Clinical Nutrition 34 (2015) 586e592

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Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Randomized control trials

Calcium plus vitamin D supplementation affects glucose metabolism

and lipid concentrations in overweight and obese vitamin D deficient
women with polycystic ovary syndrome
Zatollah Asemi a, Fatemeh Foroozanfard b, Teibeh Hashemi b, Fereshteh Bahmani a,
Mehri Jamilian c, Ahmad Esmaillzadeh d, e, *
a
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran
b
Department of Gynecology and Obstetrics, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran
c
Department of Gynecology and Obstetrics, School of Medicine, Arak University of Medical Sciences, Arak, Iran
d
Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
e
Department of Community Nutrition, School of Nutrition and Food Science, Isfahan University of Medical Sciences, Isfahan, Iran

a r t i c l e i n f o s u m m a r y

Article history: Background & aims: Few studies have examined the effects of calcium plus vitamin D supplementation
Received 18 July 2014 on glucose metabolism and lipid concentrations in overweight and obese vitamin D deficient women
Accepted 22 September 2014 with polycystic ovary syndrome (PCOS). This study was conducted to determine the effects of calcium
plus vitamin D supplementation on glucose metabolism and lipid concentrations among overweight and
Keywords: obese vitamin D deficient women with PCOS.
Calcium
Methods: This randomized double-blind placebo-controlled clinical trial was conducted among 104
Vitamin D
overweight and obese vitamin D deficient women diagnosed with PCOS. Participants were randomly
Supplementation
Polycystic ovary syndrome
assigned into four groups to receive: 1) 1000 mg/d calcium þ vitamin D placebo (n ¼ 26); 2) 50,000 IU/
Glucose metabolism wk vitamin D þ calcium placebo (n ¼ 26); 3) 1000 mg calcium/d þ 50,000 IU/wk vitamin D (n ¼ 26) and
Lipid concentrations 4) calcium placebo þ vitamin D placebo (n ¼ 26) for 8 weeks. Fasting blood samples were taken at
baseline and after 8 weeks' intervention to measure glucose metabolism and lipid concentrations.
Results: Calcium-vitamin D co-supplementation resulted in higher levels of serum calcium (P ¼ 0.002)
and vitamin D (P < 0.001) compared with other groups. Co-supplementation, compared with other
groups, led to decreased serum insulin levels (P ¼ 0.03), homeostasis model of assessment-insulin
resistance (HOMA-IR) score (P ¼ 0.04) and a significant rise in quantitative insulin sensitivity check
index (QUICKI) (P ¼ 0.001). Furthermore, a significant decrease in serum triglycerides (P ¼ 0.02) and
VLDL-cholesterol levels (P ¼ 0.02) was seen following the administration of calcium plus vitamin D
supplements compared with the other groups. Co-supplementation with calcium and vitamin D had no
significant effects on FPG, total-, LDL-, HDL-, and non-HDL-cholesterol levels.
Conclusions: In conclusion, calcium plus vitamin D supplementation for eight weeks among vitamin D
deficient women with PCOS had beneficial effects on serum insulin levels, HOMA-IR, QUICKI, serum
triglycerides and VLDL-cholesterol levels, but it did not affect FPG and other lipid profiles.
Clinical registration numberwww.irct.ir: IRCT201309275623N10
© 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction
Polycystic ovary syndrome (PCOS) is one of the most common
heterogeneous endocrine disorders in reproductive-age women
* Corresponding author. Food Security Research Center, Isfahan University of
Medical Sciences, Isfahan, Iran. Tel.: þ98 311 7922720; fax: þ98 311 6681378.
E-mail address: Esmaillzadeh@hlth.mui.ac.ir (A. Esmaillzadeh).
[1]. The prevalence of PCOS varies between 15 and 20% in the world
depending on the criteria used [2]. In Iran, it has been reported that
15.2% of women are affected [3]. Obesity and hyperinsulinemia are
responsible factors for the metabolic abnormalities of PCOS [4].
Individuals with PCOS are at 11-fold greater risk of metabolic
syndrome [5]. However, the risk of metabolic syndrome largely
varies among the different study cohorts and depends on the
prevalence of obesity [6,7].

http://dx.doi.org/10.1016/j.clnu.2014.09.015
0261-5614/© 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592
Lifestyle modification, including diet therapy and exercise, has
strongly been recommended for management of insulin resistance,
dyslipidemia, menstrual irregularities, symptoms of androgen
excess, infertility and ovulation induction in PCOS patients [2].
Recently, some studies have indicated an association between low
serum 25-hydroxyvitamin D [25(OH) D] levels and symptoms of
PCOS including insulin resistance, hirsutism, and infertility and
ovulatory disorders [8]. In addition, the beneficial effects of vitamin
D alone as well as calcium plus vitamin D supplementation on
ovarian follicles maturation, ovulation and menstrual regularity
have earlier been shown [9,10]. In a study by Wehr et al. [11], it has
been indicated that weekly vitamin D supplementation (20,000 IU)
for 24 weeks resulted in improved glucose metabolism, tri-
glycerides, estradiol levels and menstrual frequency among PCOS
women. Vitamin D and calcium has been proposed to act jointly
rather than independently. Calcium and vitamin D supplementa-
tion might affect metabolic profiles through their effects on the
regulation of cell cycle [12], parathyroid hormone (PTH) suppres-
sion [13] and apolipoprotein gene expression [14]. However, some
investigators did not observe the beneficial effects of single or
combined supplementation of calcium and vitamin D on insulin
metabolism and lipid profiles. For instance, Pal et al. [10] found that
3-month vitamin D [daily vitamin D3 (2000 IU) and calcium
(530 mg/day) supplementation did not affect insulin resistance. In
addition, 600 mg elemental calcium plus 125 IU/day vitamin D3
supplementation did not influence serum insulin levels and lipid
profiles in overweight college students [15].
To the best of our knowledge, the beneficial effects of calcium
plus vitamin D supplementation on parameters of glucose ho-
meostasis and insulin resistance have been reported only in a small
semi-experimental study [10] without any control group. In addi-
tion, effects of co-supplementation on lipid profiles have not been
examined in previous study. The current study was, therefore, done
to investigate the effects of calcium plus vitamin D supplementa-
tion on glucose metabolism and lipid concentrations in overweight
and obese women with PCOS.
2. Subjects and methods
2.1. Participants
This randomized double-blind placebo controlled clinical trial
was performed in Kashan, Iran, during September 2013 to
December 2013. For estimating the sample size, we used a ran-
domized clinical study sample size formula where type one (a) and
type two error (b) were 0.05 and 0.20 (power ¼ 80%), respectively.
Based on a previous study [10], we also considered 5.74 as standard
deviation (SD) and 4.5 as the difference in mean (d) of homeostasis
model of assessment-insulin resistance (HOMA-IR) as the key
variable. Based on this, we needed 25 subjects in each group. The
sample size formula for the current study gave 21 patients in each
arm. To take the probable drop-outs into account, we enrolled 26
patients in each arm. Actually, we did consider all dependent var-
iables (glucose homeostasis parameters and lipid profiles) as pri-
mary outcomes in the current study. HOMA-IR was used to
estimate sample size because it is the most important variable in
PCOS patients. Furthermore, the largest sample size was obtained
when we used this variable. Therefore, the sample size obtained
based on this variable were covering the required sample size for all
other variables. In this study, we included vitamin D deficient
(<20 ng/mL) overweight or obese (BMI  25 kg/m2) women, aged
18e40 y diagnosed with PCOS based on Rotterdam criteria. We did
not include women aged<18 or >40 years, those with BMI < 25 kg/
m2, individuals with neoplastic, hepatic, renal or cardiovascular
disorders, malabsorptive disorders, those taking calcium and
587
vitamin D within the last 6 months and women who had calcium
intake more than 1500 mg per day. Those that were using hormone
therapy, antidiabetic, or anti-obesity medications within the last 6
months were not included as well. Individuals who intended to
adopt a diet and/or a specific physical activity program were not
included in this study. As levels of 25(OH) D in overweight and
obese women is lower [16], we included overweight PCOS subjects
in the present study. Earlier studies have shown that serum levels
of 25(OH) D was lower among overweight women, especially those
with visceral obesity, compared with lean PCOS subjects [17]. We
hypothesized that vitamin D deficiency in these women has led to
insulin resistance and therefore included overweight and obese
PCOS subjects in the present study. Diagnosis of PCOS was done
according to the Rotterdam criteria [18]: those with the two of the
following criteria were considered as having PCOS: oligo- and/or
anovulation, hyperandrogenism and polycystic ovaries. A total of
700 women attended gynecology clinics affiliated to Kashan Uni-
versity of Medical Sciences, Kashan, Iran, were screened for PCOS.
Females who reported menstrual irregularity and/or had a modi-
fied Ferriman Gallwey (mF-G) score of 8 were invited for a clinical
examination. Those who did not have these criteria were not
further evaluated and were deemed not to have PCOS. Taking a
medical history and focusing on symptoms of PCOS, specifically
menstrual irregularities, clinical hyperandrogenism and medica-
tion use including hormone therapy was done by trained midwifes.
Menstrual irregularity was assessed as the presence of chronic
amenorrhea or a menstrual cycle length of less than 21 days or
more than 35 days, or more than four days variation between cy-
cles. Clinical hyperandrogenism was assessed as the self-reported
degree of hirsutism using the mF-G scoring method based on a
chart displaying degree of hair growth in nine regions. All patients
attending the center were first examined by the study gynecologist
and after diagnosis of PCOS in all subjects; they were instructed to
fill mF-G scoring form. The self-reported hirsutism was re-checked
and confirmed by the study gynecologist. Polycystic ovaries were
diagnosed by Ultrasonography (US) in participants with menstrual
dysfunction and/or hirsutism. Finally, 104 women met the inclusion
criteria and were enrolled in the study (Fig. 1). Subjects were
stratified according to BMI (<30 and 30 kg/m2) and age (<30 and
30 y) and then were randomly assigned into four groups for 8
weeks. Random assignment was done by the use of computer-
generated random numbers. A trained midwife at maternity clinic
did the randomized allocation sequence and assigned participants
to the groups. The study was conducted according to the guidelines
laid down in the Declaration of Helsinki. The ethical committee of
Kashan University of Medical Sciences approved the study and
informed written consent was obtained from all participants. The
trial was registered in the Iranian website (www.irct.ir) for regis-
tration of clinical trials (IRCT code: IRCT201309275623N10).
2.2. Study design
At study baseline and after stratification for pre-intervention
BMI and age, subjects were randomly assigned to four groups
receiving: 1) 1000 mg/d calcium þ vitamin D placebo (n ¼ 26); 2)
50,000 IU/wk vitamin D þ calcium placebo (n ¼ 26); 3) 1000 mg
calcium/d þ 50,000 IU/wk vitamin D (n ¼ 26) and 4) calcium
placebo þ vitamin D placebo (n ¼ 26) for 8 weeks. Due to the lack of
evidence about the appropriate dosage of calcium and vitamin D for
PCOS women and given that all study participants were vitamin D
deficient, we used the above-mentioned doses of calcium and
vitamin D based on a previous study in vitamin D deficient non-
diabetic subjects [19]. Calcium supplements and its placebos were
manufactured by Tehran Shimi Pharmaceutical Company (Tehran,
Iran). Vitamin D supplements and its placebos were manufactured
588 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592
Fig. 1. Summary of patient flow diagram, a Individuals received 1000 mg calcium carbonate daily, b Individuals received 50000 IU vitamin D3 weekly, c Individuals received 1000 mg
calcium per day plus 50000 IU vitamin D3 weekly, IVF: In vitro fertilisation, PCOS: polycystic ovary syndrome.
by Dana Pharmaceutical Company (Tabriz, Iran) and Barij Essence
Pharmaceutical Company (Kashan, Iran). Quality control of calcium
and vitamin D supplements were done in the laboratory of Food
and Drug Administration in Tehran, Iran by enzymatic and HPLC
methods. Following quality control, we found that the amount of
calcium and cholecalciferol in the prescribed tablets was at the
range of 950e1200 mg and 47,500e52,500 IU. Individuals were
asked not to alter their routine physical activity, and not to receive
any lipid-lowering medications as well as medications that might
affect their reproductive physiology during the 8-wk intervention.
Compliance to the calcium and vitamin D supplementation was
assessed through quantification of serum calcium and vitamin D
levels. The use of calcium and vitamin D supplements and placebos
throughout the study was checked through asking participants to
bring the medication containers. All participants provided three
dietary records (one weekend day and two weekdays) and three
physical activity records to make sure that they maintained their
usual diet and physical activity during intervention. Both dietary
and physical activity records were taken at week 2, 4 and 6 of
intervention. The dietary records were based on estimated values
in household measurements. To obtain nutrient intakes of partici-
pants based on these three-day food diaries, we used Nutritionist IV
software (First Databank, San Bruno, CA) modified for Iranian foods.
2.3. Assessment of anthropometric variables
Weight was assessed at baseline and after 8 weeks of inter-
vention in gynecology clinics by trained midwifes. Body weight was
measured in an overnight fasting status without shoes in a minimal
clothing state by the use of a digital scale (Seca, Hamburg, Ger-
many) to the nearest 0.1 kg. Height was measured using a non-
stretched tape measure (Seca, Hamburg, Germany) to the nearest
0.1 cm. BMI was calculated as weight in kg divided by height in
meters squared. Waist circumference was measured at the mini-
mum circumference between the iliac crest and the last rib. Hip
circumference was measured at the maximum protuberance of the
buttocks. All measurements were done by the same person to
reduce subjective errors.
2.4. Assessment of biochemical variables
Fasting blood samples (10 mL) were taken at baseline and after
8-wk intervention at Kashan reference laboratory in an early
morning after an overnight fast. Blood samples were immediately
centrifuged (Hettich D-78532, Tuttlingen, Germany) at 3500 rpm
for 10 min to separate serum. Then, the samples were stored
at 70  C before analysis at the KUMS reference laboratory. Serum
25-hydroxyvitamin D concentrations were assayed using a com-
mercial ELISA kit (IDS, Boldon, UK). The inter- and intra-assay CVs
for serum 25-hydroxyvitamin D assays ranged from 4.6 to 6.8%.
Commercial kits were used to measure fasting plasma glucose
(FPG), serum calcium, cholesterol, triglycerides, VLDL-, LDL- and
HDL-cholesterol concentrations (Pars Azmun, Tehran, Iran). The
total-to HDL-cholesterol ratio was obtained by dividing total
cholesterol by HDL-cholesterol. Non-HDL-cholesterol was calcu-
lated as total cholesterol minus HDL-cholesterol. The intra- and
inter-assay CVs for FPG were 2.5 and 3.5%, respectively. All inter-
and intra-assay CVs for lipid profile were less than 5%. Serum in-
sulin was assayed by ELISA kit (DiaMetra, Milano, Italy). The intra-
and inter-assay CVs for serum insulin were 2.5 and 5.4%, respec-
tively. The homeostatic model assessment for HOMA-IR and the
quantitative insulin sensitivity check index (QUICKI) was calculated
based on suggested formulas [20]. Measurements of glucose, lipid
concentrations and insulin were done in a blinded fashion, in
 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592 589

duplicate, in pairs (before/after intervention) at the same time, in
the same analytical run, and in random order to reduce systematic
error and inter-assay variability.
2.5. Statistical analysis
We used KolmogroveSmirnov test to examine the normal dis-
tribution of variables. Log transformation was conducted for non-
normally distributed variables. After log-transformation, the
normality of variables was re-checked. The analyses were done
based on intention-to-treat approach. Missing values were treated
based on Last-Observation-Carried-Forward method. One-way
analysis of variance was used to detect differences in general
characteristics, dietary intakes and metabolic profiles at study
baseline between the four groups. To determine the effects of cal-
cium plus vitamin D supplementation on glucose metabolism and
lipid profiles, we used repeated measures analysis of variance. The
changes across four groups were compared using one-way analysis
of variance with Bonferoni post hoc pair-wise comparisons. To
assess if the magnitude of the change depended on the baseline
values, we adjusted all analyses for the baseline values, age and
baseline BMI to avoid the potential bias that might have resulted.
P < 0.05 was considered as statistically significant. All statistical
analyses were done using the Statistical Package for Social Science
version 17 (SPSS Inc., Chicago, Illinois, USA).
3. Results
Among individuals in the calcium group, 3 women [withdraw
(n ¼ 2) and health problems (n ¼ 1)], in the vitamin D group, 3
women [withdraw (n ¼ 1), health problems (n ¼ 1) and the use of
medications (n ¼ 1)] and in the calcium plus vitamin D group, 3
women [in vitro fertilisation (IVF) treatment (n ¼ 1), health prob-
lems (n ¼ 1) and became pregnant (n ¼ 1)] were excluded. The
exclusions in the placebo group were 3 persons [IVF treatment
(n ¼ 1), became pregnant (n ¼ 1) and the use of medications
(n ¼ 1)]. Finally, 92 participants [calcium (n ¼ 23), vitamin D
(n ¼ 23), calcium plus vitamin D (n ¼ 23) and placebo (n ¼ 23)]
completed the trial (Fig. 1). However, as the analysis was done
based on intention-to-treat approach, all 104 women (26 in each
group) were included in the final analysis.
Mean age and height of study participants were not statistically
different between the four groups. Baseline weight and BMI as well
as their means after intervention were not significantly different
comparing the groups; however, waist circumference (P ¼ 0.03)
and hip circumference (P ¼ 0.02) were significantly different at
study baseline and end-of-trial comparing the four groups
(Table 1).
Based on the three-day dietary records obtained throughout the
intervention, no statistically significant difference was seen be-
tween the four groups in terms of dietary intakes of energy, car-
bohydrates, proteins, fats, saturated fatty acids (SFA),
polyunsaturated fatty acids (PUFA), monounsaturated fatty acids
(MUFA), cholesterol, total dietary fiber (TDF), calcium and vitamin
D (Table 2).
No significant differences were observed between the four
groups in terms of baseline values of lipid profiles; however, serum
calcium (P ¼ 0.01), vitamin D (P ¼ 0.008), FPG (P < 0.001), HOMA-IR
(P ¼ 0.04) and QUICKI (P ¼ 0.01) were significantly different at
study baseline comparing the four groups (Table 3).
Calcium-vitamin D co-supplementation resulted in higher levels
of serum calcium (P ¼ 0.002) and vitamin D (P < 0.001) compared
with other groups (Table 4). Co-supplementation, compared with
other groups, led to decreased serum insulin levels (P ¼ 0.03),
HOMA-IR score (P ¼ 0.04) and a significant rise in QUICKI index
(P ¼ 0.001). Furthermore, a significant decrease in serum tri-
glycerides (P ¼ 0.02) and VLDL-cholesterol levels (P ¼ 0.02) was
seen following the administration of calcium plus vitamin D sup-
plements compared with the other groups. Co-supplementation
with calcium and vitamin D had no significant effects on FPG, to-
tal-, LDL-, HDL-, and non-HDL-cholesterol levels. When we
adjusted the analyses for baseline values of the corresponding
measures, the above-mentioned findings remained significant
except for serum triglycerides (P ¼ 0.12) and VLDL-cholesterol
levels (P ¼ 0.12) (Data not shown). Adjustment for age and base-
line BMI did not affect our findings except for HOMA-IR score
(P ¼ 0.05).
4. Discussion
The current study demonstrated that calcium plus vitamin D
supplementation for 8 weeks among vitamin D deficient women
with PCOS resulted in a significant decrease in serum insulin levels,
HOMA-IR score, serum triglycerides and VLDL-cholesterol levels
and a significant rise in QUICKI index. We did not find any signifi-
cant effect of calcium plus vitamin D supplementation on FPG and

Table 1
General characteristics of study participants.a

Placebo (n ¼ 26) Calciumb (n ¼ 26) Vitamin Dc (n ¼ 26) Calcium þ vitamin Dd (n ¼ 26) Pe

Age (y) 24.3 ± 5.2 25.0 ± 6.7 25.6 ± 4.4 24.9 ± 5.1 0.87
Height (cm) 161.9 ± 4.5 159.0 ± 4.5 160.8 ± 5.0 160.5 ± 5.4 0.20
Weight at study baseline (kg) 71.9 ± 13.8 71.5 ± 11.9 75.6 ± 10.4 70.9 ± 16.9 0.57
Weight at end-of-trial (kg) 72.1 ± 14.3 71.4 ± 12.2 75.2 ± 10.7 70.4 ± 16.2 0.60
Weight change (kg) 0.2 ± 1.8 0.1 ± 0.9 0.4 ± 1.9 0.5 ± 2.0 0.48
BMI at study baseline (kg/m2) 27.5 ± 5.2 28.3 ± 4.7 29.3 ± 3.9 27.3 ± 5.3 0.43
BMI at end-of-trial (kg/m2) 27.5 ± 5.4 28.3 ± 4.8 29.1 ± 3.9 27.1 ± 5.0 0.48
BMI change (kg/m2) 0.06 ± 0.7 0.03 ± 0.4 0.2 ± 0.9 0.2 ± 0.8 0.49
Waist circumference at study baseline (cm) 80.7 ± 8.9 81.7 ± 9.9 88.0 ± 11.4 80.7 ± 11.0 0.03
Waist circumference at end-of-trial (cm) 80.8 ± 9.3 81.5 ± 10.4 87.6 ± 11.5 80.0 ± 10.1 0.03
Waist circumference change (cm) 0.1 ± 1.8 0.3 ± 1.6 0.4 ± 1.9 0.8 ± 1.9 0.42
Hip circumference at study baseline (cm) 98.3 ± 8.9 98.9 ± 10.7 105.0 ± 10.6 96.6 ± 11.6 0.02
Hip circumference at end-of-trial (cm) 98.5 ± 9.3 98.6 ± 10.7 104.5 ± 10.8 95.8 ± 10.9 0.02
Hip circumference change (cm) 0.2 ± 1.6 0.3 ± 1.4 0.5 ± 2.1 0.8 ± 2.1 0.31
a
Data are means ± standard deviations.
b
Receiving 1000 mg calcium carbonate per day plus a weekly placebo for vitamin D.
c
Receiving 50,000 IU vitamin D3 per week plus a daily placebo for calcium.
d
Receiving 1000 mg calcium carbonate per day plus 50,000 IU vitamin D3 per week.
e
Obtained from ANOVA test.
590 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592

Table 2
Dietary intakes of study participants throughout the study.a

Placebo (n ¼ 26) Calciumb (n ¼ 26) Vitamin Dc (n ¼ 26) Calcium þ vitamin Dd (n ¼ 26) Pe

Energy (kcal/d) 2364 ± 273 2343 ± 295 2464 ± 247 2367 ± 305 0.41
Carbohydrates (g/d) 322.3 ± 55.7 318.7 ± 51.2 345.4 ± 40.2 324.8 ± 59.7 0.25
Protein (g/d) 86.3 ± 13.6 87.5 ± 20.2 87.0 ± 12.9 87.9 ± 18.7 0.98
Fat (g/d) 84.4 ± 13.4 83.7 ± 14.0 81.1 ± 14.1 83.2 ± 16.2 0.85
SFAf (g/d) 24.3 ± 5.2 26.5 ± 5.3 25.2 ± 4.7 26.4 ± 6.3 0.43
PUFAg (g/d) 25.3 ± 6.1 26.2 ± 6.1 25.5 ± 6.9 25.9 ± 6.3 0.96
MUFAh (g/d) 23.2 ± 7.5 23.4 ± 5.8 22.5 ± 5.2 23.4 ± 6.4 0.95
Cholesterol (mg/d) 218.9 ± 123.2 217.4 ± 114.9 199.9 ± 91.8 208.1 ± 108.7 0.91
TDFi (g/d) 19.4 ± 4.5 19.5 ± 4.2 21.4 ± 4.5 19.1 ± 4.5 0.24
Calcium (mg/d) 1129.2 ± 210.0 1136.6 ± 189.5 1166.1 ± 184.3 1110.9 ± 167.1 0.76
Vitamin D (mg/d) 2.8 ± 0.9 2.9 ± 0.9 2.8 ± 0.8 2.7 ± 0.7 0.77
a
Data are means ± standard deviations.
b
Receiving 1000 mg calcium carbonate per day plus a weekly placebo for vitamin D.
c
Receiving 50,000 IU vitamin D3 per week plus a daily placebo for calcium.
d
Receiving 1000 mg calcium carbonate per day plus 50,000 IU vitamin D3 per week.
e
Obtained from ANOVA test.
f
SFA: Saturated fatty acid.
g
PUFA: Polyunsaturated fatty acid.
h
MUFA: Monounsaturated fatty acid.
i
TDF: Total dietary fiber.

other lipid profiles. To the best of our knowledge, this study is
among the first investigations that reported the effect of calcium
plus vitamin D supplementation on glucose metabolism and lipid
profiles in women with PCOS.
Women with PCOS are susceptible to several complications
including insulin resistance, hyperinsuli-naemia and dys-
lipidemia [8]. We showed that calcium and vitamin D supple-
mentation for 8 weeks among PCOS women led to a significant
reduction in serum insulin levels, HOMA-IR score and a significant
elevation in QUICKI index. Previous studies have indicated the
effects of single calcium or vitamin D supplementation on glucose
metabolism in patients with PCOS, the combined effects of calcium
and vitamin D supplementation in these patients have been
understudied. In agreement with our findings, Harinarayan et al.
[19] found improved HOMA-B score after supplementation
with 1000 mg/d calcium and 9570 IU/d vitamin D for 2 months
in vitamin D deficient non-diabetic subjects. Short-term
supplementation with cholecalciferol (2000 IU once daily) with
calcium carbonate (400 mg twice daily) or without calcium car-
bonate has also resulted in improved b cell function among adults
at high risk for diabetes [20]. The same findings were also seen
after taking combined 500 mg calcium citrate and 700 IU vitamin
D per day in non-diabetic adults [21]. In contrast to these findings,
some studies did not find the effect of calcium or vitamin D sup-
plementation on insulin function. For instance, Pal et al. [10] found
no significant difference in glucose metabolism following the
intake of vitamin D (daily dose of 3533 IU, increased to 8533 IU
after the first 5 participants entered into the study) and calcium
supplements (530 mg elemental calcium per day) in overweight
vitamin D deficient women with PCOS after 3 months. In addition,
the effect of vitamin D on pancreatic b cell function might be
explained through its effect on regulation of serum calcium, that in
turn, affects insulin secretion, which is a calcium-dependent pro-
cess [22]. In peripheral cells, the active form of vitamin D may

Table 3
The baseline metabolic profiles of study participants.a

Placebo (n ¼ 26) Calciumb (n ¼ 26) Vitamin Dc (n ¼ 26) Calcium þ vitamin Dd (n ¼ 26) Pe

Calcium (mg/dL) 9.1 ± 0.6 9.5 ± 0.4 9.3 ± 0.6 9.4 ± 0.4 0.01
Vitamin D (ng/mL) 14.0 ± 4.1 13.9 ± 2.0 11.6 ± 4.7 15.1 ± 3.6 0.008
FPGf (mg/dL) 67.6 ± 11.7 71.9 ± 11.7 87.3 ± 16.4 81.6 ± 10.0 <0.001
Insulin (mIU/mL) 12.0 ± 5.6 8.5 ± 4.1 13.5 ± 9.9 11.1 ± 14.2 0.28
HOMA-IRg 2.0 ± 1.1 1.5 ± 0.6 3.1 ± 2.8 2.2 ± 2.8 0.04
QUICKIh 0.35 ± 0.02 0.36 ± 0.02 0.33 ± 0.03 0.35 ± 0.03 0.01
Triglycerides (mg/dL) 98.3 ± 41.7 135.7 ± 56.0 143.2 ± 89.8 129.2 ± 64.1 0.07
VLDL-cholesteroli (mg/dL) 19.7 ± 8.3 27.1 ± 11.2 28.6 ± 17.9 25.8 ± 12.8 0.07
Total cholesterol (mg/dL) 159.6 ± 40.9 175.5 ± 40.2 178.4 ± 35.6 178.8 ± 27.1 0.18
LDL-cholesterolj (mg/dL) 95.6 ± 35.3 101.8 ± 34.6 105.8 ± 31.0 108.2 ± 21.0 0.48
HDL-cholesterolk (mg/dL) 44.3 ± 7.6 46.6 ± 8.4 44.0 ± 7.2 44.8 ± 6.7 0.60
Total-/HDL-cholesterol 3.7 ± 1.1 3.8 ± 1.0 4.1 ± 1.0 4.1 ± 0.8 0.33
Non-HDL-cholesterol (mg/dL) 115.3 ± 40.7 128.9 ± 39.0 134.4 ± 35.6 133.9 ± 27.7 0.19
a
Data are means ± standard deviations.
b
Receiving 1000 mg calcium carbonate per day plus a weekly placebo for vitamin D.
c
Receiving 50,000 IU vitamin D3 per week plus a daily placebo for calcium.
d
Receiving 1000 mg calcium carbonate per day plus 50,000 IU vitamin D3 per week.
e
Obtained from ANOVA test.
f
FPG: Fasting plasma glucose.
g
HOMA-IR: Homeostasis model of assessment-insulin resistance.
h
QUICKI: Quantitative insulin sensitivity check index.
i
VLDL-cholesterol: Very low density lipoprotein-cholesterol.
j
LDL-cholesterol: Low density lipoprotein-cholesterol.
k
HDL-cholesterol: High density lipoprotein-cholesterol.
 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592 591

Table 4
The effect of calcium plus vitamin D supplementations on glucose metabolism and lipid profiles in PCOS women.a

End-of-trial Changes from baseline

Placebo Calciumb Vitamin Dc Calcium þ Pe Placebo Calciumb Vitamin Dc Calcium þ Pe

(n ¼ 26) (n ¼ 26) (n ¼ 26) vitamin Dd (n ¼ 26) (n ¼ 26) (n ¼ 26) vitamin Dd
(n ¼ 26) (n ¼ 26)

Calcium (mg/dL) 8.9 ± 0.7 9.8 ± 0.4 9.5 ± 0.6 9.7 ± 0.3 <0.001 0.2 ± 0.7 0.3 ± 0.4l 0.2 ± 0.4l 0.3 ± 0.3l 0.002
Vitamin D (ng/mL) 14.4 ± 4.7 14.2 ± 2.2 23.4 ± 7.1 26.8 ± 7.8 <0.001 0.4 ± 2.6 0.3 ± 2.0 11.8 ± 6.0l,m 11.7 ± 8.1l,m <0.001
f
FPG (mg/dL) 73.5 ± 23.8 71.2 ± 14.7 86.8 ± 16.1 76.4 ± 13.3 0.009 5.9 ± 20.8 0.7 ± 15.4 0.5 ± 14.8 5.2 ± 8.3 0.08
Insulin (mIU/mL) 15.1 ± 7.1 7.1 ± 3.4 12.4 ± 5.5 7.8 ± 3.6 <0.001 3.1 ± 6.1 1.4 ± 3.0 1.1 ± 8.5 3.3 ± 11.4l 0.03
HOMA-IRg 2.8 ± 1.9 1.3 ± 0.8 2.8 ± 1.6 1.5 ± 0.7 <0.001 0.8 ± 1.9 0.2 ± 0.6 0.3 ± 2.5 0.7 ± 2.4l 0.04
QUICKIh 0.33 ± 0.02 0.37 ± 0.02 0.33 ± 0.03 0.36 ± 0.02 <0.001 0.02 ± 0.02 0.01 ± 0.02l 0.0004 ± 0.02 0.008 ± 0.02l 0.001
Triglycerides (mg/dL) 117.7 ± 52.3 125.2 ± 69.0 131.2 ± 71.2 107.2 ± 65.7 0.57 19.4 ± 45.7 10.5 ± 45.9 12.0 ± 65.7 22.0 ± 41.9l 0.02
VLDL-cholesteroli (mg/dL) 23.5 ± 10.5 25.0 ± 13.8 26.2 ± 14.2 21.4 ± 13.1 0.57 3.8 ± 9.1 2.1 ± 9.2 2.4 ± 13.1 4.4 ± 8.4 l
0.02
Total cholesterol (mg/dL) 162.1 ± 36.3 165.1 ± 32.6 180.3 ± 30.8 171.9 ± 30.8 0.20 2.5 ± 35.3 10.4 ± 38.4 1.9 ± 24.0 6.9 ± 22.3 0.34
j
LDL-cholesterol (mg/dL) 93.4 ± 32.1 94.2 ± 26.0 109.9 ± 26.2 103.7 ± 26.1 0.10 2.2 ± 31.7 7.6 ± 30.1 4.1 ± 18.8 4.5 ± 20.8 0.42
HDL-cholesterolk (mg/dL) 45.2 ± 19.6 45.9 ± 8.9 44.1 ± 7.7 46.7 ± 7.0 0.87 0.9 ± 17.8 0.7 ± 8.3 0.1 ± 4.5 1.9 ± 6.7 0.83
Total-/HDL-cholesterol 3.8 ± 1.1 3.7 ± 0.9 4.2 ± 1.0 3.8 ± 1.0 0.28 0.1 ± 1.0 0.1 ± 0.6 0.1 ± 0.5 0.3 ± 0.7 0.09
Non-HDL-cholesterol (mg/dL) 116.9 ± 37.6 119.3 ± 30.9 136.1 ± 31.7 125.1 ± 32.7 0.16 1.6 ± 35.9 9.6 ± 33.1 1.7 ± 21.7 8.8 ± 22.9 0.30
a
Data are means ± standard deviations.
b
Receiving 1000 mg calcium carbonate per day plus a weekly placebo for vitamin D.
c
Receiving 50,000 IU vitamin D3 per week plus a daily placebo for calcium.
d
Receiving 1000 mg calcium carbonate per day plus 50,000 IU vitamin D3 per week.
e
Obtained from repeated measures ANOVA.
f
FPG: Fasting plasma glucose.
g
HOMA-IR: Homeostasis model of assessment-insulin resistance.
h
QUICKI: Quantitative insulin sensitivity check index.
i
VLDL-cholesterol: Very low density lipoprotein-cholesterol.
j
LDL-cholesterol: Low density lipoprotein-cholesterol.
k
HDL-cholesterol: High density lipoprotein-cholesterol.
l
Significant difference with the placebo group.
m
Significant difference with the calcium group.

enhance insulin sensitivity through transcriptional activation of
the human insulin receptor gene [23].
It must be considered that mean dietary calcium intake was
higher in our study participants than the recommended levels but
lower than upper limits (2500 mg). Such a high intake of dietary
calcium intake in these women remains unexpected for us. How-
ever, it might be explained by the use of a non-Iranian food
composition table for analyzing dietary data in the current study
due to the lack of Iranian food database. Data on the effects of
calcium supplementation on health status in subjects with high
dietary calcium intake are conflicting. For instance, Michaelsson
et al. [24] indicated that dietary calcium intake above 1400 mg/day
for 19 years were associated with higher death rates from all causes.
However, a significant reduction of all-cause mortality was
observed following the consumption of dietary calcium intake
higher than 1599 mg for 9 years [25]. In addition, some studies have
suggested that short-term intervention with calcium supplements,
even among those with high dietary calcium intake, is beneficial for
health. Furthermore, the cardiovascular risks of excessive calcium
intake appear to be more closely related to calcium supplements
than dietary calcium [26]. Nonetheless, further studies are required
on the cardiovascular risks of long-term increased calcium intake. It
must also be taken into account that there was a significant dif-
ference in serum calcium, vitamin D, plasma glucose levels, HOMA-
IR and QUICKI between the calcium, vitamin D, calcium þ vitamin D
and placebo groups at study baseline. This difference might have
been occurred due to several reasons. Random assignment to four
groups was done after stratification for pre-intervention BMI (<30
and 30 kg/m2) and age (<30 or 30 years) and random assign-
ment was done by the use of computer-generated random
numbers. Therefore, the difference in above mentioned variables
between the four groups might have been occurred by random. In
addition, when we adjusted the analyses for baseline values, no
significant changes in our findings except for HOMA-IR score were
observed.
Findings from the current study revealed that administration of
calcium plus vitamin D supplements among vitamin D deficient
PCOS women led to a significant reduction in serum triglycerides
and VLDL-cholesterol levels, but did not affect other lipid profiles.
Few studies have examined joint calcium-vitamin D supplemen-
tation on lipid profiles. In line with our findings, a significant
decrease in serum triglyceride levels was seen following the
administration of 500 mg/d calcium and 200 IU/d vitamin D for 9
weeks among pregnant women at risk for pre-eclampsia, but did
not influence other lipid profiles [27]. Furthermore, 15-wk sup-
plementation with 600 mg/d calcium plus 200 IU/d vitamin D
resulted in a significant reduction in serum triglycerides among
healthy overweight women [28]. However, some investigators did
not find any significant effect of combined calcium-vitamin D or
single calcium supplementation on lipid profiles in healthy over-
weight adults after 12 weeks [15]. Different study designs, different
dosages of calcium and vitamin D used, lack of considering baseline
levels of dependent variables along with characteristics of study
participants might provide some reasons for discrepant findings.
Several mechanisms can explain the favorable effects of joint
calcium-vitamin D supplementation on serum triglycerides and
VLDL-cholesterol levels. Calcium supplements might result in
reduced absorption of fatty acids and increased fecal fatty acid
content through formation of insoluble calcium-fatty soaps in the
gut [29]. Furthermore, increased intracellular calcium in liver leads
to stimulating microsomal triglycerides transfer protein (MTP)
which is implicated in the formation and secretion of VLDL, and
then results in decreased serum triglycerides and VLDL-cholesterol
levels [30]. The effects of vitamin D on apolipoprotein gene
expression [14] and serum triglycerides concentrations are still
controversial.
Some limitations must be considered in the interpretation of our
findings. One of the limitations of the current study is the duration
of intervention in this study. We were unable to administer calcium
and vitamin D supplements for more than 8 weeks. Long-term
592 Z. Asemi et al. / Clinical Nutrition 34 (2015) 586e592
interventions might lead to greater changes. Although examining
the effect of supplementation on hormonal picture of PCOS women,
including androgens levels, would be of value, we could not assess
the effect of calcium and vitamin D supplementation on these
variables and body fat due to limitations in funding. In addition, we
were unable to perform oral glucose tolerance test to examine in-
sulin function. Future studies are recommended to assess this to
obtain more detailed information about relevant parameters.
In conclusion, calcium plus vitamin D supplementation for eight
weeks among vitamin D deficient women with PCOS had beneficial
effects on serum insulin levels, HOMA-IR, QUICKI, serum tri-
glycerides and VLDL-cholesterol levels, but it did not affect FPG and
other lipid profiles.
Funding
The study was supported by a grant (no. 92088) from Kashan
University of Medical Sciences.
Authors' contributions
Z.A. and A.E. contributed in conception, design, statistical anal-
ysis and drafting of the manuscript. F.F., T.H., F.B. and M.J. contrib-
uted in data collection and manuscript drafting. A.E. supervised the
study. All authors approved the final version for submission.
Conflicts of interest
None of the authors had any personal or financial conflict of
interest.
Acknowledgment
The present study was supported by a grant from the Vice-
chancellor for Research, KUMS, and Iran. The authors would like
to thank the staff of Naghavi Clinic (Kashan, Iran) for their assis-
tance in this project.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.clnu.2014.09.015.
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