prevent femoral [31, 32] and ver-
tebral [32] bone loss in rats. Furthermore, soy protein
isolate increased bone formation by stimulating insulin-
like growth factor 1 messenger RNA synthesis in rats
[33] or moderately increasing bone turnover in favor of
bone formation [31–33]. In an ovariectomy-induced
boneloss rat model it has been demonstrated that
daidzein and estradiol were equally effective and better
than genistein for prevention of bone loss [34], bearing
in mind that all rats are equol producers. Mechanisms
for genistein effects on bone are suggested to involve an
increase in osteoprotogerin mRNA expression from os-
teoblasts, which in turn neutralizes the receptor-activa-
tor for NF-kB needed for osteoclast formation and bone
resorption [35]. Likewise it has been shown in a rodent
model that genistein exibits estrogenic actions in bone
and bone-marrow, but not in the uterus [36]. Similar
studies have not been performed for daidzein or equol.
In humans three short-term human studies of 6–9
months duration have examined the effect of soy protein
(40 g/d with 80–90 mg/d of isoflavones) on bone mineral
density, two of them showing a bone-sparing effect in
the lumbar spine [14, 15] in a post- and a peri-
menopausal group of women,respectively.Both used ca-
sein as the control product.
The study by Gallagher using soy as the control [20]
did not show any effect in any measuring site, whereas
the study by Clifton-Bligh (2001) using a cover-derived
isoflavone preparation showed a significant increase in
BMD in cortical bone (radius and ulna) with dosages
ranging 57–85.5 mg/d,but not 28.5mg/d [16]. The most
recent study compared HRT with pure genistein 54 mg/d
or placebo, for one year and demonstrated similar pro-
tective effects from genistein and HRT on BMD at lum-
bar spine, femoral neck and wards triangle with in-
creases of 3 and 3.6% for genistein, 3.8 and 2.4% for
HRT and –1.6 and –0.65% for placebo in the lumbar
spine and femoral neck,respectively [17]. This is the first
study done with an isolated isoflavone supplement, ad-
ditionally demonstrating both reduced excretion of re-
sorption markers (PYR and DPYR) as well as increased
serum levels of bone-specific ALP and osteocalcin. In
the HRT group findings were similar to genistein for the
Fig. 3 Bone marker changes over 2 years, % change from baseline (PINP procolla-
gen type I, N-terminal polypeptide; ICTP collagen type I, C-terminal-telopeptide)
E. Lydeking-Olsen et al. 255
Soy or progesterone for bone conservation
resorption markers, but formation markers did not in-
crease.
A variety of factors determine the presence and ac-
tivity of the so far little known gut flora bacteria re-
sponsible for the conversion of daidzein to equol: in-
testinal flora acquired from childhood, dietary factors
and antibiotic history and use, being the two most likely
contributors. Due to the small numbers, results are not
statistically significant but indicate that equol could be
the major isoflavone metabolite responsible for a clini-
cally important effect in bone.
We could, however, not identify any specific dietary
compounds or patterns related to equol production,but
Rowland found that equol-producers consumed less fat
and more carbohydrate as percentage of energy
(26± 2.3 % vs. 35± 1.6% for fat and 55± 2.9 % vs.
47± 1.7 % for carbohydrate) compared to non-equol
producers [38] and Lampe likewise showed that equol
producers consumed more carbohydrate, fiber and
plant protein than non-equol producers [39].
Compliance of our subjects was excellent as reflected
by isoflavone levels in blood and self-reported intake of
soymilk. A limitation of the study was relatively small
group sizes, large enough to show an overall effect, but
too small to have enough statistical power for the sub-
group analysis. Keeping the compliance level high in
larger studies is an educational task whose importance
should not be neglected.
The other 2y study by Vitolins examined whole body
bone-mineral density with soy and two different
isoflavone levels. There was no difference between the
groups – all 3 groups lost less than 1% over 2 years in-
dicating that soy protein itself has a calcium sparing ef-
fect, which is likely t o be m ea sur ed best in the total skele-
ton or the hip where cortical bone is dominant [21].
Earlier studies have found that women eating a vegetar-
ian diet with a sufficient calcium intake (lacto-ovo veg-
etarians, eating a mixture of plant and cow’s milk/egg
protein) had the same bone mass as omnivores up to age
50, and that the omnivores at age 80 had lost 35% corti-
cal bone, compared to the vegetarians losing only 18%
[40, 41]. The mechanism for this finding was proposed
to be the lower amount of sulphur-containing amino
acids in plant proteins,tipping the acid-alkaline balance
towards the alkaline side,thus sparing calcium loss [41].
One study found higher mid-radius BMC in vegetarians
age 60–98 y [42], whereas others have found similar
bone densities in caucasian vegetarians and omnivores,
both pre- and postmenopausal [43,44].
The role of progesterone in preventing bone loss was
proposed by Prior [22, 23] who observed that female
athletes developed spinal bone loss coincident with
anovulatory cycles that had normal estrogen levels, but
progesterone deficiency [22].She suggested that preven-
tion of osteoporosis begins with detection and treat-
ment of amenorrhea and ovulation disorders [24]. Pre-
vious data on progesterone are few and conflicting as
only a case series and one 1-year study has been pub-
lished [25, 26].
For progesterone the difference between our study
and the study by Leonetti showing no effect on bone is
that our subjects had lower bone mass at baseline, that
the progesterone was given cyclically with a one-week
break every month, thus mimicking a natural cycle bet-
ter, possibly maintaining receptor sensitivity, as well as
this being a two-year vs. a one-year study [26].
Our results indicate that progesterone can have a role
in the prevention of osteoporosis, but further studies are
needed to clarify absorption kinetics from the transder-
mal preparations, dose-response relationships and to
clarify if there is a subgroup with special responsiveness
to progesterone, or if it is the combination of two natural
hormones – Premarin and progesterone given together,
that was responsible for the bone gains reported by Lee,
rather than progesterone per se [25].
The negative interaction between soy+ and proges-
terone exits and may partly be influenced by the fact that
the combined group had the largest proportion of smok-
ers and the lowest body weight, although not signifi-
cantly different from the other groups.The negative in-
teraction persists after exclusion of the smokers and
indicates that the two interventions should not be
mixed. The negative interaction was similar in equol and
non-equol producers.No previous studies on this issue
exists.
Studies in pre-menopausal women have shown a
~45% decrease in total-cycle progesterone levels during
a 1 l/d soymilk diet [46], whereas in post-menopausal
women endogenous estrogens, thyroid, pancreatic and
adrenal hormones have been studied, but not proges-
terone [47].
The negative interaction cannot fully be explained by
existing studies; it could be the progesterone acting as
an antiestrogen on the isoflavones or the opposite – the
isoflavones lowering the circulating levels of the supple-
mented progesterone.
The minimal changes observed in the bone resorp-
tion marker, ICTP, across the groups suggests that soy
protein may carry an antiresorptive effect, whereas the
isoflavones and progesterone maintaining levels of the
bone formation marker,PINP, suggest a different mech-
anism of action than conventional antiresorptive med-
ications.
In summary the results of this study suggest that
progesterone can spare bone loss and that soy foods
with isoflavones can prevent bone loss of the lumbar
spine in postmenopausal women,who may otherwise be
expected to lose 1.5–3% of bone/y. This prevention of
bone loss, particularly if continued into old age, could
translate into a decrease in lifetime risk of osteoporosis
and a lowering of fracture rates. Further and much
larger scale studies are needed to address this issue, as
256 European Journal of Nutrition (2004) Vol. 43, Number 4
© Steinkopff Verlag 2004
well as to clarify the possible calcium sparing effect of
soy protein regardless of isoflavone level on [cortical]
bone – especially in relation to cows milk.
In the planning of future studies it is important to
examine the metabolism of the isoflavones, especially
regarding equol status and antibiotic treatment his-
tory/current use, as we are apparently dealing with two
distinct subgroups, where different clinical responses
are to be expected.
Thus,data suggest that the inclusion of soy foods at a
level providing at least 15 g protein/d could have a gen-
eral sparing effect of postmenopausal calcium loss with
the largest benefit expected to be in cortical bone, like
the whole skeleton and hip. Choosing isoflavone-rich
soy food varieties with isoflavones in the 50–90mg
range (aglycone equivalent) further adds to this by pro-
viding an endocrine effect, with the benefit targeted at
the more endocrine-sensitive trabecular bone in the
spine. This is in principle important on a general popu-
lation level and specially important for women who are
at high risk, are poor candidates to HRT, choose not to
receive it, or live in areas of the world where access to
pharmaceutical prevention is scarce or not generally
available due to socio-economic factors. Further and
larger studies are needed to address the long-term ef-
fects and possible side-effects of soy foods and their
isoflavones,especially the risk of breast and uterine can-
cer. Further studies regarding gut flora metabolism
(“bacterio-typing”) with respect to equol-producer sta-
tus as well as fracture studies are now needed.
Our findings indicate that isoflavone-rich soy foods
or progesterone prevent bone loss in the lumbar spine
and offer a natural alternative,or adjuvant intervention,
during menopause and in older age for women who are
poor candidates for traditional HRT. The relatively sim-
ple inclusion of soy foods offers a cheap and non-phar-
macological intervention for prevention of osteoporo-
sis.
Acknowledgements We wa nt to express our gratitude first of all to
the study participants, who maintained compliance and commitment
at a high level throughout the study.Also special thanks to the nutri-
tion students and staff who undertook the diet-analysis and coding:
Mette Munkholm,Jeanette Joergensen,Karna Riis Larsen and Louise
Fisker as well as the research assistants conducting the bone scans:
Ulla Falktoft and Vibeke Hellesen and Mia Damhus,who undertook
randomization. Soy milk and placebo were provided by ALPRO,Bel-
gium, Proderma® and placebo by Phillips Nutritionals, Nevada, USA
and Osforte® by Genese A/S, Denmark. The study was supported by
grants from The Danish Ministry of Health, Fondation Idella, Glunz
and Jensen Foundation,Civilingenieur Frode V. Nyegaard and Wife’s
Foundation, Director E. Danielsen and Wife Foundation and IMK
Foundation. The sponsors and the suppliers of the study had no role
in the study design, data collection,data analysis, data interpretation
or writing the report.
Contributors
Eva Lydeki ng-Olsen wrote the protocol and report with contributions
from KDR Setchell and J-E Beck Jensen. E Lydeking-Olsen and T
Holm-Jensen investigated subjects, J-E Beck Jensen’s group con-
ducted bone scans and bone marker analysis. E Lydeking-Olsen and
J-E Beck Jensen performed statistical analyses. KDR Setchell analyzed
isoflavone levels.
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Objective: To determine effectiveness of transdermal progesterone cream for controlling vasomotor symptoms and preventing postmenopausal bone loss. Methods: We randomly assigned 102 healthy women within 5 years of menopause to transdermal progesterone cream or placebo. Study subjects and investigators were masked until data analysis was completed. An initial evaluation included complete history, physical examination, bone mineral density determination, and serum studies (TSH, FSH, lipid profile, and chemistry profile). Subjects were instructed to apply a quarter teaspoon of cream (containing 20 mg progesterone or placebo) to the skin daily. Each woman received daily multivitamins and 1200 mg of calcium and were seen every 4 months for review of symptoms. Bone scans and serum chemistries were repeated after 1 year. Results: Thirty of the 43 (69%) in the treatment group and 26 of the 47 (55%) in the placebo group complained initially of vasomotor symptoms. Improvement or resolution of vasomotor symptoms, as determined by review of weekly symptom diaries, was noted in 25 of 30 (83%) treatment subjects and five of 26 (19%) placebo subjects (P < .001). However, the number of women who showed gain in bone mineral density exceeding 1.2% did not differ (α = 05, power of 80%). Conclusion: Although we found no protective effect on bone density after 1 year, we did see a significant improvement in vasomotor symptoms in the treated group. Progesterone from diosgenin, extracted from Mexican yams, is identical to the natural progesterone of the human ovary or placenta. 1 Transdermal progesterone has been used cosmetically for over 20 years; however, recently those preparations have been used as alternatives to traditional hormone replacement therapy (HRT). Because of its organic origin and lack of side effects, use of transdermal progesterone cream has increased dramatically. Studies using cell culture and animal models suggest that progesterone might function as an osteotrophic hormone. 2 Anecdotal evidence and human noncontrolled trials suggested daily use of transdermal progesterone cream might increase lumbar spine bone mineral density in postmenopausal women and improve overall well-being. Lee noted, in reviewing his personal files, an increase in spinal bone mineral density in 63 of 100 menopausal women treated daily with 20 mg of transdermal progesterone cream. 3 He also reported an increase in well-being and excellent compliance, owing to ease of use and lack of side effects. Recent data also suggest that natural progesterone might have theoretical advantages over oral medroxyprogesterone acetate based on lipid profiles and coronary vascular experiments in primates. 4,5 We designed a randomized, double-masked, placebo-controlled trial to investigate the effectiveness of transdermal progesterone cream for controlling vasomotor symptoms and preventing menopausal bone loss.