The association of vitamin D, semen parameters, and reproductive hormones with male infertility: A cross-sectional study

Abstract Background The prevalence of vitamin D deficiency and male infertility is high in Iran. Objective The present research aimed to examine the association between serum vitamin D [25(OH)D], parameters of semen including semen volume, sperm count, motility and morphology, and reproductive hormones in infertile Iranian men. Materials and Methods This was a cross-sectional study on 119 infertile men conducted between September 2018 and May 2019. Subjects were divided into 3 groups based on serum vitamin D levels: deficient ( < 10 ng/mL), insufficient (10 ng/mL ≤ 25(OH)D ≤ 30 ng/mL), and sufficient ( > 30 ng/mL). Body mass index and waist circumference were measured. 25(OH)D, testosterone, sex hormone-binding globulin, luteinizing hormone (LH), follicle-stimulating hormone and estradiol levels, and semen parameters were assessed. Results The semen volume, sperm counts, total and progressively motile sperm, normal sperm morphology, testosterone levels, and testosterone/estradiol ratio were substantially higher in the sufficient group compared to the other groups (p < 0.001). Also, in the sufficient group, serum LH (p < 0.001) and estradiol (p < 0.001, p = 0.01) were notably lower and serum sex hormone-binding globulin (p < 0.001) and the testosterone/LH ratio (p < 0.001) were considerably higher compared to the insufficient and deficient groups. Conclusion Our study showed a positive relationship between serum vitamin D levels, and seminal parameters and sex hormones in Iranian infertile males.


Introduction
Vitamin D deficiency is a common serious public health challenge worldwide (1). It has been considered serum 25-hydroxy vitamin D (25[OH]D) < 50 nmol/l (20 ng/ml) as deficient, 25(OH)D between 50-75 nmol/l (20-30 ng/ml) as insufficient, and a serum 25(OH)D > 75 nmol/l (30 ng/ml) as a sufficient level (2). The main source of this vitamin is its synthesis in the skin through sunlight exposure (1). It has been reported that the existence of vitamin D receptors and its metabolizing enzymes in the prostate, testis, ejaculated spermatozoa, and Sertoli cells may play some significant roles in spermatogenesis and maturation of spermatozoa.
In some cross-sectional studies, 25(OH)D deficient men were reported to have lower sperm motility, total numbers of motile sperm, sex hormonebinding globulin (SHBG), testosterone/estradiol ratio, and free androgen index (FAI) compared to 25(OH)D sufficient male subjects (3,4). On the other hand, some studies have had results that contradict these, reporting no significant relationship between 25(OH)D and parameters of semen and sex hormones (1,5,6). Therefore, the association between vitamin D and male reproductive function remains unclear.
The link between male infertility and vitamin D should be studied, especially in countries with high prevalence rates of vitamin D deficiency and male infertility like Iran. The infertility rate in Iranian couples has been reported to be 24.9%, which is higher than the global average (7). If the positive relationships between vitamin D deficiency and infertility parameters are confirmed, vitamin D as a simple, non-aggressive and cheap clinical tool could be used in future infertility treatment and assisted reproductive technologies.
One limitation of the studies that have examined vitamin D is the variability in the vitamin D reference ranges and authors' definitions of vitamin D status, which can consequently affect the obtained results.
In most of these studies, serum levels of 25(OH)D < 20 ng/mL have been considered as deficient (8)

Study population
This cross-sectional study was conducted on 119 infertile men (ranged between 20-50 yr) who were referred to Jahad Daneshgahi Fertility Clinic in Ahvaz, Iran, from September 2018 to May 2019.

Semen collection and analysis
Semen samples were taken from the included participants after 2-7 days of sexual abstinence, and these were then transferred immediately to the laboratory. These were analyzed in terms of semen volume, sperm count, progressive and total sperm motility, and normal sperm morphology parameters according to the World Health Organization 2010 criteria (10). The examination of the semen samples was done using the methods described in our previous article (9) by trained clinical technicians.

Ethical considerations
The Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran approved this study (Code: IR.AJUMS.REC.1398.313) and it was carried out in accordance with the Declaration of Helsinki.
An informed consent form was signed by all participants.

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) version 22 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The median (range), mean ± standard deviation, or number (percentage) were used to present the data. The Kolmogorov-Smirnov statistical test was used to test for normality. One-way analysis of variance (ANOVA) and the Kruskal-Wallis test were used to compare the means of groups to determine any significant differences. The Chi-square test was also used for testing relationships between categorical variables. Moreover, post hoc pairwise comparisons (for variables with normal distribution) and the Mann-Whitney test (for variables without normal distribution) were used to determine differences between means. P-values < 0.05 were considered statistically significant.

Semen analysis
Sperm volume and count, total and progressively motile sperm and normal sperm morphology were significantly different between groups.
The pairwise comparison showed that sperm volume, sperm count, motile sperm and normal sperm morphology were considerably higher  (Table II).

Hormone analysis
Serum levels of LH, SHBG, testosterone, the testosterone/estradiol ratio, the testosterone/LH ratio, and estradiol were significantly different  (Table III).

Discussion
The results of the present research showed that men with vitamin D deficiency had lower semen volume, lower sperm counts, less motile and progressive spermatozoa, and lower normal sperm morphology, SHBG levels, testosterone, testosterone/estradiol ratio and testosterone/LH ratio; however, they had higher LH and estradiol levels than men with adequate vitamin D levels.  However, no association between vitamin D levels and sperm motility was found in a previous study (15), whereas a negative correlation was found between both low and high levels of vitamin D and semen parameters in young healthy men in another study (3). Some studies have reported a positive relationship between vitamin D and sperm count (4,11) or morphology (11), while other studies have reported that vitamin D3 is neither related to total sperm count (1) nor sperm morphology (1,4).
Therefore, the association between vitamin D deficiency and the parameters of semen is still unclear. In terms of the influence of vitamin D on serum estradiol levels, no significant association was observed in some cross-sectional studies conducted on young healthy men (3,15). However, a negative relationship was found among serum levels of estradiol and 25(OH)D and ionized calcium in several studies (21,22). As vitamin D suppresses aromatase function in adipose tissue, so it may subsequently lead to a decrease in bioavailable estradiol (9). In accordance with those studies reporting a negative relationship between serum estradiol levels and 25(OH)D, in the present research, serum estradiol levels and the ratio of testosterone/estradiol were lower in the vitamin D sufficient group compared to in the sufficient and deficient vitamin D groups.
This study had some limitations such as a small sample size and the absence of a fertile control group. In addition, the levels of reactive oxygen species, calcium, phosphorus and osteocalcin were not measured in the obtained samples.
Moreover, seasonal differences in the levels of 25(OH)D across the groups were not considered in our study.

Conclusion
In conclusion, our study showed a relationship between vitamin D levels, and semen parameters and sex hormones in Iranian infertile men.
However, further studies with larger sample sizes are needed to improve our knowledge on the association between vitamin D and male infertility as well as on the underlying mechanisms.