{"paper_id":"46c59156-a4c3-4b66-9bbe-b9bdc163ed97","body_text":"Pharmaceutical Sciences Asia   Pharm Sci Asia 2022; 49(4), 333-339 \n  DOI:10.29090/psa.2022.04.22.051 \n \n333 \n \nEvaluation of vitamin D-deficiency effects on the incidence \nof uterine fibroids\n \nShafq Al-azzawi*, Dhafir Masheta, Rawnaq Kadhim \n \n1 College of Pharmacy, University of Babylon, Iraq \n \n \n1. INTRODUCTION \n \nUterine fibroid (UF), also known as “small burden”, \nis a localized proliferation of the uterine smooth muscle \ncells. It is considered a benign tumor that commonly oc-\ncurred in a woman’s genital tract and its aetiology remains \nunclear. Tumor necrosis factor α (TNF-α) is suggested to \nbe one of the key factors in the transformation of the \nuterine smooth muscle cells into abnormal and immortal \ncells, that lead to clonal division and consequently to UF \ntumor1. The mechanisms that control the growth of UF are \nsophisticated and still not well understood2. It is suggested \nthat their growth results from the increased cellular \nproliferation and abnormal and excessive deposition of \n“extracellular matrix” (ECM)3. The main hormones that \ncan simulate UF development and growth are oestrogen \nand progesterone2,4-5. Cell signaling dysregulation, cyto-\ngenetic abnormalities, and miRNA expression have also \nbeen implicated in UF etiology. Mutations of the mediator \ncomplex gene subunit (MED12) may also result in fibroid \nformation6-7. Fibroids is mainly composed of smooth \nmuscle cells and various amounts of fibrous tissue \nenclused by a neurofibrovascular networks 8. Typically, \nthe symptoms include; heavy menstrual bleeding or men-\nstrual disorders, pelvic pain, anemia and bulky abdomen \nsymptoms. Moreover, fibroids in the uterine could lead \nto infertility, abortion and several adverse gynecologic \nand obstetric outcomes  with different debilitating mor -\nbidities9-10. \nThe main applicable treatment of uterine fibroid is \ntheir removal by surgical intervention, either myomectomy \nor hysterectomy and are considered expensive11. Ulipristal \nacetate has recently been introduced as “a selective \nprogesterone receptor modulator” to be a new treatment \nstrategy for uterine fibroids 12. However, idiosyncratic \nliver injury was reported after its using as treatment for \nUF13. Obviously, a therapeutic strategy for uterine fibroids \nshould be effective and with low cost and risk. \nThence, it is imperative to search for nonsurgical \nand  efficient  alternative  strategies  for  uterine  fibroids \n \n*Corresponding author: \n*Shafq Al-azzawi Email: Phar.shafaq.kadhim@uobabylon.edu.iq \n \nPharmaceutical Sciences Asia © 2022 by  \nFaculty of Pharmacy, Mahidol University, Thailand is licensed under CC BY-NC-ND 4.0. To view a copy of this license, visit  \nhttps:// www.creativecommons.org/licenses/by-nc-nd/4.0/ \n \nABSTRACT \n \nUterine fibroids are a very common benign tumor in women of reproductive age due to unknown aetiology. \nThere is growing evidence that vitamin D deficiency has a potential role in developing uterine fibroids. This \nstudy aims to investigate the effect of vitamin D levels in patients with uterine fibroids in Hilla city (a sunny \ncity). The study was conducted on 70 women diagnosed with uterine fibroids that visited private clinics and were \nreferred to biochemical laboratories. Vitamin D levels were assessed in patients’ sera, and questionnaire data on \nage, body mass index, symptoms and drugs used were analysed. In addition, information from their ultrasound \nscans of the fibroids, including the size and the number of fibroids, were also recorded. The results demonstrated \nthat more than half of patients had vitamin D deficiency and 25% of them were with insufficient vitamin D levels. \nThe age group, 41-45 year, showed the lowest values, whereas no significant differences in vitamin D levels were \nobserved in body mass index and severity of symptoms parameters. The results also revealed that a significant \ndecrease in vitamin D levels was associated with large size and multiple fibroids. It can be concluded that vitamin \nD has a great implication on the incidence of uterine fibroids, even in sunny cities. \n \nKeywords:  \nVitamin D, Uterine fibroid, Hypovitaminosis, Small burden, Leiomyoma \n \nResearch Article \n\nS. Al-azzawi et al.  Pharm Sci Asia 2022; 49(4), 333-339 \n \n334 \nmanagement, and in the first place among these strategies \nis the prevention of its occurrence. It is known that \nvitamin D3 acts as a regulator of calcium homeostasis \nin addition to its strong antifibrotic activity14. Moreover, \nstudies have reported vitamin D3 as a potent antitumor \nfactor that efficiently inhibits uterine fibroid in cell culture \nand shrinks fibroid in animal studies. However, there are \nnot enough clinical trials conducted in the human uterine \nregarding this area of research1,12,15-16. \nVitamin D deficiency is associated with enormous \nunfavorable conditions, in particular, osteomalacia  and \nrickets17. Low levels of vitamin D have previously been \ndetected in women with osteoporosis and metabolic \nsyndrome, which in turn emphasizes the value of vitamin \nD in women’s general health18-19. \nVitamin D can be found within the diet, such as cod \nliver oil, oily fish and dairy products. However, the main \nvitamin D source is sun exposure, where ultra-violet light \ntransforms 7-dehydrocholesterol in the skin to vitamin \nD via a nonenzymatic isomerization. After that, vitamin \nD is subjected to metabolic biotransformation in the liver \nby 25α-hydroxylase to 25 -hydroxyvitamin-D; which is \nthen converted in the kidney to the active form (1,25 -\ndihydroxyvitamin-D) (vitamin D3) by the action of 1α-\nhydroxylase20. Vitamin D3 exerts its biological activities \nin cells by interacting with its specific receptor, vitamin \nD receptor (VDR). VDR is “a nuclear transcription \nfactor”, it plays an important role in gene expression \nmodulation and trans cription. VDR affects the cell \nsignalling such as “growth arrest, differentiation and/or \ninduction of apoptosis”, which in turn inhibits cell \ngrowth1,21. \nIt has been recently suggested that vitamin D defi-\nciency is associated with increasing uterine fibroids risk \n1-2,4,22. Vitamin D acts as a potent antiprogesteronic and \nantiestrogenic compound and can inhibit leiomyoma cell \nproliferation. Furthermore, vitamin D was found to inhibit \nthe growth of fibroid cells and also promotes cell apoptosis \nin in vitro culture, whereas in in vivo animal models, it \nreduced the fibroid size. \nSeveral previous epidemiological studies conducted \nin different populations emphasized the importance of \nvitamin D deficiency in the development of UFs 24-26. \nNevertheless, no study so far has ever addressed this \nissue in the Iraqi population (country in the Middle East) \nespecially in Hilla city (located in the middle area of Iraq) \nwhere it is considered a hot region with high levels of \nsunlight exposure. Therefore, this study aims to investi-\ngate the relationship of occurrence of fibroids in women \ndiagnosed with uterine fibroid with vitamin D deficiency \nin Hilla city. \n \n2. SUBJECTS AND METHODS \n \nThe cross -section study was conducted in 2021 \nduring the summer season, from June to October, to ex-\nclude deficiency of vitamin D associated with the winter \nseason, on a sample of 70 subjects. The participants were \nrecruited among the female patients attended private \ngynecological clinics and were referred to biochemical \nanalytical laboratories in Hilla city. \nA female subject was considered eligible to partici-\npate in the study if she presented with uterine fibroid that \nwas detected by ultrasound scan (transvaginal or abdo -\nminal) and all types of fibroids (subserosal, submucosal, \nintramural and pedunculated fibroids) as appeared in the \nreports were included. Information from their ultrasound \nscan reports were considered as variables in the study \nincluding the size of fibroids; small (<1.9 cm), medium \n(2-2.9 cm) and large (>3 cm) 27 and number of fibroids \n(single or multiple). \nThe medical history and clinical features for each \npatient were recorded as a questionnaire to include the \nfollowing parameters: age, “body mass index (BMI)”, \nprevious or current intake of vitamin D supplements or  \nnot and a history of disease. Fibroids-related symptoms \nsuch as menstrual disorders and pelvic pain (which are \nclassified as minor, mild or severe) were also reported. \nWomen with a history of intake of vitamin D supple -\nments and those who followed a special diet regimen due \nto weight lowering or disease were excluded from the \nstudy. In addition, women with chronic disease (such as \ncardiovascular and central nervous system disorders, \ndiabetes mellitus, autoimmune diseases) were also \nexcluded. \nVitamin D3 lev els were obtained from laboratory \nbiochemical analysis reports which were measured in \nthe serum of participants using MINI VIDAS system \n(France)28 to study the effect of vitamin D3 levels on the \nincidence of UF. Vitamin D deficiency was deemed \nwhen serum levels were <20 ng/mL and considered \ninsufficient if the levels were 20 -30 ng/mL and normal \nwhen the levels were >30 ng/mL29. Data of the patients \nwith vitamin D3 deficiency and insufficiency were \nanalyzed to evaluate the relationship between vitamin D \nand the tested variables (age, BMI, severity of symptoms \nand the size and number of fibroids). To study the \nrelationship of vitamin D concentration with age factor, \nparticipants were stratified into four groups (<35, 35-40, \n41-45 and >45 years) whereas for BMI factor, they were \ngrouped into four groups (<25, 25-30, 31-40 and >40). \n \n2.1. Statistical analysis \n \nThe data in this work were collected and expressed \nas percentages or means±standard deviation (represented \nby error bars) using Excel Microsoft. The data were \nanalyzed by ANOVA an d Tukeys test using Minitab \nsoftware, and probability <0.05 is considered for the \nsignificant differences. \n \n \n\nPharmaceutical Sciences Asia \n \n335 \n3. RESULTS AND DISCUSSION \n \nIn this study, 70 women diagnosed with fibroids \nwere recruited and their vitamin D3 serum levels were \nevaluated. The results revealed that 56% of the parti ci-\npants were with deficient vitamin D3 values (<20 ng/  \nmL), while 25% of them were found with insufficient \nvitamin D3 values and only 19% were normal (>30 ng/ \nmL) (Figure 1). \n \n \nFigure 1.  Percentages of women having normal, insufficient and \ndeficient vitamin D levels associated with uterine fibroids. \n \nTo evaluate the association of vitamin D deficiency \nwith the age factor, women were stratified into four age \ngroups. It is of note that the youngest women (<35 years) \nhave the highest levels of vitamin D3 while its lowest \nlevels are accompanied with older women. Furthermore, \nboth age groups, 41-45 and >45 showed significant dif-\nferences (p<0.05) in vitamin D levels from the <35 group. \nSignificant differences (p<0.05) were found in compari-\nson with the group of control women matched per age \n(Figure 2). \n \n \nFigure 2. Vitamin D levels within age subgroups for women with \nuterine fibroids with comparison to control groups. Significant diffe-\nrences (p<0.05) were found between each subgroup with the corres-\nponding age in control group(*), and between >40 subgroups and \n<40 subgroups(+). \n \nThis  is  consistent  with  the  previous  findings  that \nhave documented a positive correlation between age and \nserum vitamin D concentrations31-32. It has been reported \nthat low vitamin D levels associated with increasing age \nis due to several factors including reduction in calcium \nabsorption with increasing “intestinal resistance” to \nvitamin D31, low expression of VDR5, and reduced renal \nproduction of vitamin D by the kidneys 31. Obesity and \nlack of outdoor activity by the older women may also \ncontribute to the vitamin D deficiency33. Therefore, con-\nsumption of vitamin D-rich food with increasing sunlight \nexposure are required for people aged more than 41 years \nto avoid vitamin D deficiency. \nThe data regarding BMI showing the lowest vita -\nmin D3 levels were noticed with high BMI (>30) group, \nwhereas the highest levels of the vitamin (19.1±5 ng/mL) \nwere observed with low BMI (<25) group, however there \nwas no significant differences (p>0.05) within the tested \nsubgroups (Figure 3). \n \n \nFigure 3. Vitamin D levels within BMI subgroups  for women with \nuterine fibroids. \n \nConsidering other studies, it has been shown that \nobesity is “independently correlated” with vitamin D \ndeficiency34. A previous study has demonstrated that \nvitamin D deficiency was found in more than 50% of \nobese individuals (BMI ≥40)35. The relationship between \nobesity and vitamin  D is not clear and still unknown, \nand may be explained by the presence of “excess body \nfat tissue” that retain vitamin D resulting in an increase \nin the volume of distribution of the vitamin, which in \nturn causes a decrease in the circulatory vitamin D levels. \nAlternatively, metabolic syndromes could also be  the \nreason behind the vitamin D deficiency as overweight is \nconsidered as one of its main components34. It has been \nfound that vitamin D deficiency is associated with meta-\nbolic syndrome especially in postmenopausal women18. \nFurther explanation may link obesity with vitamin D \ndeficiency is that obese individuals have higher cortical \ntissue mineral density and cortical thickness than thin  \nones have or may due to differences between obese and \nnormal subjects in liver enzymes expression that activate \nvitamin D33. \nThe severity of symptoms associated with UF was \n\n\nS. Al-azzawi et al.  Pharm Sci Asia 2022; 49(4), 333-339 \n \n336 \nalso evaluated to detect any suspected effects of vitamin \nD deficiency. In this regard, women with mild to mode-\nrate symptoms were associated with the lowest values \nof vitamin D (16.3±2 ng/mL) while highest values (20.2± \n4 ng/mL) were detected in individuals with minor symp-\ntoms (Figure 4). However, there were no significant \ndifferences (p>0.05) within groups of severity of symp-\ntoms (minor, mild or severe). These controversial results \ncould be attributed to the inaccurate information given \nby some patients or some of them have tolerated these \nsymptoms or underestimated them (pain-threshold varia-\ntions from patient to patient). \n \n \nFigure 4. Vitamin D levels according to severity of symptoms asso-\nciated with uterine fibroids. \n \nTo identify any potential correlation between vita-\nmin D deficiency and the incidence of UF, serum levels \nwere investigated according to the size and number of \nfibroids in the involved subjects. As expected, th e con-\ncentrations of vitamin D were found to be significantly \n(p<0.05) lower in patients with large size fibroids (14.8 \n±2 ng/mL) than in those have small ones (20.9±3 ng/mL) \n(Figure 5). Interestingly, the serum levels of vitamin D \nwere found at its lowest concentrations (12.9±4 ng/mL) \nin the cases with multiple number of fibroids with signi-\nficant differences (p<0.05) when compared to that of the \nsingle fibroid cases (22.6±3 ng/mL) (Figure 5). \n \n \nFigure 5. Vitamin D levels according to size and number of fibroids. \nSignificant differences (P<0.05) were found between small and large \nsize subgroups and between single and multiple fibroids subgroups \n(+). \n \nThence, current findings demonstrated the crucial \nrole of vitamin D deficiency in developing fibroids and \nincreasing cell growth and proliferation. As previously \nreported, vitamin D has potent anti -tumor activity by \ninhibiting “leiomyoma cell proliferation”4,24, this is also \nsupported by other studies that have proposed its \npotential role in the non-surgical management of uterine \nleiomyoma9,14,16. It has been previously postulated that \nvitamin D could exert an antiproliferative action on \nuterine cells through arresting cell growth and inhibition \nof “Wnt/β -catenin pathway”, suggesting its effective \noption in stabilizing leiomyoma size and preven ting its \ngrowth36. It has been demonstrated that increasing \nvitamin D concentration was correlated with inhibition \nof UF cell growth 15,23. Further study has shown the \ninhibitory effect of vitamin D3 on the “transforming \ngrowth factor beta” (TGF-β) pathway that is deemed to \nbe the main fac tor in the developing of “fibrosis -asso-\nciated diseases” confirming the role of this vitamin in \nthe fibroid pathogenesis. Furthermore, vitamin D may \nact as a potent antiprogesteronic and antiestrogenic \ncompound, and upon vitamin D supplementation, the \nprogesteronic and estrogenic receptors expression \ndecreased and the VDR up-regulation increased resulting \nin reducing disease progression 9. In addition, it is \nbelieved that “human uterine leiomyoma” contains lower \nvitamin D3 concentrations than its adjacent myome -\ntrium30. Another study has suggested that vitamin D3 \nreduces UF growth via modulating the up -regulation \nand activities of “matrix metalloproteinase-2 and -9 and \nit is considered a promising natural substance with anti-\nuterine fibroid properties3. Other several in vivo and in \nvitro studies have shown the effectiveness of vitamin D \nin reducing the size and the frequency of leiomyomas  \n25,37. Recent studies have demonstrated that a combined \nsupplementation of vitamin D plus epigallocatechin \ngallate could reduce myomas’ volume and improve \nwomen’s quality of life38. \nAs a result, vitamin D3 supplementation seems to \nbe beneficial to ameliorate the difficulties associated \nwith, or even preventing, UFs, which in turn may \nimprove women’s health. Vitamin D can regulate cells \nproliferation and differentiation, inhibit angiogenesis, \nand stimulate cells apoptosis, consequently, this leads to \ninhibition of tumorous tissue growth and of neoplastic \nformation associated with fibroids of the uterus 9,36. \nAnother explanation for the role of vitamin D supple -\nments on reduction of fibroids progression by recove-\nring the damaged DNA, including suppression of the \nUF's phenotype via orchestrated addressing at multiple \nmolecules of the DNA repa iring pathways 39. Vitamin \nD3 is found able to shrink the UF growth and reverse \nseveral abnormal biological pathways by ameliorating \n\n\nPharmaceutical Sciences Asia \n \n337 \nthe developmental exposure -induced DNA damages \nand inflammatory pathways in primed myometrial stem \ncells40. \nVitamin D deficiency has become a common \ndilemma associated with many health problems and its \nconsequences could not be  underestimated. Chronic \nconditions are usually multifactorial, yet they could \neasily be overcome; however, vitamin D is considered \nan effective and potent, safe and low-cost treatment that \ncan also be taken as a prophylactic agent 14,23. Although \nsun exposure is the “ideal source” of vitamin D, most \nindividuals, in reality, have hypovitaminosis D and need \nsupplementation. Vitamin D deficiency was found pan-\ndemic even in “sun-drenched” countries and associated \nwith tremendous negative health consequences. The \nskin is capable of production of vitamin D supplying 80-\n100% of the body’s requirements of vitamin D. However, \nage, time of day, latitude, season and pigmentation can \ninfluence the production of vitamin D in the skin 41. \nGenerally, people avoid the sun because of the overex -\nposure dangers. Moreover, many of “life’s obligations” \nforce us to spend most of our time inside under fluores-\ncent lights, keeping us away from natural sunlight. In \naddition, the cultural differences in clothing and Muslim \ndress style could have impact on sunlight exp osure, as \nmany Iraqi women cover their bodies with hejab or \nabaya that limits the skin’s exposure to sunlight and \nlowers the ability to synthesize vitamin D 42. Therefore, \nhealth in ge neral and vitamin D levels, in particular, \nmust be checked routinely to unearth any latent disease \nor even to prevent its manifestation via modifying the \nrisk factors. In Iraq, the temperature in summer may \nreach over 50○C that prevents most people from getting \noutside or doing any outdoor activities. Hence, vitamin \nD supplementation might be considered especially for \npeople at high risk factors and for women at their \nreproductive age to avoid occurrence or progression of \nfibroids. \nTo reach sufficient serum vitamin D3 concentration \n(75 nmol/L), according to previous studies, different daily \nvitamin D supplemental intakes were  recommended \ndepending on region, status and age of individuals. For \ninstance, the dose of 2250 IU/day for pregnant women \nand of 2026 IU/day for adults in Middle East area is \nsuggested37,43. Adverse effects associated with vitamin D \nself-administration such as hypercalciuria and hypercal-\ncemia are rare, and usually due to taking it for a long time \nwith extremely high doses44. In addition, many nutrients \nand dietary habits can be associated with myoma develop-\nment risk, so dietary supplements should be considered \nfor women with UF such as food rich with fibres, and \nfish oil as well some fruits and vegetables, especially the \ncoloured ones, these can help in improving health and \npotentiating the action of vitamin D i n preventing the \nprogression of diseases45-46. \n \n4. CONCLUSION \n \nVitamin D3 plays a potential role in UF pathoge -\nnesis and its severity. In spite of the fact that the region \nwhere the study was conducted is considered sunny, \npeople may suffer from vitamin D deficiency due to life-\nstyle and low sun exposure. Natural vitamin D sources \nand supplements seem to be promising, b eneficial, and \ninexpensive tools in the fight against UFs and in allevia-\nting their progression. A recent study has found the \nadministration of vitamin D supplements could reduce \nthe recurrence rates of UFs and the size of recurrent UFs \n47. Vitamin D with specific anti UF drugs could provide \na synergistic effect and is an interesting issue to be further \nstudied. \n \n5. ACKNOWLEDGEMENT \n \nAuthors would like to thank private clinics and cli-\nnical biochemistry laboratories staff in Hilla, in particular \nAhmed Al -khafaji and Zaid Jawdet for their helpful \nparticipation during collection and analysis of samples \nof this study. \n \nAuthor contributions \nSA designed the whole study, collected the data and \nwrote the manuscript. DM and RK collected the data, \nanalyzed the study data and edited the manuscript. \n \nConflict of interest \nNone to declare. \n \nFunding \nNone to declare. \n \nList of abbreviations \nBMI= Body Mass Index \nECM= Extracellular matrix \nTNF-α= Tumor necrosis factor α \nUF= Uterine fibroid \nVDR= Vitamin D receptor \n \nEthics approval and consent to participate \n \nAll patients in this study were asked for their consent to \nparticipate and the study protocol was approved by the \nlocal ethics committee. Permission was authorized by the \nCommittee of Research Ethical Approval, COP, Uni -\nversity of Babylon, Iraq, on 20/April/2021 (Reference \nno.: COPSEC 2021-O3). \n \nArticle info: \nReceived February 2, 2022 \nReceived in revised form April 11, 2022 \nAccepted May 17, 2022 \n \n\nS. Al-azzawi et al.  Pharm Sci Asia 2022; 49(4), 333-339 \n \n338 \nREFERENCES \n1. Al-Hendy A, Badr M . Can vitamin D reduce the risk of uterine \nfibroids?. 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