Insufficiency of vitamin D is one of the utmost general health issues all over the globe, including Pakistan. Incompatible data are present on the possible relationship among serum vitamin D values and quality of semen with sperm motility. Lack of vitamin D was common amongst male subjects with infertility. Additional vitamin D and calcium may be the suitable treatment for subjects with post washed out total progressively motile sperm count (TPMSC) less than 5 million/ml with documented Vitamin D and Calcium deficiency  .
Insufficiency of vitamin D is well thought-out as the most important community health issue in both developed and under developing nations. In the tropical regions of the Persian Gulf as Iran, Bahrain and Saudi Arabia this is considered as widely spread health issue; because of changes in dietary habits, lifestyle alterations and also due to industrialization  .
Daylight exposure, clothing, skin pigmentations, geographical latitude, seasons, daily nutritional intake of Vitamin D, air contamination as well as overweight are the different factors related with different levels of vitamin D between different populations    .
In tropical regions the incidence of vitamin D deficiency (≤20 ng/ml) ranges between 30% and 90%   . In human spermatozoa Vitamin D Receptor (VDR) and other metabolic enzymes are located    . There is a relationship among different parameters of semen with low or high levels of vitamin D    . The semen quality depends upon the levels of vitamin D in serum; lack of this vitamin can alter the reproductive system through calcium dependent mechanism  . Studies had been conducted all over the world regarding this issue but our setup is lacking it. In current study vitamin D3 levels will be assessed in infertile male subjects to assess the occurrence of low levels of vitamin D3 in these subjects.
The present research was aimed to evaluate the association of low levels of vitamin D3 with sperm count and sperm motility in Pakistani infertile males.
1.2. Operational Definitions
1) Male Infertility:
Failure to conceive after one year of unprotected intercourse with the same partner is defined as male infertility  .
2) Vitamin D3 deficiency: 
Analysis of serum concentrations of 25(OH) D through laboratory.
Normal > 30 ng/ml,
Insufficiency > 20 and < 30 ng/ml,
Deficiency < 20 ng/ml.
2. Material and Methods
1) Design of study: The current research was cross sectional.
2) Place of study: Peoples Medical College Hospital Nawabshah.
3) Duration of study: January 2016 to December 2017.
4) Inclusion criteria: All young and middle aged males with history of infertility and semen detailed report, showing evidence of infertility in its different parameters. Having no co morbid in relation to vitamin D related like liver diseases endocrine disease metabolic disease etc. All the male patients who were reported for semen analysis due to infertility and having semen abnormality were included for vitamin D levels. Simultaneously control group was added of same age and gender for vitamin D analysis.
5) Exclusion criteria: Subjects with age less than 20 and more than 60 years, not willing to participate in the study. Subjects having history of mumps, trauma, surgery, un-descended testis, history of radiation, chemotherapy, drugs affecting sperm count and motility, sex and pituitary hormone use, brain surgery, pituitary surgery, renal disease and surgery, sexually transmitted infections.
6) Data collection:
After ethical consideration and permission this study was performed at PMCH Nawabshah. This was cross-sectional type of study; 243 male subjects who presented to clinic for assessment of infertility were included in this study from January 2106 to December 2017.
Rao-software for sample size calculation was used to calculate sample number. Total population of district Shaheed Benazirabad is approximately 1,600,000, with confidence interval of 95%, 50% response distribution a sample size of 243 subjects with infertility were selected for study after fulfilling the inclusion criteria.
141 normal fertile subjects of same age and sex were selected as control group for to compare the level of vitamin D3 in control subjects and patients having no co morbid state.
According to WHO (World Health Organization) 2010 criteria the contestants were with abnormal sperm count. Samples for semen analysis were taken after sexual abstinence of 2 - 5 days as established upon World Health Organization (WHO) criterion (2010)  .
Data was collected on pre designed proforma; every contestant provided informed written consent before the beginning of study. Questionnaires for dietary habits were also completed by subjects.
Samples of blood and semen were collected for analysis.
Results for semen analyses and vitamin D3 were entered on a proforma simultaneously to check the relation of sperm count and quality with vitamin D levels.
Blood was collected from a vein and was propelled to laboratory for the assessment of vitamin D3. Mini Vidas Biomerieux Global Company France was used for serum vitamin D3 assessment. Hypovitaminosis D was labeled when serum vitamin D levels were <30 ng/ml.
The semen was collected and analyzed for gross and microscopic examination for different parameters of semen.
7) Data analyses:
All the collected data were assessed by SPSS (Statistical Package for Social Science) version 20.0.Variables (categorical) as infertile males and serum levels of vitamin D were computed for assessment of frequency and percentages. Clinical, qualitative and quantitative variables were calculated for Mean ± SD, median and percentages for sperm count.
Mean and standard deviation were aimed for quantitative variables like, age and levels of vitamin D3. The t-test, one-way Anova were used to compare in between groups. Pearson correlation analyses were done to evaluate the relationship among various parameters.
Importance of serum levels of vitamin D were perceived for age, infertility, semen analyses with different parameters to see the impact of these on outcomes. P < 0.05 was taken statistically significant. Chi-square test was used for the assessment of differences in ratios. Association amongst serum levels of vitamin D3, male infertility and semen analysis were explored through bivariate correlation analysis.
3.1. Descriptive Statistics
In this current study 243 subjects presented with infertility, all were male, the minimum age was 20 years while maximum age was 45 years, mean age was 31.94 years with standard deviation of 7.52 years. The mean age of controls was 32.2 years with SD 8.42 years, age ranged between 20 years as minimum to 46 years as maximum.
The minimum marriage duration was 1 year while maximum 25 years, mean marriage duration was 31.94 years and standard deviation was 5.70. The minimum vitamin D level was 7.00 ng/dl while maximum 130 ng/dl, mean was 26.20 and standard deviation was 22.06 ng /ml.
The mean vitamin D level of controls was 49.40 with SD of 35.98 ng/ml. 28% of controls had also vitamin D levels below the normal range, which indicates the prevalence of vitamin D deficiency in general population here in our setup.
The minimum serum calcium level was 07.2 while maximum 10.80, mean was 08.82 and standard deviation was 0.96 mg%. The mean serum calcium of control was 09.4 with SD of 0.88 mg%.
The minimum semen quantity was 1ml while maximum 5ml, mean was 2.07 ml and standard deviation was 1.40 ml. The minimum total sperm count was 0.00 million while maximum was 120 millions, mean was 25.91 and standard deviation was 26.41. The minimum percentage of motile sperms was 0.00% while maximum was 80%, mean was 29.73 and standard deviation was 26.33. The minimum percentage of rapid linear progression of sperms was 0.00% while maximum 20.00% mean was 2.41 and standard deviation was 3.75. The minimum percentage of slow non linear progression was 0.00% while maximum was 60%, mean was 12.11 and standard deviation was 13.11. The minimum percentage of non motile non progressive sperms was 0.00% while maximum was 100%, mean was 61.60 and standard deviation was 36.83. The minimum percentage of dead sperms was 0.00% while maximum was 95%, mean was 48.00 and standard deviation was 33.70. The minimum percentage of normal forms of sperms was 0.00% while maximum 100% mean was 49.40 and standard deviation was 35.98. The minimum percentage of abnormal sperms was 0.00% while maximum was 95%, mean was 28.78 and standard deviation was 29.02. The minimum germ cell per high power field (HPF) was 0.00 while maximum was 2.0, mean was 0.32 and standard deviation was 0.65. The minimum RBCs per high power field (HPF) was 0.00 while maximum was 10.00, mean was 3.20 and standard deviation was 2.14. The minimum pus cells per high power field (HPF) was 2 while maximum was 10.0, mean was 4.78 and standard deviation was 2.07. The minimum average sun exposure duration/24 hours was 01 hour while maximum was 05 hours, mean was 1.71 and standard deviation was 1.08 (Table 1).
3.2. Demographic Statistics
In Table 2 it is observed that 187 (77%) males were belonging to young age group while 56 (23%) were belonging to middle age group. History of sun exposure was <01 hour in 147 (60.5%), 2 - 3 hours in 49 (20.2%), 3 - 4 hours in 26 (10.7%), 4 - 5 hours in 11 (4.5%) and >5 hours in 10 (4.1%) subjects. A total of 219 (90.1%) belonged to rural setup while 24 (9.9%) were urban. Socioeconomic class had shown dominant ratio of lower economic class 221 (90.9%), while 15(6.2%), 7 (2.9%) belonged middle and upper class respectively. Occupation wise percent had shown that 126 (51.9%) were indoor workers while 90 (37%)
Table 1. Descriptive statistics n = 243.
Table 2. Frequency and percentage of demographic variables n = 243.
were out door workers and remaining 27 claimed no occupation. Vitamin D levels were within normal or sufficient range in 79 (32.5%), it was below normal range in 164 (67.5%) cases, while insufficient in 17 (7.0%) and deficient in 147 (60.5%) cases of infertility Table 2.
3.3. Vitamin D and Semen Quantity
Numbers of valid cases were 243 infertile males. The quantity of semen in ml were counted 126 cases have semen quantity 1 ml, 46 cases 2 ml, 29 cases 3 ml, 11 cases 4 ml and 31 cases 5 ml. Out of 126 cases having semen quantity 1 ml, 51 had normal range vitamin D level, while 75 cases were below normal range. From 46 cases having 2ml semen per ejaculate 9 cases has normal vitamin D levels, while 37 with lower than normal. From 29 cases having 3ml semen per ejaculate 6 cases has normal vitamin D levels, while 23 with lower than normal. From 11 cases having 4ml semen per ejaculate 4 cases has normal vitamin D levels, while 7 with lower than normal. From 31 cases having 5 ml semen per ejaculate 9 cases has normal vitamin D levels, while 22 with lower than normal. Vitamin D levels were normal in 79 cases of infertility while they were insufficient and deficient in 17 and 147 cases respectively. Pearson chi square was 10.026, df 8, Asymp. sig. (2-sided) 0.263. Likelyhood ratio 10.277 df 8, Asymp. sig. (2-sided) 0.246. Linear by linear association was 2.202, df 1, Asymp. sig. (2-sided) 0.138. Interval by interval pearsons R value was 0.095, Approx. Sig 0.138. Ordinal by ordinal Spearman correlation value was 0.130, Approx. Sig 0.042 (Table 3).
3.4. Vitamin D and Total Sperm Count
Out of 243 cases 53 (21.8%) were having sperm count nil. While 190 (79.2%) cases having different count of sperm ranging from 02 million to 120 million. From these subjects 79/243 had normal range vitamin D levels while 164 had below normal range vitamin D levels. Pearson chi square was 175.201, DF 28, Asymp. sig. (2-sided) p < 0.001. Likelyhood ratio 182.638, df 28, Asymp. sig. (2-sided) p < 0.001 Linear by linear association was 94.038, df 1, Asymp. sig. (2-sided) p < 0.001 Interval by interval pearsons R value was −0.623, Approx.
Table 3. Vitamin D deficiency and semen quantity (normal 2 - 4 ml) n = 243.
Sig. p < 0.001 Ordinal by ordinal Spearman correlation value was −0.541, Approx. Sig p < 0.001 Table 4.
3.5. Vitamin D and Sperm Motility
Regarding the sperm motility normal range was 70% - 90%. Over all Pearson chi square was 103.788, df 22, Asymp. sig. (2-sided) p < 0.001. Out of 243 cases 53 (21.8%) were having sperm count nil. While 190 (79.2%) cases having different percentage of sperm motility ranging from 5% - 80%. From these subjects 79/243 had normal range vitamin D levels while 164 had below normal range vitamin D levels. Likelyhood ratio 117.412 df 22, Asymp. sig. (2-sided) p < 0.001. Linear by linear association was 44.488, df 1, Asymp. sig. (2-sided) p < 0.001. Interval by interval pearsons R value was −0.429, Approx. Sig p < 0.001. Ordinal by ordinal Spearman correlation value was −0.414, Approx. Sig p < 0.001 Table 5.
Table 4. Vitamin D deficiency and total sperm count (normal 40 - 200 M/Ml) n = 243.
Table 5. Vitamin D deficiency and motile sperm (normal 70% - 90%) n = 243.
3.6. Vitamin D and Normal Sperm Count
The normal forms were considered if they are more than 4%. In 21.8% there were no sperm at all; while in rest there were different percentages of normal sperms ranging from 5% to 100%. Only 1.2% cases were with 100% normal forms , while others were with different percentages as shown in Table 6, 79.2% cases were of normal forms but out of them 79 were with normal range of vitamin D and rest of all 164/243 were vitamin D deficient. Pearson chi square was 96.799, df 20, Asymp. sig. (2-sided) p < 0.001 Likelyhood ratio 112.519, df 20, Asymp. sig. (2-sided) p < 0.001, Linear by linear association was 37.007, df 1, Asymp. sig (2-sided) p < 0.001. Interval by interval pearsons R value was −0.391, Approx. p < 0.001. Ordinal by ordinal Spearman correlation value was −0.445, Approx. Sig p < 0.001 (Table 6).
3.7. Vitamin D and Abnormal Sperm Count
Abnormal sperm morphology had been observed in semen analysis, in context to vitamin D levels. 23.0% cases had no abnormality while 775 cases were with different percentages of abnormality ranged from 10% to 95%. Pearson chi square was 88.583, df 18, Asymp. sig. (2-sided) p < 0.001. Likelyhood ratio 103.671 df 18, Asymp. sig. (2-sided) p < 0.001 Linear by linear association was 17.195, df 1, Asymp. sig. (2-sided) p < 0.001 Interval by interval pearsons R value was .267, Approx. Sig p < 0.001. Ordinal by ordinal Spearman correlation value was .225, Approx. Sig p < 0.001 (Table 7).
3.8. Vitamin D and Dead Sperm Count
In current study there were 21.8% cases with no sperm, while 78.2 percent with different percentages of dead sperms ranged from 20% to 95% as shown in Table 8 in relation to different levels of vitamin D. Pearson chi square was 122.850, df 20, Asymp. sig. (2-sided) p < 0.001. Likelyhood ratio 135.612 df 20, Asymp. sig. (2-sided) p < 0.001 Linear by linear association was 6.869, df 1, Asymp. sig. (2-sided) p < 0.001. Interval by interval pearsons R value was 0.168, Approx. Sig p < 0.008, Ordinal by ordinal Spearman correlation value was 0.203, Approx. Sig p < 0.001 (Table 8).
Table 6. Vitamin D deficiency and normal form sperms n = 243.
Table 7. Vitamin D deficiency and abnormal sperm% n = 243.
Table 8. Vitamin D deficiency and dead sperm n = 243.
3.9. Vitamin D and Sun Exposure
A total of 65.5% of subjects were having sun exposure less than 1 hour, 20.25 having 2 - 3 hours, 10.7% having 3 - 4 hours, 4.5% having 4 - 5 hours and 4.15 have history of sun exposure more than 5hours daily. Pearson chi square was 95.257, df 8, Asymp. sig. (2-sided) p < 0.001 Likely hood ratio 104.121 df 8, Asymp. sig. (2-sided) p < 0.001 Linear by linear association was 77.130, df 1, Asymp. sig. (2-sided) p < 0.001, Interval by interval Pearsons R value was −0.565, Approx. Sig p < 0.001. Ordinal by ordinal Spearman correlation value was −0.597, Approx. Sig p < 0.001 (Table 9).
Table 9. Vitamin D deficiency and sun exposure n = 243.
3.10. Vitamin D and Rapid Linear Progression of Sperms
There was strong association and significant correlation between vitamin D levels and rapid linear progression movement of sperms as shown in graph 1. As the vitamin D level increases the linear progression of sperms also increases. Rapid linear movements were more decreased in subjects within low vitamin D (Figure 1).
3.11. Vitamin D and Semen (Paired Statistic and Correlations)
The paired sample testing and paired samples correlation were analyzed and found statistically significant. Various means and standard deviations with standard error of mean were checked in parallel to correlations and significance as shown in the p-value was statistically significant vitamin D level with motile sperms p < 0.001 calcium level p < 0.001 rapid linear progression p < 0.001 slow/non linear progression p < 0.001 non motile sperms (p < 0.590), dead sperms (p < 0.108), normal forms of sperms p < 0.001, abnormal form sperms p < 0.001 and head abnormalities (0.239) (Table 10).
Table 10. Paired samples statistics and correlations of vitamin D and semen parameters n = 243.
Figure 1. Linear and logistic Correlation of Vitamin D Level with Rapid Linear Progression of Sperms.
3.12. Vitamin D and Semen Parameters (Paired Sample Tests)
In relation to semen parameters and vitamin D levels paired sample test was performed with mean and SD, upper and lower limits, with 95% confidence interval as shown in Table 11, the p-value was statistically significant vitamin D level with motile sperms (p < 0.05), calcium level p < 0.001 rapid linear progression p < 0.001 slow/non linear progression p < 0.001 non motile sperms p < 0.001 dead sperms p < 0.001 normal forms of sperms p < 0.001 abnormal form sperms (p < 0.319) and head abnormalities p < 0.001 (Table 11).
Issue of infertility is common all over the world. It is bilateral some time on male side and some time on female side and rarely both sides. They are primary and secondary. They are correctable in some situations while not in many like azoospermia. Some genetic diseases are also responsible for infertility in male and females. A social, cultural, ethical and religious value plays a great role in infertility. When a male partner is infertile his wife is not allowed for fertility with other male due to social, cultural, ethical and religious values. Simultaneously when a female is infertile the same social, cultural, ethical and religious values allows male to marry other women.
Table 11. Vitamin D deficiency and semen parameters paired samples test n = 243.
Lot of factors are responsible for infertility in males, very recently Vitamin D has been recognized as one of contributing factor in male infertility.
In current study vitamin D level were analyzed in male subjects who reported with primary infertility. The results of current study were noticeable because the vitamin D levels were below normal range in 67% of cases as compared to control subjects where it was 28%. This indicates the relationship of vitamin D and infertility.
Pakistan is a developing nation with decreased resources and deprived population growth control. Limitations in the health management system are major issues. Current study was carried out to observe the association of serum vitamin D levels in subjects with infertility. Sun exposure is sufficient around the year in most parts of the country, but concept of sunbath is negligible. In females sun exposure is limited due to parda, but males usually remain outdoors during daytime mostly for earning purpose. Usually health problems are ignored due to unsatisfactory health education  .
Vitamin D is acquired through nutritional consumption. Ultraviolet B radiation (UV-B) from sun exposure is required for synthesis of Vitamin D in the derma. Endogenous vitamin D (cholecalciferol or D3) is biologically inactive form. UV-B rays in the skin are needed for conversion of 7-dehydroxycholesterol to cholecalciferol D3. Nutritional forms of vitamin D are D2 and D3. Fungi and yeast in the presence of UV rays are needed for synthesis of D  .
In current study 60.5% of the subjects were having history of sun exposure less than 01 hour.
This may be the strong risk factor contributing to vitamin D deficiency and creating a bias also.
Regulation of calcium is maintained by vitamin D, for performance of reproductive functions such as hyper activation, acrosomal reaction, spermatogenesis, and sperm motility calcium is the vital component   .
The ion of calcium plays vital role in the fertilization at different levels of sperm journey from ejaculate to penetration into ovum for fertilization. Almost all the steps like rapid movement, attraction towards ovum, attachment and penetration are calcium dependent  .
In current research the mean serum calcium was very low, which may be also additional factor for decreased motility of sperms and their rapid linear progression which was markedly decreased in current analysis.
Hypertension, cardiovascular disease, stroke, diabetes, and cancer as well as serum androgens had also been associated with vitamin D deficiency. The role of vitamin D in male reproduction is not yet resolved   . No any difference before and after alteration of season (spring and summer) had been noted  .
Our research supports the statement that vitamin D deficiency is also one of contributing risk factors in males with infertility.
Required concentrations of vitamin D for reproductive system and other organ systems are still not established. The role of vitamin D deficiency in decreased reproductive activity is still unclear and further studies in regard of pathogenesis and treatment in infertility are required  .
In Pakistan no organized diet schedule had been implemented to combat the vitamin D deficiency. Lot of research is needed to sort out the effect and relation of vitamin D with fertility.
Some studies had shown a weak association between sex hormone, vitamin D and phosphorus, while in other studies no relationship was observed among vitamin D and sex hormones   . It may be concluded from reports that the hypothalamic-pituitary-gonadal axis does not affect the outcome of different parameters of sperm in vitamin D deficient subjects, some studies also concluded no relationship of vitamin D deficiency with sperm count and concentrations    .
This study only covers the relation of vitamin D and infertility, no hormonal analysis was done because of limited resources. Efforts should be done on large scale in different areas of Pakistan to confirm the correlation of vitamin D and sex hormones.
In a research (cross sectional) by Yang et al. affirmative relationship among sperm motility and vitamin D levels was observed  .
In current research there was strong correlation of sperm motility and vitamin D levels p ≤ 0.000
In humans elongated and round spermatids were communicated with VDR as well as enzymes that metabolize the vitamin D  .
Calcium homeostasis influence the compromised reproductive ability of the body that may be induced by vitamin D deficiency, it indicates that the compromised reproductive ability can be corrected by supplementation of calcium and vitamin D  .
It has been observed in current research that vitamin D has a significant correlation with calcium p ≤ 0.000.
Consequently it had proposed that sperm motility might be affected by deficiency and/or insufficiency of vitamin D  .
Results of current study also favors above statement that there is linear relationship of there is strong relation of sperm motility and vitamin D levels.
The role of vitamin D had been established in various organs and tissues, mostly in male reproductive activity and spermatogenesis, but the precise mechanism by which vitamin D effects this is still unclear  .
The results of present study concluded that infertile males are vitamin D deficient as compared to controls.
The optimum sperm ability may directly depend on the consequence of vitamin D, but the indirect influence of calcium homeostasis on impaired infertility as observed in animal models was restored after correction of serum calcium levels    .
In present study the rapid linear movement of sperms was markedly lower in subjects with infertility p < 0.000 which was statistically significant.
Sperm count was observed below the WHO recommended levels in 20% of young subjects and 40% of subjects had sperm concentrations below the optimal level considered for fertility  .
The total sperm count was 00.00 to 120million but mean sperm count was 25.91 million which was below fertile levels in our subjects.
Sh. Abbasihormozi et al. noted a substantial association among sperm motility, and serum levels of calcium and vitamin D in OAT (oligo-astheno-teratozoospermia) group  .
In present study the abnormal sperm percentage was with mean level 28.78 with SD29.04, this was also matchable with other studies. There was statistically significant association and relationship among sperm count, motility, morphology, serum calcium and vitamin D levels.
This is a small study representing a local population.
The mass results of vitamin D levels are not available on large scale as reference from population defined. Few studies are available in relation to semen and vitamin D worldwide. Our setup lacks that.
Local temperature is very high sometimes at world record levels here. So it affects the sperm count and viability.
Root level causes of infertility are not searched as reference here, so we can search on large scale.
It is hard to share the reports for research purpose especially semen analyses because of social, cultural and religious values.
Vitamin D3 levels are low in infertile males and are associated with semen parameter abnormalities significantly affecting the male fertility. It does not only affect the count of sperm but morphology, chemistry and function of sperms. Low vitamin D3 levels are associated with abnormal movements of sperms with decrease rapid linear progression, slow or no progression leading to infertility in males.
The authors of this research are very grateful to the study participants for their cooperation and the health management Mr Parkash Kumar Lohana and working staff of Jhooly Lal diagnostic Centre (registered) Nawabshah. We also sincerely thank the Faculty of Medicine for their involvement in data collection.
Conflict of Interests
The authors declare no conflict of interest for this study.
There is no funding from institutional and any other Governmental and nongovernmental organizations.
Contribution of Authors
Jamali AA, Tanwani BM and Jamali GM planned the current study, also had contribution in all aspects for research as data gathering, scrutiny, explanation and in writing of the document. Other authors took part in the data gathering. The study was supervised by Jamali AA. The manuscript was checked and approved by all writers.
 Özdemir, E., Tokmak, A., Erkilinç, S., Yakut, H.I., Erkaya, S. and Yilmaz, N. (2015) Association between Vitamin D Levels and Semen Parameters in Infertile Males. Journal of Clinical and Analytical Medicine, 6, 765-769.
 Fields, J., Trivedi, N.J., Horton, E. and Mechanick, J.I. (2011) Vitamin D in the Persian Gulf: Integrative Physiology and Socioeconomic Factors. Current Osteoporosis Reports, 9, 243-250.
 Hosseinpanah, F., Pour, S.H., Heibatollahi, M., Moghbel, N., Asefzade, S. and Azizi, F. (2010) The Effects of Air Pollution on Vitamin D Status in Healthy Women: A Cross Sectional Study. BMC Public Health, 10, 519.
 LeFevre, M.L. (2015) Screening for Vitamin D Deficiency in Adults: U.S. Preventive Services Task Force Recommendation Statement. Annals of Internal Medicine, 162, 133-140.
 Overton, E.T., Chan, E.S., Brown, T.T., Tebas, P., McComsey, G.A., Melbourne, K.M., Napoli, A., Hardin, W.R., Ribaudo, H.J. and Yin, M.T. (2015) Vitamin D and Calcium Attenuate Bone Loss with Antiretroviral Therapy Initiation: A Randomized Trial. Annals of Internal Medicine, 162, 815-824.
 Holick, M.F., Binkley, N.C., Bischoff-Ferrari, H.A., Gordon, C.M., Hanley, D.A., Heaney, R.P., Murad, M.H. and Weaver, C.M. (2011) Evaluation, Treatment, and Prevention of Vitamin D Deficiency: An Endocrine Society Clinical Practice Guideline. The Journal of Clinical Endocrinology & Metabolism, 96, 1911-1930.
 Aquila, S., Guido, C., Middea, E., Perrotta, I., Bruno, R., Pellegrino, M. and Ando, S. (2009) Human Male Gamete Endocrinology: 1alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) Regulates Different Aspects of Human Sperm Biology and Metabolism. Reproductive Biology and Endocrinology, 7, 140.
 Blomberg, J.M., Nielsen, J.E., Jorgensen, A., Rajpert-De, M., Kristensen, D.M., Jorgensen, N., Skakkebaek, N.E., Juul, A. and Leffers, H. (2010) Vitamin D Receptor and Vitamin D Metabolizing Enzymes Are Expressed in the Human Male Reproductive Tract. Human Reproduction, 25, 1303-1311.
 Hammoud, A.O., Meikle, A.W., Peterson, C.M., Stanford, J., Gibson, M. and Carrell, D.T. (2012) Association of 25-Hydroxy-Vitamin D Levels with Semen and Hormonal Parameters. Asian Journal of Andrology, 14, 855-859.
 Yang, B., Sun, H., Wan, Y., Wang, H., Qin, W., Yang, L., Zhao, H., Yuan, J. and Yao, B. (2012) Associations between Testosterone, Bone Mineral Density, Vitamin D and Semen Quality in Fertile and Infertile Chinese Men. International Journal of Andrology, 35, 783-792.
 Tak, Y.J., Lee, J.G., Kim, Y.J., Park, N.C., Kim, S.S., Lee, S., Cho, B.M., Kong, E.H., Jung, D.W. and Yi, Y.H. (2015) Serum 25-Hydroxyvitamin D Levels and Testosterone Deficiency in Middle-Aged Korean Men: A Crosssectional Study. Asian Journal of Andrology, 17, 324-328.
 Cooper, T.G., Noonan, E., von Eckardstein, S. Auger, J., Baker, H.W., Behre, H.M., et al. (2010) World Health Organization Reference Values for Human Semen Characteristics. Human Reproduction Update, 16, 231-245.
 Shen, L. and Ji, H.F. (2015) Associations between Vitamin D Status, Supplementation, Outdoor Work and Risk of Parkinson’s Disease: A Meta-Analysis Assessment. Nutrients, 7, 4817-4827.
 Jamali, A.A., Jamali, G.M., Tanwani, B.M., Jamali, N.H. and Bhatia, M.R.. (2018) Frequency of Low Vitamin D3 Levels in Subjects with Parkinson’s Disease. A Study Conducted at PMCH, a Tertiary Care Hospital, Nawabshah. Advances in Parkinson’s Disease, 7, Article ID: 82630.
 Benoff, S., Cooper, G.W., Hurley, I., Mandel, F.S., Rosenfeld, D.L., Scholl, G.M., Gilbert, B.R. and Hershlag, A. (1994) The Effect of Calcium Ion Channel Blockers on Sperm Fertilization Potential. Fertility and Sterility, 62, 606-617.
 Rahman, M.S., Kwon, W.S. and Pang, M.G. (2014) Calcium Influx and Male Fertility in the Context of the Sperm Proteome: An Update. BioMed Research International, 2014, Article ID: 841615.
 Chowdhury, R., Warnakula, S., Kunutsor, S., Crowe, F., Ward, H.A., Johnson, L., Franco, O.H., Butterworth, A.S., Forouhi, N.G., Thompson, S.G., Khaw, K.T., Mozaffarian, D., Danesh, J. and Di Angelantonio, E. (2014) Association of Dietary, Circulating, and Supplement Fatty Acids with Coronary Risk: A Systematic Review and Meta-Analysis. Annals of Internal Medicine, 160, 398-406.
 Abbasihormozi, Sh., Kouhkan, A., Alizadeh, A.R., Shahverdi, A.H., Nasr-Esfahani, M.H., Sadighi Gilani, M.A., Salman Yazdi, R., Matinibehzad, A. and Zolfaghari, Z. (2016) Association of Vitamin D Status with Semen Quality and Reproductive Hormones in Iranian Subfertile Men. Andrology, 5, 113-118.
 Lee, D.M., Tajar, A., Pye, S.R., Boonen, S., Vanderschueren, D., Bouillon, R., O’Neill, T.W., Bartfai, G., Casanueva, F.F., Finn, J.D., Forti, G., Giwercman, A., Han, T.S., Huhtaniemi, I.T., Kula, K., Lean, M.E., Pendleton, N., Punab, M. and Wu, F.C. (2012) Association of Hypogonadism with Vitamin D Status: The European Male Ageing Study. European Journal of Endocrinology, 166, 77-85.
 Wulaningsih, W., Van Hemelrijck, M., Michaelsson, K., Kanarek, N., Nelson, W.G., Ix, J.H., Platz, E.A. and Rohrmann, S. (2014) Association of Serum Inorganic Phosphate with Sex Steroid Hormones and Vitamin D in a Nationally Representative Sample of Men. Andrology, 2, 967-976.
 Blomberg Jensen, M., Bjerrum, P.J., Jessen, T.E., Nielsen, J.E., Joensen, U.N., Olesen, I.A., Petersen, J.H., Juul, A., Dissing, S. and Jorgensen, N. (2011) Vitamin D Is Positively Associated with Sperm Motility and Increases Intracellular Calcium in Human Spermatozoa. Human Reproduction, 26, 1307-1317.
 Ramlau-Hansen, C.H., Moeller, U.K., Bonde, J.P., Olsen, J. and Thulstrup, A.M. (2011) Are Serum Levels of Vitamin D Associated with Semen Quality? Results from a Cross-Sectional Study in Young Healthy Men. Fertility and Sterility, 95, 1000-1004.
 Kidroni, G., Har-Nir, R., Menezel, J., Frutkoff, I.W., Palti, Z. and Ron, M. (1983) Vitamin D3 Metabolites in Rat Epididymis: High 24,25-Dihydroxy Vitamin D3 Levels in the Cauda Region. Biochemical and Biophysical Research Communications, 113, 982-989.
 Uhland, A.M., Kwiecinski, G.G. and DeLuca, H.F. (1992) Normalization of Serum Calcium Restores Fertility in Vitamin D-Deficient Male Rats. The Journal of Nutrition, 122, 1338-1344.
 Menegaz, D., Rosso, A., Royer, C., Leite, L.D., Santos, A.R. and Silva, F.R. (2009) Role of 1alpha, 25(OH)2 Vitamin D3 on alpha-[1-(14)C] MeAIB Accumulation in Immature Rat Testis. Steroids, 74, 264-269.