Evaluation of vitamin D-deficiency effects on the incidence of uterine fibroids

Uterine fibroids are a very common benign tumor in women of reproductive age due to unknown aetiology. There is growing evidence that vitamin D deficiency has a potential role in developing uterine fibroids. This study aims to investigate the effect of vitamin D levels in patients with uterine fibroids in Hilla city (a sunny city). The study was conducted on 70 women diagnosed with uterine fibroids that visited private clinics and were referred to biochemical laboratories. Vitamin D levels were assessed in patients’ sera, and questionnaire data on age, body mass index, symptoms and drugs used were analysed. In addition, information from their ultrasound scans of the fibroids, including the size and the number of fibroids, were also recorded. The results demonstrated that more than half of patients had vitamin D deficiency and 25% of them were with insufficient vitamin D levels. The age group, 41-45 year, showed the lowest values, whereas no significant differences in vitamin D levels were observed in body mass index and severity of symptoms parameters. The results also revealed that a significant decrease in vitamin D levels was associated with large size and multiple fibroids. It can be concluded that vitamin D has a great implication on the incidence of uterine fibroids, even in sunny cities.

Vitamin D, Uterine fibroid, Hypovitaminosis, Small burden, Leiomyoma Research Article S. Al-azzawi et al. Pharm Sci Asia 2022; 49(4), 333-339 334 management, and in the first place among these strategies is the prevention of its occurrence. It is known that vitamin D3 acts as a regulator of calcium homeostasis in addition to its strong antifibrotic activity14. Moreover, studies have reported vitamin D3 as a potent antitumor factor that efficiently inhibits uterine fibroid in cell culture and shrinks fibroid in animal studies. However, there are not enough clinical trials conducted in the human uterine regarding this area of research1,12,15-16. Vitamin D deficiency is associated with enormous unfavorable conditions, in particular, osteomalacia and rickets17. Low levels of vitamin D have previously been detected in women with osteoporosis and metabolic syndrome, which in turn emphasizes the value of vitamin D in women’s general health18-19. Vitamin D can be found within the diet, such as cod liver oil, oily fish and dairy products. However, the main vitamin D source is sun exposure, where ultra-violet light transforms 7-dehydrocholesterol in the skin to vitamin D via a nonenzymatic isomerization. After that, vitamin D is subjected to metabolic biotransformation in the liver by 25α-hydroxylase to 25 -hydroxyvitamin-D; which is then converted in the kidney to the active form (1,25 - dihydroxyvitamin-D) (vitamin D3) by the action of 1α- hydroxylase20. Vitamin D3 exerts its biological activities in cells by interacting with its specific receptor, vitamin D receptor (VDR). VDR is “a nuclear transcription factor”, it plays an important role in gene expression modulation and trans cription. VDR affects the cell signalling such as “growth arrest, differentiation and/or induction of apoptosis”, which in turn inhibits cell growth1,21. It has been recently suggested that vitamin D defi- ciency is associated with increasing uterine fibroids risk 1-2,4,22. Vitamin D acts as a potent antiprogesteronic and antiestrogenic compound and can inhibit leiomyoma cell proliferation. Furthermore, vitamin D was found to inhibit the growth of fibroid cells and also promotes cell apoptosis in in vitro culture, whereas in in vivo animal models, it reduced the fibroid size. Several previous epidemiological studies conducted in different populations emphasized the importance of vitamin D deficiency in the development of UFs 24-26. Nevertheless, no study so far has ever addressed this issue in the Iraqi population (country in the Middle East) especially in Hilla city (located in the middle area of Iraq) where it is considered a hot region with high levels of sunlight exposure. Therefore, this study aims to investi- gate the relationship of occurrence of fibroids in women diagnosed with uterine fibroid with vitamin D deficiency in Hilla city. 2. SUBJECTS AND METHODS The cross -section study was conducted in 2021 during the summer season, from June to October, to ex- clude deficiency of vitamin D associated with the winter season, on a sample of 70 subjects. The participants were recruited among the female patients attended private gynecological clinics and were referred to biochemical analytical laboratories in Hilla city. A female subject was considered eligible to partici- pate in the study if she presented with uterine fibroid that was detected by ultrasound scan (transvaginal or abdo - minal) and all types of fibroids (subserosal, submucosal, intramural and pedunculated fibroids) as appeared in the reports were included. Information from their ultrasound scan reports were considered as variables in the study including the size of fibroids; small (3 cm) 27 and number of fibroids (single or multiple). The medical history and clinical features for each patient were recorded as a questionnaire to include the following parameters: age, “body mass index (BMI)”, previous or current intake of vitamin D supplements or not and a history of disease. Fibroids-related symptoms such as menstrual disorders and pelvic pain (which are classified as minor, mild or severe) were also reported. Women with a history of intake of vitamin D supple - ments and those who followed a special diet regimen due to weight lowering or disease were excluded from the study. In addition, women with chronic disease (such as cardiovascular and central nervous system disorders, diabetes mellitus, autoimmune diseases) were also excluded. Vitamin D3 lev els were obtained from laboratory biochemical analysis reports which were measured in the serum of participants using MINI VIDAS system (France)28 to study the effect of vitamin D3 levels on the incidence of UF. Vitamin D deficiency was deemed when serum levels were 30 ng/mL29. Data of the patients with vitamin D3 deficiency and insufficiency were analyzed to evaluate the relationship between vitamin D and the tested variables (age, BMI, severity of symptoms and the size and number of fibroids). To study the relationship of vitamin D concentration with age factor, participants were stratified into four groups (45 years) whereas for BMI factor, they were grouped into four groups (40). 2.1. Statistical analysis The data in this work were collected and expressed as percentages or means±standard deviation (represented by error bars) using Excel Microsoft. The data were analyzed by ANOVA an d Tukeys test using Minitab software, and probability <0.05 is considered for the significant differences. Pharmaceutical Sciences Asia 335 3. RESULTS AND DISCUSSION In this study, 70 women diagnosed with fibroids were recruited and their vitamin D3 serum levels were evaluated. The results revealed that 56% of the parti ci- pants were with deficient vitamin D3 values (30 ng/ mL) (Figure 1). Figure 1. Percentages of women having normal, insufficient and deficient vitamin D levels associated with uterine fibroids. To evaluate the association of vitamin D deficiency with the age factor, women were stratified into four age groups. It is of note that the youngest women (45 showed significant dif- ferences (p<0.05) in vitamin D levels from the <35 group. Significant differences (p<0.05) were found in compari- son with the group of control women matched per age (Figure 2). Figure 2. Vitamin D levels within age subgroups for women with uterine fibroids with comparison to control groups. Significant diffe- rences (p40 subgroups and <40 subgroups(+). This is consistent with the previous findings that have documented a positive correlation between age and serum vitamin D concentrations31-32. It has been reported that low vitamin D levels associated with increasing age is due to several factors including reduction in calcium absorption with increasing “intestinal resistance” to vitamin D31, low expression of VDR5, and reduced renal production of vitamin D by the kidneys 31. Obesity and lack of outdoor activity by the older women may also contribute to the vitamin D deficiency33. Therefore, con- sumption of vitamin D-rich food with increasing sunlight exposure are required for people aged more than 41 years to avoid vitamin D deficiency. The data regarding BMI showing the lowest vita - min D3 levels were noticed with high BMI (>30) group, whereas the highest levels of the vitamin (19.1±5 ng/mL) were observed with low BMI (0.05) within the tested subgroups (Figure 3). Figure 3. Vitamin D levels within BMI subgroups for women with uterine fibroids. Considering other studies, it has been shown that obesity is “independently correlated” with vitamin D deficiency34. A previous study has demonstrated that vitamin D deficiency was found in more than 50% of obese individuals (BMI ≥40)35. The relationship between obesity and vitamin D is not clear and still unknown, and may be explained by the presence of “excess body fat tissue” that retain vitamin D resulting in an increase in the volume of distribution of the vitamin, which in turn causes a decrease in the circulatory vitamin D levels. Alternatively, metabolic syndromes could also be the reason behind the vitamin D deficiency as overweight is considered as one of its main components34. It has been found that vitamin D deficiency is associated with meta- bolic syndrome especially in postmenopausal women18. Further explanation may link obesity with vitamin D deficiency is that obese individuals have higher cortical tissue mineral density and cortical thickness than thin ones have or may due to differences between obese and normal subjects in liver enzymes expression that activate vitamin D33. The severity of symptoms associated with UF was S. Al-azzawi et al. Pharm Sci Asia 2022; 49(4), 333-339 336 also evaluated to detect any suspected effects of vitamin D deficiency. In this regard, women with mild to mode- rate symptoms were associated with the lowest values of vitamin D (16.3±2 ng/mL) while highest values (20.2± 4 ng/mL) were detected in individuals with minor symp- toms (Figure 4). However, there were no significant differences (p>0.05) within groups of severity of symp- toms (minor, mild or severe). These controversial results could be attributed to the inaccurate information given by some patients or some of them have tolerated these symptoms or underestimated them (pain-threshold varia- tions from patient to patient). Figure 4. Vitamin D levels according to severity of symptoms asso- ciated with uterine fibroids. To identify any potential correlation between vita- min D deficiency and the incidence of UF, serum levels were investigated according to the size and number of fibroids in the involved subjects. As expected, th e con- centrations of vitamin D were found to be significantly (p<0.05) lower in patients with large size fibroids (14.8 ±2 ng/mL) than in those have small ones (20.9±3 ng/mL) (Figure 5). Interestingly, the serum levels of vitamin D were found at its lowest concentrations (12.9±4 ng/mL) in the cases with multiple number of fibroids with signi- ficant differences (p<0.05) when compared to that of the single fibroid cases (22.6±3 ng/mL) (Figure 5). Figure 5. Vitamin D levels according to size and number of fibroids. Significant differences (P<0.05) were found between small and large size subgroups and between single and multiple fibroids subgroups (+). Thence, current findings demonstrated the crucial role of vitamin D deficiency in developing fibroids and increasing cell growth and proliferation. As previously reported, vitamin D has potent anti -tumor activity by inhibiting “leiomyoma cell proliferation”4,24, this is also supported by other studies that have proposed its potential role in the non-surgical management of uterine leiomyoma9,14,16. It has been previously postulated that vitamin D could exert an antiproliferative action on uterine cells through arresting cell growth and inhibition of “Wnt/β -catenin pathway”, suggesting its effective option in stabilizing leiomyoma size and preven ting its growth36. It has been demonstrated that increasing vitamin D concentration was correlated with inhibition of UF cell growth 15,23. Further study has shown the inhibitory effect of vitamin D3 on the “transforming growth factor beta” (TGF-β) pathway that is deemed to be the main fac tor in the developing of “fibrosis -asso- ciated diseases” confirming the role of this vitamin in the fibroid pathogenesis. Furthermore, vitamin D may act as a potent antiprogesteronic and antiestrogenic compound, and upon vitamin D supplementation, the progesteronic and estrogenic receptors expression decreased and the VDR up-regulation increased resulting in reducing disease progression 9. In addition, it is believed that “human uterine leiomyoma” contains lower vitamin D3 concentrations than its adjacent myome - trium30. Another study has suggested that vitamin D3 reduces UF growth via modulating the up -regulation and activities of “matrix metalloproteinase-2 and -9 and it is considered a promising natural substance with anti- uterine fibroid properties3. Other several in vivo and in vitro studies have shown the effectiveness of vitamin D in reducing the size and the frequency of leiomyomas 25,37. Recent studies have demonstrated that a combined supplementation of vitamin D plus epigallocatechin gallate could reduce myomas’ volume and improve women’s quality of life38. As a result, vitamin D3 supplementation seems to be beneficial to ameliorate the difficulties associated with, or even preventing, UFs, which in turn may improve women’s health. Vitamin D can regulate cells proliferation and differentiation, inhibit angiogenesis, and stimulate cells apoptosis, consequently, this leads to inhibition of tumorous tissue growth and of neoplastic formation associated with fibroids of the uterus 9,36. Another explanation for the role of vitamin D supple - ments on reduction of fibroids progression by recove- ring the damaged DNA, including suppression of the UF's phenotype via orchestrated addressing at multiple molecules of the DNA repa iring pathways 39. Vitamin D3 is found able to shrink the UF growth and reverse several abnormal biological pathways by ameliorating Pharmaceutical Sciences Asia 337 the developmental exposure -induced DNA damages and inflammatory pathways in primed myometrial stem cells40. Vitamin D deficiency has become a common dilemma associated with many health problems and its consequences could not be underestimated. Chronic conditions are usually multifactorial, yet they could easily be overcome; however, vitamin D is considered an effective and potent, safe and low-cost treatment that can also be taken as a prophylactic agent 14,23. Although sun exposure is the “ideal source” of vitamin D, most individuals, in reality, have hypovitaminosis D and need supplementation. Vitamin D deficiency was found pan- demic even in “sun-drenched” countries and associated with tremendous negative health consequences. The skin is capable of production of vitamin D supplying 80- 100% of the body’s requirements of vitamin D. However, age, time of day, latitude, season and pigmentation can influence the production of vitamin D in the skin 41. Generally, people avoid the sun because of the overex - posure dangers. Moreover, many of “life’s obligations” force us to spend most of our time inside under fluores- cent lights, keeping us away from natural sunlight. In addition, the cultural differences in clothing and Muslim dress style could have impact on sunlight exp osure, as many Iraqi women cover their bodies with hejab or abaya that limits the skin’s exposure to sunlight and lowers the ability to synthesize vitamin D 42. Therefore, health in ge neral and vitamin D levels, in particular, must be checked routinely to unearth any latent disease or even to prevent its manifestation via modifying the risk factors. In Iraq, the temperature in summer may reach over 50○C that prevents most people from getting outside or doing any outdoor activities. Hence, vitamin D supplementation might be considered especially for people at high risk factors and for women at their reproductive age to avoid occurrence or progression of fibroids. To reach sufficient serum vitamin D3 concentration (75 nmol/L), according to previous studies, different daily vitamin D supplemental intakes were recommended depending on region, status and age of individuals. For instance, the dose of 2250 IU/day for pregnant women and of 2026 IU/day for adults in Middle East area is suggested37,43. Adverse effects associated with vitamin D self-administration such as hypercalciuria and hypercal- cemia are rare, and usually due to taking it for a long time with extremely high doses44. In addition, many nutrients and dietary habits can be associated with myoma develop- ment risk, so dietary supplements should be considered for women with UF such as food rich with fibres, and fish oil as well some fruits and vegetables, especially the coloured ones, these can help in improving health and potentiating the action of vitamin D i n preventing the progression of diseases45-46. 4. CONCLUSION Vitamin D3 plays a potential role in UF pathoge - nesis and its severity. In spite of the fact that the region where the study was conducted is considered sunny, people may suffer from vitamin D deficiency due to life- style and low sun exposure. Natural vitamin D sources and supplements seem to be promising, b eneficial, and inexpensive tools in the fight against UFs and in allevia- ting their progression. A recent study has found the administration of vitamin D supplements could reduce the recurrence rates of UFs and the size of recurrent UFs 47. Vitamin D with specific anti UF drugs could provide a synergistic effect and is an interesting issue to be further studied. 5. ACKNOWLEDGEMENT Authors would like to thank private clinics and cli- nical biochemistry laboratories staff in Hilla, in particular Ahmed Al -khafaji and Zaid Jawdet for their helpful participation during collection and analysis of samples of this study. Author contributions SA designed the whole study, collected the data and wrote the manuscript. DM and RK collected the data, analyzed the study data and edited the manuscript. Conflict of interest None to declare. Funding None to declare. List of abbreviations BMI= Body Mass Index ECM= Extracellular matrix TNF-α= Tumor necrosis factor α UF= Uterine fibroid VDR= Vitamin D receptor Ethics approval and consent to participate All patients in this study were asked for their consent to participate and the study protocol was approved by the local ethics committee. Permission was authorized by the Committee of Research Ethical Approval, COP, Uni - versity of Babylon, Iraq, on 20/April/2021 (Reference no.: COPSEC 2021-O3). Article info: Received February 2, 2022 Received in revised form April 11, 2022 Accepted May 17, 2022 S. Al-azzawi et al. Pharm Sci Asia 2022; 49(4), 333-339 338

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