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OPEN Vitamin D Supplementation for

Premenstrual Syndrome-Related
inflammation and antioxidant
markers in students with vitamin D
deficient: a randomized clinical trial
Hajar Heidari1, Reza Amani2,3*, Awat Feizi   3, Gholamreza Askari4, Shahnaz Kohan5 &
Parastoo Tavasoli6
Premenstrual syndrome (PMS) is a common disorder in the reproductive age that negatively significant
impacts on women’s quality of life. This randomized clinical trial study was undertaken to investigate
the effect of vitamin D supplementation on inflammatory and antioxidant markers in 44 vitamin D
deficient (25(OH)D < 20 ng/mL) students with PMS. Participants received either 50,000 IU vitamin D3
or a placebo pearl fortnightly for 4 months. At the baseline and in the last 2 months of intervention,
participants were asked to complete the PMS Daily Symptoms Rating form along with taking the
pearls and their blood samples were collected to assess serum levels of 25(OH)D3, Interleukin10 and 12
(IL-10, IL-12) and total antioxidant capacity (TAC). In vitamin D group, serum levels of IL-10 and IL-12
significantly decreased while TAC significantly increased post-intervention. There were significant
differences regarding serum IL-12 and TAC levels between the two groups. Mean score of the total
PMS symptoms showed significant improvement in 25(OH)D. Vitamin D supplementation seems
to be an effective strategy to improve inflammation and antioxidant markers in vitamin D deficient
women with PMS. This clinical trial was registered at Iranian Registry of Clinical Trials on 20/06/2018
(IRCT20180525039822N1).

Premenstrual syndrome (PMS) is a common disorder in the reproductive age, which includes a wide group of
physical, emotional and behavioral symptoms that occur in the late luteal phase of the menstrual cycle and resolv-
ing quickly after the onset of menstruation1,2. Both physical and psychological symptoms of PMS have significant
negative impact on health and quality of life in women3. PMS is more common in young women, and according
to the WHO, PMS is a public health threat in the modern societies4. Around 70–90% of females in their produc-
tivity period show at least one of the PMS symptoms. The average prevalence of PMS in the world is 47.8%5. In
Iranian medical students prevalence of PMS has been reported 39.4% of whom 60.6% were mild, 25.1% moderate
and 14.2% severe6,7.
While, there is no special physical index or laboratory test for diagnosis of PMS, however, a prospective record
of cycle related symptoms is the gold standard for diagnosis2. The American Psychiatry Association has published
diagnostic criteria for diagnosing PMS8.

1
Department of Clinical Nutrition, School of Nutrition and Food Science, Food Security Research Center, Isfahan
University of Medical Sciences, Isfahan, Iran. 2Professor of Nutrition. Department of Clinical Nutrition, School
of Nutrition and Food Science, Food Security Research Center, Isfahan University of Medical Sciences, Isfahan,
Iran. 3Professor of Biostatistics, Department of Biostatistics and Epidemiology, School of Health, Psychosomatic
Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. 4Associate Professor of Nutrition, Department
of Community Nutrition, School of Nutrition and Food Science, Food Security Research Center, Isfahan University of
Medical Sciences, Isfahan, Iran. 5Associate Professor of Reproductive Health, Nursing and Midwifery Care Research
Center, School of Nursing and Midwifery, Isfahan University of Medical Sciences, Isfahan, Iran. 6Molecular Research
Lab,, School of Nutrition and Food Sciences, Food Security Research Center, Isfahan University of Medical Sciences,
Isfahan, Iran. *email: [email protected]

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The exact etiology of this syndrome is unknown, however, some hypotheses exist including; expanded genetic
vulnerability, sensitivity to gonadal steroid fluctuations and altered brain processes5. None of these hypotheses
have yet gained general acceptance, it is theorized that gonadal hormones appear to intervene changes in the
activity of central neurotransmitters, such as serotonin and modulation of gamma-aminobutyric acid (GABA)
receptors in the brain9,10.
PMS has a wide range of stress symptoms including psychiatric and somatic complaints11 and oxidative imbal-
ance seems to be involved in the biochemical basis of pathophysiologic mechanisms of PMS1. Several studies
have shown that luteal phase level of total antioxidant capacity (TAC) is significantly decreased in PMS women1,5.
Moreover, chronic inflammation, that occurs when cytokine producing cells remain activated, is involved in
the etiology of depression and other psychiatric and somatic disorders known as common features of PMS. A
cross-sectional study on PMS women revealed that total symptom score was positively associated with IL-2,
IL-4, IL-10 and IL-12 and associations were higher for IL-12 and IL-10 suggesting that there are elevated levels of
inflammatory factors in PMS12.
Although some treatments for PMS are suggested, due to their side effects, high costs and the absence of com-
plete effectiveness of medicines for treatment, most women tend to experience alternative therapies like dietary
supplements, minerals and vitamins3,5.
It has recently been reported that abnormality of calcium metabolism may be responsible for emotional and
physical symptoms of PMS13. Regarding the role of vitamin D in calcium absorption and its metabolism regula-
tion, it is proposed that vitamin D deficiency can be associated with PMS8.
Vitamin D is a steroid hormone that its biologic actions are mediated through vitamin D receptor (VDR).
VDR has been identified not only in calcium-regulating tissues, but also in many other reproductive organs
representing a potential role for vitamin D in female reproductive physiology14. Moreover, scientific evidence
offers that maintenance of an adequate level of 25(OH)D is essential in the prevention of a wide variety of health
disorders15.
Antioxidant role of vitamin D was shown in 1993 through its ability to inhibit iron-dependent liposomal lipid
peroxidation. Recently, studies have also recognized the antioxidant properties of this vitamin. Others have pro-
posed other functions of vitamin D such as its anti-inflammatory and immunomodulatory actions16.
Researchers have explained vitamin D deficiency (VDD) as a worldwide epidemic16. Women with PMS have
lower serum levels of vitamin D in the luteal phase compared to their normal controls8. Moreover, the highest
levels of vitamin D intake (706IU/day) were associated with 41% reduction in PMS vs. the lowest levels (112 IU/
day) due to affecting calcium levels, periodic fluctuations in sex hormones and/or modulating neurotransmitters.
Therefore, vitamin D deficiency appears to be related to PMS17. Cellular studies have demonstrated that vita-
min D supplementation decreased the production of inflammatory cytokines and increased anti-inflammatory
markers18.
Looking at the mechanisms of the effect of vitamin D on PMS and the observed effects of its administration
on inflammation and oxidative stress biomarkers which are mediated through Apo-lipoprotein gene expression,
parathyroid hormone suppression, repressing NF-kB activation and upregulation of antioxidant systems19,20, it
is postulated that improving vitamin D status may improve the inflammatory factors and antioxidant capacity
and, hence, decrease the incidence and severity of PMS symptoms. According to the best of our knowledge, no
study has been conducted in this regard so far. Thus, this study was undertaken to investigate the effect of vitamin
D supplementation on inflammation and antioxidant markers in vitamin D deficient (25(OH)D < 20 ng/mL)
women with PMS.

Results
Baseline characteristics.  During the four months of the study, two participants from the vitamin D group
and four participants from the placebo group dropped out of the study (Fig. 1) and data analysis carried out on
38 participants (20 from vitamin D group and 18 from placebo group). Both vitamin D and placebo supplements
were well tolerated and no complaints were observed during the study period.
The mean ± SD of students’ age in vitamin D and placebo groups were 21.3 ± 1.6, 21.7 ± 1.8, respectively. As
show in Table 1, there were no statistically significant differences between the groups in terms of sociodemo-
graphic and obstetrics characteristics as well as the serum 25(OH)D, IL-10, IL-12 and TAC concentrations at the
baseline (Table 1).
Dietary intakes of the participants in each group pre- and post-intervention and between the groups were
evaluated (Table 2). No significant differences were seen in dietary intakes of energy, macronutrients and micro-
nutrients between the groups, however, since there were marginal significant differences in energy intake and
vitamin E between the two groups, all analyzes were adjusted for the levels of these nutrients further on.

Effect of vitamin D supplementation on serum 25(OH)D levels.  As described, serum 25(OH)D lev-
els increased by 47% from baseline following supplementation and reached the normal levels (p < 0.001, Table 3).

Effect of vitamin D supplementation on inflammation and antioxidant markers.  In vitamin D

group, the serum levels of IL-10 and IL-12 decreased by 18.7% and 60.41% after intervention (p < 0.001 and
p:0.001, respectively). Moreover, mean serum levels of TAC increased by 36.67% (p < 0.001, Table 3). While, in
placebo group, serum IL-12 levels elevated (p = 0.36 and p = 0.01, respectively) and serum TAC levels signifi-
cantly decreased post-intervention (p = 0.02, Table 3).

Effect of vitamin D supplementation on mean score of the total PMS symptoms.  Mean score of
the total PMS symptoms showed significant improvements in vitamin D group (p < 0.001, Table 4).

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100 participants assessed for

Enrolment
eligibility

Excluded (n=35):
-Not meeting inclusion criteria (n-
30)
-Refused to participate (n=5)

65 participants fulfilled PMS Daily

Symptoms Rating form within 2
consecutive cycles

Excluded (n=11):
Failure to submit the form (n=3)
Failure to diagnose the disease
(n=8)

54 patients eligible:

Vitamin D deficiency checked

Excluded (n=10):
Allocation

-Serum 25(OH)D3<10ng/mL
(n=4)
-Serum 25(OH)D3>30ng/mL
(n=6)
44 patients were randomized

22 patients assigned to 22 patients assigned to

Follow-up

vitamin D group placebo group

Discontinued intervention (n=4):

-Lost to fallow-up (n=2)
-Got married (n=2)

Discontinued intervention (n=2):

-Lost to fallow-up (n=2)
analysis

20 patients completed 18 patients completed

follow up and analyzed follow up and analyzed

Figure 1.  Flowchart of the participants through the study.

Discussion
This study was designed to investigate the effect of vitamin D on inflammation and antioxidant markers in vita-
min Ddeficient students with PMS in a randomized clinical trial.
The findings indicated that vitamin D supplementation has noticeable effects on both inflammatory mark-
ers (IL-10, IL-12) and a significant increase was shown in serum levels of TAC compared to the placebo group.
Moreover, improvements in clinical PMS scores were indicated in vitamin D group.
Vitamin D administration fortnightly has been shown to improved serum 25(OH)D concentrations in
humans, and none of the treated participants were 25(OH)D deficient (<20 ng/mL) while no adverse or side
effects like hypercalcemia were reported21. Therefore, we decided to use 50,000 IU vitamin D fortnightly to ensure
that there would be no possible interference.
To the best of knowledge, this is the first clinical trial that has investigated the effect of vitamin D supplemen-
tation on inflammation and antioxidant markers in PMS.
There are plenty of findings supporting the role for inflammation in PMS. Azizieh et al. pointed out to a
possible role of pro-inflammatory cytokines IL-8 and TNF-α as a contributing factor in symptoms of PMS15. In
humans and animals, pro-inflammatory cytokine release results in illness behavior and symptoms of depression,
anorexia, anhedonia, paralysis, cognitive dysfunction and reduced social interaction, all of which are common
symptoms of PMS. Cytokine expression was shown in the endometrium, ovarian tissue and granulosa cells.

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Vitamin D group Placebo group

Group N = 20 N = 18 P-valuea
Age, years 21.3 ± 1.6 21.7 ± 1.8 0.41
BMI, kg/m2 20.7 ± 1.2 21.4 ± 2.01 0.21
Waist circumference, Cm 77 ± 8.4 80 ± 10.01 0.59
Menarche age, n (%) 0.51
   <12 years 9(40.9) 9(45)
   ≥12 years 13(59.1) 11(55)
Having previous history of PMS (%) 12(54.5) 12(60) 0.48
Habit of adding salt to the table, yes(%) 10(47.6) 9(45) 0.55
Coffee consumption, n (%) —
   ≤2 cups/day 22(100) 20(100)
   >2 cups/day 0 0
Sun exposure (min/day), n (%) 0.78
   <30 6(27.3) 8(40)
   30–60 11(50) 9(45)
   60–120 4(18.2) 2(10)
   >120 1(4.5) 1(5)
Using sunscreen, yes(%) 15(68.2) 17(85) 0.18
Body on sun expose, n (%) 0.20
   Only face and hands 18(81.8) 19(95)
   More 4(18.2) 1(5)
Serum 25(OH)D3, ng/mL 21.4 ± 7.6 21.1 ± 6.8 0.86
Serum IL-10, Pg/mL 92 ± 8 84 ± 35 0.49
Serum IL-12, ng/mL 18 ± 3 19 ± 24 0.89
Serum TAC, U/mL 13 ± 2 19 ± 11 0.064

Table 1.  Baseline characteristics in the two study groups. aResulted from independent t-test for quantitative
and chi-square test for categorical variables Q uantitative variables: mean ± SD Qualitative variables: frequency
(percentage). BMI, body mass index.

Vitamin D group Placebo group

N = 20 N = 18
Nutrients Baseline Post-intervention P-value Baseline Post-intervention P-value P-valuea
Energy intake (kcal/day) 1970 ± 150 2010 ± 150 0.001 2070 ± 190 2110 ± 210 0.004 0.06
Carbohydrate (g/day) 170 ± 40 170 ± 40 0.28 170 ± 50 170 ± 50 0.51 0.60
Protein (g/day) 48 ± 10 47 ± 10 0.04 50 ± 13 50 ± 10 0.002 0.30
Fat (g/day) 45 ± 10 44 ± 10 0.01 50 ± 20 47 ± 20 0.18 0.87
Linolenic acid (gr) 0.2 ± 0.1 0.3 ± 0.12 0.05 0.2 ± 0.1 0.2 ± 0.1 0.54 0.87
Vitamin A (µg/day) 410 ± 230 400 ± 210 0.21 460 ± 270 461 ± 260 0.82 0.70
Vitamin C (mg/day) 60 ± 19 60 ± 18.5 0.11 67 ± 30 60 ± 30 0.30 0.84
Vitamin E (mg/day) 12 ± 8 12 ± 8 0.99 8 ± 7 8 ± 6 0.81 0.04
Vitamin D (µg/day) 0.8 ± 0.8 0.7 ± 0.6 0.10 1 ± 1 1 ± 1 0.29 0.82
Selenium (µg/day) 0.05 ± 0.03 0.05 ± 0.02 0.85 0.04 ± 0.02 0.04 ± 0.02 0.56 0.29
Dietary fiber (g/day) 8 ± 2.5 8.1 ± 2.6 0.38 8 ± 2 8 ± 2 0.81 0.83
Soluble fiber (g/day) 0.4 ± 0.2 0.4 ± 0.3 0.27 0.3 ± 0.1 0.4 ± 0.2 0.09 0.12
Insoluble fiber (g/day) 2.2 ± 0.9 2.3 ± 0.75 0.37 1.9 ± 0.1 2 ± 0.9 0.02 0.13

Table 2.  Dietary intakes of the two study groups. Data are shown as mean ± SD. aResulted from independent
t-test.

The role of inflammation in PMS was further supported by studies of premenstrual exacerbation of asthma,
in which increased levels of airway inflammation during the late luteal phase were associated with worsening of
symptoms12. CRP, TNF-α and IL-6 have been positively associated with migraine headache, a usual symptom of
PMS22.
According to Johnson et al., there was the strongest correlation between the total score of symptoms and IL-10
and IL-12 levels among inflammatory indices in PMS women12.
The correlation analysis between IL-10 and anxiety in Foster et al. study showed a positive correlation in the
luteal phase in female soccer players with PMS post-game, while this correlation was negative in the group with-
out PMS9.

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Vitamin D group Placebo group Adjusted

N = 20 N = 18 P-valuea P-valueb
Baseline 21 ± 8 21 ± 7
Post-intervention 40 ± 8 23 ± 7
Serum of 25(OH)D (ng/mL) Mean <0.001 <0.001
19(14.5,23.4) 2.09(0.12,4.05)
difference(%95 CI)
P-value <0.001 0.03
Baseline 92 ± 8 84 ± 35
Post-intervention 75 ± 5 91 ± 37
Serum IL-10 (Pg/mL) Mean 0.007 0.017
−17.18(−25.4,−8.9) 7.36(−9.4,24.16)
difference(%95 CI)
P-value <0.001 0.36
Baseline 18 ± 3 19 ± 24
Post-intervention 7 ± 1 26 ± 24
Serum IL-12 (Pg/mL) Mean <0.001 <0.001
−11.3(−17.5,−5.09) 7.04(1.90,12.2)
difference(%95 CI)
P-value 0.001 0.01
Baseline 13 ± 2 19 ± 11
Post-intervention 21 ± 2 16 ± 12
Serum TAC (U/mL) Mean <0.001 <0.001
7.6(4.3,10.9) −3.3(−6.5,−0.5)
difference(%95 CI)
P-value <0.001 0.02

Table 3.  Within- and between-group comparison of changes in serum levels of 25(OH)D, inflammatory and
antioxidant markers in the two groups of study. aResulted from independent samples t-test based on comparing
mean difference between two groups. bResulted from ANOCOVA based on comparing post-intervention values
after adjustment for baseline values of outcomes, energy and vitamin E intake. Data are shown as mean ± SD.
*Significant differences between groups (p < 0.05).

Vitamin D group Placebo group Adjusted

N = 20 N = 18 P-valuea P-valueb
Baseline 39 ± 8 35 ± 10
Post-intervention 21 ± 6 28 ± 8
<0.001 <0.001
Mean difference(%95 CI) −18 (−21.5,−14.8) −7.0 (−9.5,−4.5)
P-value <0.001 <0.001

Table 4.  Within- and between-group comparison of changes in mean score of the total PMS symptoms in the
two study groups. aResulted from independent samples t-test based on comparing mean difference between two
groups. bResulted from ANOCOVA based on comparing post-intervention values after adjustment for baseline
values of outcomes, energy and vitamin E intake. Data are shown as mean ± SD.

The Mechanism by which vitamin D decreases inflammation remains poorly understood, but based on find-
ings, it seems that the effect of vitamins D on Interleukin is mediated through the action of 1α,25-dihydroxy vita-
min D3 that has been reported to inhibit IL-12 production in activated macrophages. Moreover, inhibitory effect
of 1α,25(OH)2D3 on IL-12 expression has been presented, while no functional vitamin D response elements
(VDREs) has been found in the genes IL-12A or IL-12B23. The suppressing effect of 1α,25(OH)2D3 has been
associated with the proximal IL-12B promoter, but no VDR direct binding to this area has been found24.
This suggests that 1α,25(OH)2D3 appears to have double effect on the expression of IL-12B. At the onset of
inflammation, the early effect of 1α,25(OH)2D3 is cyclical down-regulation of IL-12B expression, until maintaining
the immune response on passible level, and later a secondary effect by IL-10 occurs that turns off the IL-12B gene23,25.
It is important to note that IL-10, in addition to its anti-inflammatory immune function, has effects on the
brain and behavior, taking part in the modulation of mood, anxiety and depression symptoms. Therefore, along
with a significant improvement inflammation marker, we observed that mean score of the total PMS symptoms
also significantly improved.
There are various arguments on the effect of antioxidants on PMS. Balat et al. and Kalia et al. assessed the anti-
oxidants levels such as glutathione, ceruloplasmin, SOD and lipid peroxidation product-MDA in PMS. They did
not find any significant differences between the control and the PMS women in terms of antioxidant levels26,27. On
the other hand, some studies reported that TAC levels in luteal phase were decreased in PMS women1,5.
Different results seem to be due to the selection of different markers in assessing the effect of antioxidants on
PMS and TAC, that includes a set of several antioxidant indices1.
Although estrogen is a potent antioxidant, its excessive activity in the luteal phase of PMS women may stim-
ulate its pro-oxidant property leading to reduced TAC levels in these women27,28. Therefore, neuronal membrane
dysfunction can be secondary to free radical-mediated pathology through toxic effects of free radicals on high
ratio of polyunsaturated fatty acid in neuronal membrane, and may affect GABAergic system in PMS women1.

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Based on our results, increased serum levels of vitamin D after treatment with vitamin D resulted in significant
increase in serum TAC levels, reflecting an improvement in antioxidant status in PMS women.
The slight increment in serum vitamin D levels in the controls was possibly due to more sun exposure in the
summer, however, controls did not reach the normal range (Table 3). Interestingly, this elevation in vitamin D
levels might have led to significant improvement in mean score of the total PMS, again not comparable to the
intervention group (Table 4).
Abdollahi et al. have indicated that 2,000 IU vitamin D supplementation in vitamin D-deficient young girls
with PMS every other day for 12 weeks had no significant impact on PMS symptoms29. On the other hand,
according to a meta-analysis3, vitamin D could exert significant clinical effects on PMS symptoms30.
Finally, looking at the possible effects of placebo on premenstrual syndrome, some studies have shown placebo
to have significant effects on symptoms of PMS. The response rate to placebo has normally been indicated as
30–40%. It becomes clear that obtaining attention could positively affect the mental status of PMS31.
As the limitations of this study, we did not measure serum 1α,25-dihydroxy vitamin D3, calcium and PTH
levels to support some aspects of mechanism. We also did not examine the effect of vitamin D on the severity of
symptoms and quality of life in women with PMS, which can be new areas for further investigations.
Therefore, results obtained in this study indicate the beneficial effects of 50,000 IU vitamin D3 fortnightly on
symptoms and inflammatory and antioxidant markers in vitamin D-deficient students with PMS. Moreover, no
adverse effects of supplementation were reported in our participants.

Methods
Study design and participants.  This study was a randomized, parallel, placebo-controlled, double-blind
clinical trial to study the effect of 4 months supplementation with vitamin D (50,000 IU fortnightly) on inflamma-
tory and antioxidant markers and clinical symptoms of premenstrual syndrome in vitamin D deficient university
students from December 2017 to August 2018.
Considering the significance level of 0.05 with a statistical power of 80% for detecting at least standardized
effect size of Δ = 1 for TAC levels, depression and anxiety scores based on previous studies8,19, sample size was
determined as 17 participants in each group, an addition of 20% to cover up the possible drop outs. Totally, sam-
ple size was considered to be 22 participants in each group.

Study participants recruitment.  At the beginning, 100 female students aged 18–25 years old residing at
dormitories of the Isfahan University of Medical Sciences were randomly selected. Participants were included
if they had normal body mass index (18.5 ≤ BMI < 25), regular menstrual cycles between 24 to 35 days, being
single, not doing exercise professionally, having no evidence of acute or chronic illness and anemia, not using con-
traceptives, antipsychotics or anti-inflammatory medications and also not using vitamin D supplementation in
the last 3 months. Participants were excluded if they got married, were experiencing mental or physical diseases,
had serum 25(OH)D levels below 10 ng/mL and not willing to complete the study.
After confirming the participation and filling out the questionnaire regarding the symptoms of PMS, women
were assessed for depression and anxiety using Beck Depression Short Inventory (BDI-S) and Beck Anxiety
Inventory (BAI), respectively. If a participant was not affected by depression or anxiety disorder, she was asked to
fill out the PMS Daily Symptoms Rating form. Sixty-five students were then asked to record their symptoms on a
Daily Symptoms Rating form within 2 consecutive menstrual cycles. The questionnaire addressed the most prev-
alent symptoms of the psychological and physical domains. The participants marked the intensity of their daily
symptoms from 0 (not having the symptom), 1 (mild: the symptoms are present, but do not interfere with daily
activities such as education and work), 2 (moderate: the symptoms affect the daily activities moderately), or 3
(severe: the symptom prevents the person from taking part in normal daily activities). PMS cases were diagnosed
based on the diagnostic criteria of the American Psychiatry Association8.
After 2 months, 60 students returned the forms and 54 of them were diagnosed with PMS. Then, a blood
sample was taken from each individual to detect the vitamin D deficiency. Finally, 44 students who were simul-
taneously affected with PMS and vitamin D deficiency (serum 25(OH)D levels 10–30 ng/mL) were selected
as subjects and enrolled in the study. The study protocol was approved by the Medical Ethical Committee at
Isfahan University of Medical Sciences on 22/01/2017 under grant no. 395734 and ethical code no. IR.MUI.
REC.1395,3.734 and Iranian Registry of Clinical Trials (Registration No: IRCT20180525039822N1). All subjects
signed the written informed consent to participate in the study.

Intervention.  Participants who were eligible for the study received a randomization number generated by
software and randomly divided into intervention and control groups. All participants and researchers were blind
to the treatment groups until the statistical analysis was completed. In the intervention group (n = 22), all partic-
ipants received one oral pearl containing 50,000 IU vitamin D3 (D-Vitin 50,000; Zahravi Pharm Co, Tabriz, Iran)
and control group (n = 22) received one placebo pearl (Zahravi Pharm Co) every 2 weeks for the 4 months16,32.
Placebo pearls contained edible paraffin quite identical to the vitamin D3 ones in appearance, color, smell, size and
package to guarantee the blindness. In the last 2 months of intervention, participants were asked to complete the
PMS Daily Symptoms Rating form along with taking the pearls.

Data collection.  A validated semi-quantitative food frequency questionnaire (FFQ) containing 86 items
was applied to compare the amount of vitamin D intake before commencing and during the study period (totally
6 months)33. The amount of vitamin D received from sunlight was also checked by self-report questionnaire34.
Anthropometric parameters including height, weight and body mass index (BMI) were measured using standard
methods pre- and post-intervention.

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Biochemical analysis.  In both groups, blood samples were collected at the baseline and the end of the study
in order to assess serum levels of 25(OH)D3, Interleukin10 and 12 (IL-10, IL-12) and total antioxidant capacity
(TAC). Each time, 5 mL of blood sample was drawn from each participant within one week before the onset of
her menstrual period (luteal phase). Serums were collected and stored at −80 °C until further laboratory analyses.
Serum 25(OH)D levels were determined using ELISA kits (bioactiva diagnostica GmbH, Homburg,
Germany); according to the manufacturer protocol. Serum concentrations of IL-10, IL-12 and TAC were analyzed
using ELISA kits (HANGZHOU EASTBIOPHARM, Torrance, USA) according to the manufacturer protocol in
the research laboratory at the School of Nutrition and Food Sciences, Isfahan University of Medical Sciences.

Statistical analysis.  The nutritional status was analyzed by customized Nutritionist IV software. All statis-
tical analyses were carried out using SPSS statistical software version16 (IBM, Chicago, IL, USA). Quantitative
variables were presented as mean ± SD while qualitative variables were as frequency (percentage). Normality of
quantitative data was evaluated using Kolmogorov–Smirnov test and Q-Q plot. Positive skewed data were sub-
jected to logarithmic transformation. To compare quantitative variables between the groups, independent sam-
ples t-test and analysis of covariance (ANCOVA) with adjusting the baseline values of measured outcomes and
other confounding variables were used and for categorical variables chi-square test was applied. Within-group
comparisons were made using a paired samples t test. The observed effect size for study outcomes were presented
as 95% confidence intervals (CI). For all tests, two-sided p-values less than 0.05 were considered statistically
significant.

Ethical approval and informed consent

Approval.  This is to certify that the MSc thesis of Ms. Hajar Heidari that was conducted under the supervi-
sion of Dr. Reza Amani had been reviewed by the Research Council of Isfahan University of Medical Science on
22/01/2017 and approved with grant no. 395734 and ethical code no. of IR.MUI.REC.1395,3.734 and registered at
Iranian Registry of Clinical Trials on 20/06/2018 (Registration No: IRCT20180525039822N1).

Accordance.  In this study, the methods were carried out in accordance with the confirmed relevant guide-
lines and regulations.

Informed consent.  All subjects signed the written informed consent to participate in the study.
Received: 15 April 2019; Accepted: 2 October 2019;

Published: xx xx xxxx

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Acknowledgements
This study was extracted from MSc dissertation of Hajar Heidari. We wish to thank Isfahan University of Medical
Sciences for supporting this research and all students who kindly participated in this study. This Clinical Trial
Study was approved and financially granted by the Vice-Chancellor for Research, Isfahan University of Medical
sciences, Isfahan, Iran (Grant No. 395734).

Author contributions
R.A. designed the concept, supervised the project and revised the manuscript. H.H. did the data gathering,
analyzed the data and wrote the first draft. A.F. helped with data analysis. P.T. helped with lab analyses. GhR.A
and Sh.K helped with clinical interpretation and patients’ management. All authors have read and approved the
final version of the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to R.A.
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