major depressive disorder ( mdd ) is a common mental disorder that affects persons health related quality of life accompanied by reduced productivity . the disorder was known as the second global most important cause of disability in 2020 ( 1 ) . the causes of depression are suspected to be a combination of biological , environmental , and psychological factors . recent studies have given an increasing amount of attention to a possible role of vitamin d in cognitive function and mental health ( 3 , 4 ) . a unique neurosteroid hormone may have also an important role in the development of depression . vitamin d receptor ( vdr ) and vitamin d activating enzyme , 1-alpha - hydroxylase , are widely distributed in human brain , particularly in the hypothalamus and also in the limbic system , which is assumed to be involved in pathophysiology of the depression ( 5 , 6 ) . vitamin d is also involved in numerous brain processes including neuroimmunomodulation , regulation of neurotrophic factors , neuroprotection , neuroplasticity and brain development making it biologically plausible that this vitamin might be associated with the depression ( 7 ) . over one billion people have either vitamin d insufficiency or deficiency ( 8) . improving epidemiologic evidences have indicated a possible correlation between vitamin d levels and depression ( 9 ) , but clinical evidence is limited . therefore , this study was designed to investigate the association between serum levels of vitamin d and depression in patients with mdd as compared to control subjects . the study conducted during december to june 2013 and consisted of 85 participants ( aged 1863 y ) , 44 drug - free patients with mdd ( 9 men and 35 women ) and 41 healthy subjects as control ( 9 men and 32 women ) . not all the patients were under any antidepressant treatments for at least 3 month prior the initial assessment . healthy participants were characterized with no history of past or current chronic physical or mental disorders , including neurological diseases and were not taking any specific medications . patients depression level and mental health of the healthy controls was diagnosed according to dsm iv ( 10 ) by an experienced psychiatrist . the study was approved by ethical committee at tabriz university of medical sciences , tabriz , iran . the medical history included questions pertaining to any previous medications used to manage depression , anxiety , mood and other related issues , clinical manifestations of the disease , diagnosis history and smoking status . physical activity level was determined by using a validated international physical activity questionnaire ( ipaq ) and expressed as a standard metabolic energy turnover in met - minutes / week ( 11 ) . three levels of physical activity were classified : low ( < 600 met - minutes / week ) , moderate ( from 600 to 2999 met - minutes / week ) and high ( 3000 met - minutes / week or more ) . dietary intake of vitamin d was evaluated by a 3-day food record , completed on non - consecutive days . body weight was measured using a digital scale , with the examinee wearing a light gown . height was measured barefoot using a wall - mounted stadiometer to the nearest 0.5 cm . body mass index ( bmi ) was calculated as body weight ( kg ) divided by the square of height ( m ) . waist circumference ( wc ) was measured by a tape measure at the midpoint between the lower costal margin and iliac crest to the nearest 0.5 cm . hip circumference was measured over light clothing at the widest point over the buttocks when viewed from the side . sunlight exposure was assessed by a question relating to the amount of daily exposure during weekdays . the stress level of participants studied was assessed by means of the holmes and rahe stress scale . stress level was defined as mild ( score < 100 ) , moderate ( score= 100150 ) and severe ( score > 150 ) . to assess the severity of depression in patients , the 17-item hamilton rating scale for depression ( hrsd ) ( 12 ) was used by a well - trained rater . the severity of depression was categorized to mild ( score= 1013 ) , moderate ( score= 1417 ) and severe ( score > 17 ) . fasting blood samples were collected and sera were immediately collected after centrifugation at 5000 rpm for 3 minutes . serum 25(oh ) d levels of < 20 ng / ml defined as deficiency , > 20 as desirable level and < 10 as severe vitamin deficiency ( 13 ) . samples collected and measured from december to march considered as winter samples and samples from april to june as spring samples . demographic characteristics of the participants were summarized by either mean  standard deviation ( sd ) for continuous data , or frequency ( percentage % ) for proportional data . differences between the groups were assessed by chi - square or fisher s exact test for categorical variables , student s t test and mann - whitney u test for continuous variables . the study conducted during december to june 2013 and consisted of 85 participants ( aged 1863 y ) , 44 drug - free patients with mdd ( 9 men and 35 women ) and 41 healthy subjects as control ( 9 men and 32 women ) . not all the patients were under any antidepressant treatments for at least 3 month prior the initial assessment . healthy participants were characterized with no history of past or current chronic physical or mental disorders , including neurological diseases and were not taking any specific medications . patients depression level and mental health of the healthy controls was diagnosed according to dsm iv ( 10 ) by an experienced psychiatrist . the study was approved by ethical committee at tabriz university of medical sciences , tabriz , iran . the medical history included questions pertaining to any previous medications used to manage depression , anxiety , mood and other related issues , clinical manifestations of the disease , diagnosis history and smoking status . physical activity level was determined by using a validated international physical activity questionnaire ( ipaq ) and expressed as a standard metabolic energy turnover in met - minutes / week ( 11 ) . three levels of physical activity were classified : low ( < 600 met - minutes / week ) , moderate ( from 600 to 2999 met - minutes / week ) and high ( 3000 met - minutes / week or more ) . dietary intake of vitamin d was evaluated by a 3-day food record , completed on non - consecutive days . body weight was measured using a digital scale , with the examinee wearing a light gown . height was measured barefoot using a wall - mounted stadiometer to the nearest 0.5 cm . body mass index ( bmi ) was calculated as body weight ( kg ) divided by the square of height ( m ) . waist circumference ( wc ) was measured by a tape measure at the midpoint between the lower costal margin and iliac crest to the nearest 0.5 cm . hip circumference was measured over light clothing at the widest point over the buttocks when viewed from the side . sunlight exposure was assessed by a question relating to the amount of daily exposure during weekdays . the stress level of participants studied was assessed by means of the holmes and rahe stress scale . stress level was defined as mild ( score < 100 ) , moderate ( score= 100150 ) and severe ( score > 150 ) . to assess the severity of depression in patients , the 17-item hamilton rating scale for depression ( hrsd ) ( 12 ) was used by a well - trained rater . the severity of depression was categorized to mild ( score= 1013 ) , moderate ( score= 1417 ) and severe ( score > 17 ) . fasting blood samples were collected and sera were immediately collected after centrifugation at 5000 rpm for 3 minutes . serum 25(oh ) d levels of < 20 ng / ml defined as deficiency , > 20 as desirable level and < 10 as severe vitamin deficiency ( 13 ) . samples collected and measured from december to march considered as winter samples and samples from april to june as spring samples . demographic characteristics of the participants were summarized by either mean  standard deviation ( sd ) for continuous data , or frequency ( percentage % ) for proportional data . differences between the groups were assessed by chi - square or fisher s exact test for categorical variables , student s t test and mann - whitney u test for continuous variables . average ages of patients and healthy subjects were 36.93  12.05 and 36.97  12.9 years , respectively . there was no statistical significant difference in age , gender , anthropometric parameters , physical activity level , educational level , marital status , dietary vitamin d intake , and daily sun exposure between the two groups ( table 1 ) . in patients , the median of disease duration was 24.00 with a range of 12.00 to 60.00 months . descriptive data of depressed patients and healthy controls data were expressed as frequency ( percent ) , and the stress score was discovered to be significantly higher in patients with depression as compared to the healthy controls ( p = 0.03 ) ( table 2 ) . percentage of subjects with severe stress was significantly greater in depressed patients ( 31.8% , 14 of 44 ) as compared to the healthy subjects ( 14.6% . 54.8% ( n= 23 ) were recognized with severe depression level ( table 2 ) . as shown in table 3 , a positive correlation was observed between depression severity and stress levels ( r= 0.32 , p= 0.03 ) . there were no significant correlations between anthropometric parameters with stress level or severity of the depression ( table 3 ) stress mean score and rating in depressed patients and healthy controls hamilton rating scale for depression p < 0.05 as compared to mean stress score of controls ( total ) using man - witney u test p < 0.05 as compared to percentage of controls with severe levels of stress using chi - square test correlation of serum 25(oh)d level , depression severity and stress with potential confounding factors hrsd= hamilton rating scale for depression , p values less than 0.05 were considered statistically significant . as shown in table 4 , median ( interquartile range , iqr ) serum 25(oh ) d level of patients with depression was 10.54 ( 6.16 , 19.35 ) ng / ml compared to 8.95 ( 6.33 , 17.80 ) ng / ml in healthy controls with no statistically significant difference . serum 25(oh ) d levels did not differ between winter and spring subjects ( table 4 ) . the vitamin level was non - significantly lower in currently depressed patients , patients with depression episodes less than 2 year ( 14 ) , as compared to those who had depression 2 y ( past depression ) ( table 4 ) . totally , serum 25(oh ) d levels below 20 ng / ml were observed in 77.6% ( n= 66 ) of the participants . vitamin d deficiency was observed in 75% ( n= 33 ) of depressed patients and 80.5% ( n= 33 ) of healthy subjects with no statistically significant difference ( table 4 ) . interestingly , percentage of male subjects ( 94.4% , 17 of 18 ) with vitamin d deficiency ( < 20 ng / ml ) was remarkably greater than female subjects ( 73.1% , 49 of 67 ) ( p = 0.06 ) . vitamin d status on the base of disease duration , season , and gender data were expressed as frequency ( percent ) , and however , a higher percentage of females ( 52.2% , 35 of 67 ) had severe vitamin d deficiency ( < 10 ng / ml ) as compared to the males ( 22.2% , 4 of 18 ) ( p = 0.03 ) ( table 4 ) . totally , in patients , there were no significant correlations between disease severity and serum 25(oh)d levels , but in currently depressed patients a mild negative correlation was observed between the two variables ( r= 0.38 ) ( p = 0.08 ) compared to those who had depression 2 y ( table 5 ) . in addition , a strong negative correlation was detected between the disease severity and serum 25(oh ) d levels in the samples measured in winter ( r= 0.62 ) ( p = 0.004 ) as compared to those measured in spring ( table 5 ) . correlation of serum 25(oh ) d level with depression severity ( hrsd ) in depressed patients hrsd= hamilton rating scale for depression patients with depression episodes < 2 year ; patients with depression episodes 2 y as shown in table 3 , a mild positive association was observed between age and serum vitamin d levels ( r= 0.24 , p = 0.03 ) . there were no significant correlations between anthropometric parameters , stress level , and disease duration with serum 25(oh ) d levels ( table 3 ) . average ages of patients and healthy subjects were 36.93  12.05 and 36.97  12.9 years , respectively . there was no statistical significant difference in age , gender , anthropometric parameters , physical activity level , educational level , marital status , dietary vitamin d intake , and daily sun exposure between the two groups ( table 1 ) . in patients , the median of disease duration was 24.00 with a range of 12.00 to 60.00 months . descriptive data of depressed patients and healthy controls data were expressed as frequency ( percent ) , and the stress score was discovered to be significantly higher in patients with depression as compared to the healthy controls ( p = 0.03 ) ( table 2 ) . percentage of subjects with severe stress was significantly greater in depressed patients ( 31.8% , 14 of 44 ) as compared to the healthy subjects ( 14.6% . 54.8% ( n= 23 ) were recognized with severe depression level ( table 2 ) . as shown in table 3 , a positive correlation was observed between depression severity and stress levels ( r= 0.32 , p= 0.03 ) . there were no significant correlations between anthropometric parameters with stress level or severity of the depression ( table 3 ) stress mean score and rating in depressed patients and healthy controls hamilton rating scale for depression p < 0.05 as compared to mean stress score of controls ( total ) using man - witney u test p < 0.05 as compared to percentage of controls with severe levels of stress using chi - square test correlation of serum 25(oh)d level , depression severity and stress with potential confounding factors hrsd= hamilton rating scale for depression , p values less than 0.05 were considered statistically significant . as shown in table 4 , median ( interquartile range , iqr ) serum 25(oh ) d level of patients with depression was 10.54 ( 6.16 , 19.35 ) ng / ml compared to 8.95 ( 6.33 , 17.80 ) ng / ml in healthy controls with no statistically significant difference . serum 25(oh ) d levels did not differ between winter and spring subjects ( table 4 ) . the vitamin level was non - significantly lower in currently depressed patients , patients with depression episodes less than 2 year ( 14 ) , as compared to those who had depression 2 y ( past depression ) ( table 4 ) . totally , serum 25(oh ) d levels below 20 ng / ml were observed in 77.6% ( n= 66 ) of the participants . vitamin d deficiency was observed in 75% ( n= 33 ) of depressed patients and 80.5% ( n= 33 ) of healthy subjects with no statistically significant difference ( table 4 ) . interestingly , percentage of male subjects ( 94.4% , 17 of 18 ) with vitamin d deficiency ( < 20 ng / ml ) was remarkably greater than female subjects ( 73.1% , 49 of 67 ) ( p = 0.06 ) . vitamin d status on the base of disease duration , season , and gender data were expressed as frequency ( percent ) , and however , a higher percentage of females ( 52.2% , 35 of 67 ) had severe vitamin d deficiency ( < 10 ng / ml ) as compared to the males ( 22.2% , 4 of 18 ) ( p = 0.03 ) ( table 4 ) . totally , in patients , there were no significant correlations between disease severity and serum 25(oh)d levels , but in currently depressed patients a mild negative correlation was observed between the two variables ( r= 0.38 ) ( p = 0.08 ) compared to those who had depression 2 y ( table 5 ) . in addition , a strong negative correlation was detected between the disease severity and serum 25(oh ) d levels in the samples measured in winter ( r= 0.62 ) ( p = 0.004 ) as compared to those measured in spring ( table 5 ) . correlation of serum 25(oh ) d level with depression severity ( hrsd ) in depressed patients hrsd= hamilton rating scale for depression patients with depression episodes < 2 year ; patients with depression episodes 2 y as shown in table 3 , a mild positive association was observed between age and serum vitamin d levels ( r= 0.24 , p = 0.03 ) . there were no significant correlations between anthropometric parameters , stress level , and disease duration with serum 25(oh ) d levels ( table 3 ) . overall , we observed low serum 25(oh ) d levels in the participants studied and over three - quarters of them ( 77.6% ) were vitamin d deficient . 72.1% of male and 75.1% of female subjects in iran are living with vitamin d deficiency ( 15 ) . reported low serum 25(oh ) d levels in 79.6% of subjects , aged 20 to 69 years , in the capital city of tehran ( 16 ) . environmental conditions such as geographical location , latitude and seasons along with personal characteristics such as age , gender , race , skin color , sun protective behaviors , inadequate dietary intakes , lifestyle and cultural factors can potentially affect the serum 25(oh ) d level ( 1723 ) . given that geographical location ( 38.0667  n , 46.3000  e ) of tabriz city provides wavelength necessary to get enough sunlight , therefore , other possible factors might play a role in serum vitamin d status . limited exposure to uv due to lifestyle preferences , work schedule , and the fear of skin damage and cancer , skin - covering clothes and sunscreens are effective barriers for adequate exposure to uv rays , which impedes production of vitamin d in the skin . hence , intake of vitamin d from fortified food sources to achieve adequate levels of vitamin d appear to be necessary . the present study has also investigated association of serum levels of 25(oh)d with depressive symptoms in patients with mdd who attended to a psychiatry clinic and showed that , in total , serum 25(oh)d levels were relatively comparable in the depressed patients and healthy controls with any correlations between the vitamin levels and the disease severity . there are limited similar clinical trials in this matter and most of the studies were conducted on large general population samples with inconsistent findings . a growing body of epidemiological evidence suggests that hypovitaminosis d is associated with depressive disorders and its related clinical characteristics . in a large cohort study on population aged 1865 years indicated that low levels of 25(oh)d were associated to presence and severity of the depressive disorders ( 24 ) . in a population - based cohort study on older persons suggested that hypovitaminosis d is a risk factor for development of depressive symptoms ( 25 ) . jaddou et al . in a national population - based household sample of 4,002 jordanian participants , aged 25 years , demonstrated a significant association between depression and serum 25(oh ) d level ( 26 ) . pan et al . in a population - based study on 3262 community residents , aged 5070 , found that depressive symptoms were not associated with 25(oh ) d concentrations in middle - aged and elderly chinese ( 27 ) . zhao et al . in a survey on 3916 participants aged  20 years could not determine any associations between serum concentrations of 25(oh ) d and any rating of depression ( 28 ) . given that these studies have been performed on specific population groups in terms of age and race , together with variations in sample size , the measure used for diagnosis of depression and more importantly cut - off points used for vitamin d deficiency are key contributors to inconsistency of the findings . for example , the institute of medicine suggested serum concentration of equal or higher than 50 nmol l ( 20 ng ml ) as optimal level of 25(oh ) d. in addition , serum 25(oh ) d lower than 25 nmol l ( 10 ng ml ) and 2550 nmol l is considered as is considered vitamin d deficient and insufficient , respectively ( 13 ) . hoang et al . considered 25(oh ) d levels below than 20 ng / ml as deficient and levels between 2030 ng / ml as insufficient ( 30 ) in addition , milaneschi et al . divided 25(oh)d status into four quartiles : adequate , > 75 nmol l ; desirable , 7550 nmol l ; insufficient , set 25(oh ) d data in quartiles as follows : < 15 ng / ml ; , 15 20 ng / ml ; 20 26 ng / ml ; 26 ng / ml ( 28 ) . however , we could not show any significant associations between vitamin d and depression , but when sub - analyses was conducted on the data obtained from the participants with current depression ; we observed a remarkable negative correlation between the disease severity and serum 25(oh ) d levels as compared to the subjects with history of depression . the result is in agreement with previous findings indicating higher prevalence of vitamin d deficiency in currently depressed persons and inverse association of 25(oh ) d with symptom severity ( 14 , 24 ) . the relationship observed between low levels of vitamin d and current depression episodes suggests that hypovitaminosis d may represent an underlying biological vulnerability in initial stage of mdd . in the present study , more analysis of data collected from patients in winter season showed a strong correlation between disease severity and serum 25(oh ) d levels as compared to spring samples . however , when they did analysis by season , differential association emerged implying that a lower prevalence of depressive symptoms among persons with high serum 25(oh)d concentrations in assessments performed in november as compared to july ( 31 ) . an earlier study has been reported that vitamin d supplementation might improve depressive symptoms during winter period ( 5 ) . our finding suggests a protective effect of vitamin d on depressive symptoms and might mitigate the disease severity . results of this study did not support a protective role of vitamin d in pathogenesis of depression . however , an inverse relationship observed between the levels of vitamin d and depressive symptoms in current depression episodes , albeit statistically non - significant , and in sun - deprived seasons warrants further investigation . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
background : we investigated the association between serum 25(oh ) d levels and depressive symptoms in patients with major depressive disorder ( mdd).methods : eighty - five adults , 44 drug free patients with mdd and 41 apparently healthy controls , participated in the study . 
 the hamilton depression rating scale was used to assess severity of major depression . 
 mental health of the controls was assessed according to dsm - iv criteria . 
 stress level of the participants was assessed by the holmes and rahe stress scale . 
 serum 25(oh ) d levels was measured by immunochemiluminescence assay . 
 vitamin d deficiency was defined as a serum 25(oh ) d concentration of lower than 20 ng / ml.results : depressed patients had the higher levels of stress . 
 there was a positive correlation between stress level and disease severity ( r= 0.32 , p= 0.03 ) . 
 in total participants , mean percentage of vitamin d deficiency was 77.6% with 75% in patients and 80.5% in the healthy subjects . 
 there were no differences between the two groups in serum 25(oh ) d levels and percentage of subjects with the vitamin deficiency . 
 a negative correlation was observed between disease severity and serum 25(oh ) d level of patients with depression episodes < 2 y ( r= 0.38 , p = 0.08 ) and winter samples ( samples collected and measured from december to march , r= 0.62 , p = 0.004).conclusion : serum 25(oh ) d levels were not associated with depression . 
 however , the inverse relationship between levels of vitamin d and depressive symptoms in current depression episodes and in sun - deprived season warrants further investigation .