{"paper_id":"245761d4-9751-4a3d-8252-1bcf6347b53f","body_text":"Uterine fibroids are non-cancerous tumors of the myometrium that cause\nsignificant morbidity including menorrhagia and pelvic pain, often requiring medical\nor surgical intervention ( 1 ). Estimates based\non ultrasound screening suggest that fibroids develop in over 70% of women. Black\nwomen have a 10-year earlier tumor onset compared with White women ( 2 ); they also have larger tumors, more debilitating\nsymptoms, and an increased need for surgery ( 3 ). Existing medical and surgical treatments for fibroids can impact\nchildbearing goals and, apart from hysterectomy, most treatments do not preclude\nfibroid recurrence ( 1 ). Most individuals with\nfibroids prefer non-invasive treatments and preservation of the uterus ( 4 ).\nDespite the high prevalence of fibroids, few modifiable risk factors have\nbeen identified. None explain differences in disease severity between Black and\nWhite individuals ( 3 ,  5 ). Vitamin D concentration, which is lower on average\namong self-identified Black women, has been proposed as a possible modifiable risk\nfactor for the development of fibroids ( 6 – 9 ).  In\nvitro  and animal studies support a protective effect of vitamin D. In\nthese models, vitamin D reduces fibroid tissue proliferation ( 8 ,  10 ,  11 ), changes the expression of estrogen and\nprogesterone receptors ( 12 ), alters gene\nexpression in proliferation and apoptosis pathways ( 13 – 15 ), and reduces\nexpression of extracellular matrix proteins ( 16 – 18 ). Human\nobservational studies report lower fibroid prevalence in women with higher vitamin D\n( 6 ,  11 ). Most ( 19 – 22 ) but not all ( 23 ) human intervention studies document reduced fibroid\ngrowth when participants are treated with vitamin D, but studies were small (30 to\n205 participants).\nAlthough highly suggestive, the existing laboratory and epidemiologic\nliterature has limitations. Animal and  in vitro  studies usually\nstudy the active form of vitamin D [1,25-dihydroxyvitamin D\n(1,25(OH) 2 D)]. These models may not reflect tissue concentrations in\nhumans who obtain vitamin D through ultraviolet-B (UV-B) light exposure\n(D 3 ), diet (D 2  or D 3 ), or vitamin supplements\n(D 2  or D 3 ) with conversion to 25-hydroxyvitamin D\n(25(OH)D) in the liver and final conversion to 1,25(OH) 2 D in the kidney\nor target tissue. In addition, most epidemiologic studies have relied on clinical\ndiagnosis or ultrasound detection of prevalent fibroids, but initial fibroid onset\noccurs many years before clinical detection ( 6 ,  11 ).\nWe hypothesize that higher concentrations of serum 25(OH)D will be\nassociated with reduced fibroid growth and incidence. We investigated this\nhypothesis with four repeated standardized ultrasound examinations over five\nyears.\n\nThe Study of Environment, Lifestyle and Fibroids (SELF) is a prospective\ncohort study designed to evaluate fibroid growth and incidence ( 24 ). Due to earlier fibroid onset in Black/African\nAmerican women in the United States ( 2 ),\nSELF enrollment was limited to those who self-identified as ‘Black or\nAfrican American’ among a list of racial and ethnicity categories.\nDetails of study recruitment and activities are provided in  Appendix 1 . Briefly, recruitment\nwas conducted in the Detroit, Michigan area from 2010 to 2012 in collaboration\nwith Henry Ford Health (HFH). Eligible participants were aged 23–34, who\nreported no prior diagnosis of fibroids at recruitment. The participants\ncompleted baseline questionnaires and attended a clinic visit that included an\nultrasound examination, anthropometric measurements, and non-fasting blood\ncollection. Of 1693 participants enrolled, 1610 (95%) attended ≥1 of the\nthree follow-up visits during which the same study activities were completed.\nThe four study visits occurred approximately 20 months apart and were completed\nin 2018. Participants who missed a visit were encouraged to attend the next.\nVisits for pregnant participants were delayed until 3–4 months\npost-partum for better ultrasound imaging. Study retention was high; 91% of the\nenrolled cohort attended the final visit, 95% attended at least 2 visits, and\n79% attended all 4 study visits ( Supplemental Figure 1 ). The 5% who\nonly attended the enrollment visit tended to be older with lower income, lower\nbody mass index and were more likely to have had a pregnancy. Those participants\nwho attended all 4 visits tended to older and were more likely to be employed\ncompared to those who attended only 2 or 3 visits, but otherwise the data showed\nno clear pattern of differences in baseline characteristics ( Supplemental Table 1 ).\nSELF was approved by the institutional review boards of the National\nInstitutes Health and HFH. All participants provided written informed consent\nand received compensation.\nSerum from each visit was aliquoted and stored at −80C until\nassayed. Analysis of 25(OH)D was conducted in three batches (serum from\nbaseline, follow-up 1, and follow-up 2). Details of the vitamin D measurement\nand quality control procedures are described in  Appendix 2 . In brief, total serum\n25(OH)D for the baseline visit was measured using LIAISON, a competitive\nchemiluminescence immunoassay ( 25 ,  26 ). For subsequent visits serum\n25-hydroxyvitamin D 2  (25(OH)D 2 ) and 25-hydroxyvitamin\nD 3  (25(OH)D 3 ) were measured with liquid\nchromatography-tandem mass spectrometry (LC-MS/MS) and summed to create a total\n25(OH)D measure. Blinded quality control serum samples were included in all\nassays, and based on these samples all intra-assay coefficients of variation\n(CV) were <5% and inter-assay CVs were 9% for LIAISON and <5% for\nLC-MS/MS. The total 25(OH)D for 77 samples run on both platforms showed a mean\ndifference of 0.6 ng/ml (25 th  to 75 th  percentiles:\n−1.8 to 1.1). We did not adjust for different assay methods.\nTo account for seasonal variation in 25(OH)D, we estimated the overall\nannual average 25(OH)D using a cosinor model ( 27 ). We modeled the natural log of 25(OH)D using the sine and cosine\nof the day of the year of blood sample collection (first and second harmonic) as\npredictors ( 27 ,  28 ). We then created individual values for annual\naverage 25(OH)D by adding the residual for each participant/visit to the model\nintercept and transforming back to the original scale (ng/ml). This\nseason-adjusted 25(OH)D is denoted 25(OH)D hereafter.\nFor 90 samples, the serum volume was insufficient for the 25(OH)D assay\n(1.2–2.6% of samples at a given visit). We imputed the majority (N=80) of\nthese values using the mean of measured values from adjacent visits, or\ncarryover of the value from an adjacent visit if there was only one. The\nremaining observations (N=10) were dropped from the analyses.\nWe used clinically relevant cut points based on recommendations from the\nInstitute of Medicine (IOM) ( 29 ) and the\nEndocrine Society (ES) ( 30 ) to categorize\nindividual season-adjusted 25(OH)D values for analyses: <12 ng/ml\n(vitamin D deficient) (IOM and ES), <20 ng/ml (vitamin D inadequacy (IOM)\nor deficient (ES)), <30 ng/ml (vitamin D insufficient (ES)).\nExperienced sonographers, trained on the SELF protocol, used\ntransvaginal ultrasound to count, localize and measure fibroids ≥0.5 cm\nin diameter. Sonographers made three separate passes through the uterus,\nrecording the 3 diameters of a given tumor at each pass. We calculated the\nfibroid volume for each pass with the ellipsoid formula, and the three volumes\nwere averaged for analysis. Per protocol, sonographers noted any problems with\nvisualization (e.g., calcifications, shadowing) that may have limited\nvisualization. Video and still images were archived and an 8% sample for each\nsonographer, oversampled for fibroid cases, was reviewed every month by the lead\nsonographer (details in  Appendix 3 ).\nThe primary outcomes of interest were fibroid growth and fibroid\nincidence. Given the observed reduction in fibroid growth, fibroid loss was\nexplored as a secondary outcome. Every fibroid outcome analysis had different\neligibility criteria given that fibroid growth and loss require a fibroid to be\ndetected and fibroid incidence is estimated among those who are fibroid-free at\nenrollment ( Supplemental\nFigure 2 ).\nThe lead sonographer (TC) and one other author (DB) identified\nfibroids that could be seen across two successive visits using archived\nimages and fibroid locations. The growth analyses used data from 434\nparticipants (n=1357 interval growth measurements from successive visits).\nWe defined fibroid growth as the change in the natural logarithm of the\ntumor volume (ln-volume). Change in ln-volume was scaled to a growth rate\nper 18-months (median time between visits=19 months;\n25 th -75 th  percentiles: 18–21) by\ncalculating daily growth rates and multiplying by 540. To compare growth of\nfibroids from participants in high versus low categories of 25(OH)D we\nestimated percent difference in volume per 18 months ( Appendix 4 ).\nWe identified incident fibroid cases (first appearance of any\nfibroids) among participants confirmed via ultrasound not to have fibroids\nat enrollment (N=1246). If sonographers noted factors which impaired\ndetection of fibroids (e.g., calcifications or shadowing, only a\ntransabdominal ultrasound) the data were excluded from analysis (~0.5% of\nultrasounds), resulting in incidence data for 1232 participants. After\nexcluding observations with insufficient serum for 25(OH)D, the incidence\nanalysis included 1230 participants ( Supplemental Figure 2 ).\nFibroid loss was assessed among participants with prevalent fibroids\nat enrollment that had not been clinically diagnosed previously, or\nparticipants who developed incident fibroids. For analysis of fibroid loss,\ndefined as a decrease in fibroid number between two successive visits, we\nexcluded intervals including or following a myomectomy, hysterectomy, or\nuterine artery embolization. When there are numerous fibroids, it can be\ndifficult to accurately count tumors ( 31 ), therefore we restricted this analysis to the 539\nparticipants who had ≥2 successive visits with ≤4 fibroids at\nthe earlier visit and no sonographer report of difficulty with visualization\n( Supplemental Figure\n2 ). Fibroids that become undetectable by ultrasound between\nvisits include those that shrink below the limit of ultrasound detection\n(0.5 cm for any diameter); therefore, this outcome cannot be interpreted as\ncomplete resolution of fibroids.\nWe identified covariates of interest based on prior fibroid research\n( 5 ), previous work in this cohort\n( 32 ,  33 ), and availability of SELF data. We measured height during the\nfirst clinic visit, and weight at every visit to calculate body mass index (BMI\nkg/m 2 ) for each visit. We collected other covariate data via\ntelephone or computer assisted questionnaires at every visit including\nreproductive and hormonal variables (years since last birth, parity, age at\nmenarche, years since last use of depot medroxyprogesterone acetate (DMPA)),\ndemographic variables (household income, educational attainment, current\nemployment), physical activity and current smoking. All factors of interest were\nexplored in adjusted models and those that did not affect observed associations\nbetween 25(OH)D and the fibroid outcome were not included ( 34 ) in final models. Parameterization of covariates\n(categorization or continuous) for modelling is noted in the table footnotes for\neach fibroid outcome. Categorical covariates were usually modelled using\nindicator variables to allow for non-linear associations. Covariates were\nupdated at the beginning of each interval, except for years since last birth and\nparity which were updated at the end of the interval between visits to\nincorporate events during the interval ( 35 ). There were minimal missing covariate data (<0.5%);\ntherefore, when adjusting, we conducted complete-case analyses.\nWe used linear mixed models to account for correlated growth among\nfibroids from the same participant and for the same fibroid over time as\npreviously described ( 32 ,  33 ,  36 ). Minimally adjusted growth models included time-varying\n25(OH)D, continuous age, and categorical values for fibroid volume and\nnumber. Fully-adjusted models included the minimal adjustment set plus age\nat menarche and time-varying measures of income, employment, BMI, years\nsince last birth, and years since last DMPA use ( Appendix 4 ).\nWe used Cox regression with age as the time-scale to estimate hazard\nratios (HR) and 95% confidence intervals (95% CI) for associations between\n25(OH)D categories and fibroid incidence. Participants were included from\nthe age at enrollment until the age at fibroid detection, with censoring for\nloss to follow-up, non-fibroid-related hysterectomy or the final study\nvisit, whichever occurred first. Age-adjusted models examining the\nassociation between 25(OH)D and fibroid incidence were estimated first\nwithout covariates, and then were further adjusted for BMI, current smoking,\nincome, parity, years since last birth, and years since last DMPA use. There\nwas no evidence that the proportional hazards assumption was violated.\nWe used Poisson regression accounting for multiple observations per\nparticipant to estimate risk ratios (RR) and 95% CI with robust standard\nerrors for fibroid loss. Minimally adjusted models included time-varying\n25(OH)D, continuous age, months between visits, and categorical values for\nlargest fibroid volume and number of fibroids. Fully adjusted models\nincluded the minimal adjustment set plus time-varying education, BMI, years\nsince last birth, and years since last DMPA use.\nFor all outcomes, we removed observations with imputed serum\n25(OH)D. Because use of estrogen-containing oral contraceptives is\nassociated with increased serum concentrations of 25(OH)D ( 28 ), we explored possible confounding by\nincluding current use (yes, no) as a covariate. For fibroid incidence we set\nthe incidence of fibroids to the mid-point of the interval rather than end.\nFor fibroid growth and loss we removed fibroid-number and fibroid-volume\ncovariates to evaluate the possible influence of exposure-related\ndifferences in fibroid number and size. For fibroid growth we excluded\nstatistical outliers (positive or negative growth beyond 3SD). (Details in\n Appendix\n5 ).\nAll analyses used SAS 9.4 (Cary, NC).\n\nAt enrollment, among the 1610 participants who had ≥1 follow-up\nvisit, mean age was 29.2±3.4 years, 78% had educational attainment beyond\nhigh school, 62% were employed, and 45% had a household income below $20,000. Almost\na quarter (24%) had a BMI ≥40 kg/m 2  ( Table 1 ). Participants had low 25(OH)D (median 25(OH)D=\n15.3 ng/ml, 25 th -75 th  percentiles: 11.1–20.6). Compared\nwith those with 25(OH)D <20ng/ml, those with 25(OH)D ≥20ng/ml had\nhigher educational attainment and income, lower BMI, and were more likely to be\nusing oral contraceptives and be non-smokers ( Table\n1 ). There was no time trend in 25(OH)D concentrations across the\nstudy’s duration (2010–2016) ( Supplemental Figure 3 ). Median 25(OH)D\nconcentrations were 15.3 ng/ml at enrollment, 14.8 ng/ml at follow-up 1, and 15.3\nng/ml at follow-up 2. Among 25(OH)D measures across the study, 32% were deficient\n(<12 ng/ml), 41% were 12-<20 ng/ml, 27% were ≥20 ng/ml. Only 7%\nexceeded the Endocrine Society ( 30 ) cut point\nfor sufficiency (≥30 ng/ml) ( Supplemental Figure 4 ). Very few (1.7%)\nof the participants had this higher concentration at all three visits. Participants\nhad a median length of study participation of 4.8 years\n(25 th -75 th  percentiles: 4.7–5.0 years). Baseline\ncharacteristics were similar for the participants in each of the fibroid outcome\ngroups (growth, incidence and loss analyses), except for age, percent nulliparous,\nrecency of birth and use of DMPA. These are known factors associated with fibroid\nprevalence and are expected to differ because the growth and loss analyses require\nparticipants with fibroids while the incidence analysis requires participants\nwithout fibroids ( Supplemental\nTable 2 ).\nMost fibroids, including undiagnosed fibroids detected at enrollment and\nincident fibroids that developed during the study, were small. Median volume of\nincident fibroids at time of first detection was 0.56 cubic centimeters\n(cm 3 ) (25 th -75 th  percentiles: 0.23–1.4\ncm 3 ). Median volume for those followed for growth was 2.2\ncm 3  (25 th -75 th  percentiles: 0.7–8.6\ncm 3 ). At enrollment, 23% of participants had ≥1 fibroid\n(median: 1 fibroid, 75 th  percentile: 2 fibroids), while by the\nstudy’s end, 32% had ≥1 fibroid (median: 2 fibroids, 75 th \npercentile: 3 fibroids).\nOverall estimated fibroid growth averaged 77.0% (95% CI: 68.6%, 85.9%)\nvolume increase per 18 months. Serum 25(OH)D ≥20 ng/ml was associated with a\nreduced fibroid growth rate. Fibroids among those with 25(OH)D ≥20 ng/ml had\n9.7% less growth per 18 months (95% CI: −17.3%, −1.3%) than fibroids\nfrom participants with 25(OH)D <20 ng/ml ( Table 2 ).\nFibroid incidence averaged 9.6% within each study interval. Only at 25(OH)D\nconcentrations ≥30 ng/ml was there any indication of a decrease in incidence\n( Table 3 ). Hazard ratios for serum\n25(OH)D ≥30 ng/ml suggested an estimated 22% reduction in fibroid incidence\n(HR 0.78, 95% CI: 0.47, 1.30) when compared with serum 25(OH)D <30 ng/ml.\nAssociations were imprecise given the small number of 25(OH)D measures ≥30\nng/ml.\nFibroid loss during an interval was common (24.3% of eligible intervals had\nloss of at least one fibroid between study visits). Participants with 25(OH)D\nconcentration ≥30 ng/ml had 32% higher fibroid loss in an observation\ninterval than those with 25(OH)D <30 ng/ml (RR 1.32, 95% CI: 0.95, 1.83)\n( Table 4 ).\nSensitivity analyses showed very similar associations to those in the\nprimary analyses ( Appendix\n4 ,  Supplemental Table\n3 ).\n\nUsing prospectively collected ultrasound data and repeated measures of serum\n25(OH)D, we observed slower fibroid growth in participants with 25(OH)D ≥20\nng/ml. For participants with serum 25(OH)D ≥30 ng/ml, we found evidence for a\ngreater likelihood of fibroid loss, but only limited evidence for reduced incidence\ngiven the wide confidence interval.\nIn this cohort, there were few 25(OH)D observations ≥20 ng/ml (27%)\nor ≥30 ng/ml (7%). Only 1.7% of participants maintained 25(OH)D\nconcentrations ≥30 ng/ml for all three measures. Thus, we had limited power\nto detect associations at higher concentrations of 25(OH)D. The optimal range of\n25(OH)D is debated ( 37 ). While the Institute\nof Medicine has identified ≥20 ng/ml as needed for skeletal health ( 29 ), the Endocrine Society argues that\nconcentrations ≥30 ng/ml are needed ( 30 ). Observational and clinical trials for improved reproductive\noutcomes including the success of fertility treatment ( 38 ), fecundability in natural conceptions ( 39 ), preterm birth ( 40 ), menstrual cycle characteristics ( 41 ), and breast cancer risk ( 42 ) suggest that concentrations of 25(OH)D ≥40\nng/ml may be required.\nAlthough most fibroids in this cohort were small and might not warrant\nimmediate clinical attention, our study still has important clinical implications.\nSymptoms ( 43 ) and major interventional\ntreatments ( 44 ) are more likely with larger\nfibroids. Therefore, minimizing fibroid growth and increasing fibroid loss when\nfibroids are small can delay severe morbidity and prevent the need for surgical or\nradiologic treatments.\nOur findings for reduced growth of fibroids from participants with higher\n25(OH)D are consistent with results of small clinical trials that treated\nparticipants with vitamin D supplements ( 19 – 22 ), though most of\nthose studies ( 19 ,  21 ,  22 ) included\nparticipants with 25(OH)D concentrations ranging up to 30 ng/ml at enrollment, and\ntreated with supplementation that would have resulted in higher concentrations than\ngenerally seen in our sample. Fibroid loss was not included as an outcome in the\ntrials of fibroid growth. However, if our finding of reduced fibroid growth with\nhigher vitamin D results in some fibroids shrinking below the ultrasound detection\nlimit, increased fibroid loss would be expected.\nAlthough the association between vitamin D and fibroid incidence has not\nbeen previously studied, a recent systematic review and additional observational\nstudies, report lower concentrations of 25(OH)D among women with prevalent fibroids\ncompared with those without fibroids ( 6 ,  45 – 47 ). The observational studies rely on self-reported fibroid status, and\nthis results in undiagnosed fibroids being included in the “non-case”\ngroup ( 5 ) as well as temporal\nmisclassification of 25(OH)D concentrations. A large, randomized control trial using\nprospective ultrasound examinations is underway in China which will investigate the\neffects of daily vitamin D supplementation over 2 years on fibroid incidence in more\nthan 2000 women, and fibroid growth in 360 women ( 48 ). Trials in other populations are warranted.\nIn vitro  and animal models support our finding that vitamin\nD limits fibroid growth (reviewed in Vergara ( 10 )).  In vitro  models including those using\nimmortalized human uterine leiomyoma cells (HuLM) and cultured human cells from\nfibroid and adjacent myometrial tissue show that treatment with\n1,25(OH) 2 D inhibits their growth through changes in cell proliferation,\napoptosis, extracellular matrix composition, Wnt/β-catenin and TGFβ3\nexpression, and down regulation of estrogen and progesterone receptors ( 12 ,  13 ,\n 16 ,  18 ,  49 – 51 ). Treatment with 1,25(OH) 2 D or an analogue\nin mice and Eker rats with fibroid tumors also results in decreased tumor size\nthrough such mechanisms ( 17 ,  50 ,  52 ).\nThere have been fewer laboratory studies related to fibroid initiation and\nvitamin D. Most fibroids harbor specific somatic mutations in  HMGA2 \nor  MED12  ( 53 ). Fibroids have\nelevated DNA damage and vitamin D treatment of HuLM cells reduced evidence of DNA\ndamage with upregulation of DNA-repair proteins ( 14 ). Improved detection and repair of DNA damage could prevent the\nproliferation of cells with mutations and thus limit fibroid initiation ( 54 ).\nAn inherent limitation to studying the growth of individual fibroids is that\nthey must be observed over time, and lost fibroids are not included; to compensate,\nwe examined fibroid loss as a separate outcome. There is also measurement error in\nultrasound assessment of fibroid size, and very small fibroids may be missed.\nHowever, taking three separate passes through the uterus will maximize fibroid\ndetection, and using the mean of 3 separate volume measures has less measurement\nerror than a single measure. In addition, measurement error is greater for smaller\nfibroids ( 55 ), but we address this limitation\nby accounting for the differential error in our growth model ( 33 ). The most important limitation is that we had few\nparticipants with 25(OH)D concentrations above 30 ng/ml which limits our power to\nfind associations at the higher vitamin D concentrations recommended by the\nEndocrine Society.\nOur prospective study design with multiple standardized ultrasounds is the\nfirst that allows for the timely detection of incident fibroids, thus reducing the\nmisclassification of fibroid status which is present in prior observational studies.\nOur multiple measures of serum 25(OH)D reduce the bias introduced when a single\nmeasure of 25(OH)D is assumed to be relevant for later timepoints. In addition, our\nfocus on Black women allows us to examine individuals at high risk for both vitamin\nD deficiency and fibroid burden, although vitamin D deficiency in reproductive-aged\nwomen in the United States is widespread ( 7 ).\nVitamin D is well known to be critical for bone health ( 29 ) with growing evidence that higher concentrations of\n25(OH)D improve reproductive outcomes ( 56 )\nand reduce breast cancer risk ( 42 ). Our\nfindings add support to the existing literature suggesting vitamin D may also reduce\nfibroid development. As compared with existing medical and surgical treatments for\nfibroids that have significant side-effects and impact fertility, vitamin D is safe\nand compatible with pregnancy. The high prevalence of vitamin D deficiency in the\nSELF cohort and reproductive-aged U.S. women in general ( 47 ) suggests that further public health messaging about\nvitamin D is needed. An additional issue for U.S. women is that elevated BMI is\nassociated with lower 25(OH)D concentrations ( 57 ). Current dietary and supplement recommendations for vitamin D intake\nmay not be sufficient for individuals with high BMI who require 2–3 times\nhigher vitamin D supplementation to attain desired serum concentrations ( 58 ).\nIn conclusion, 25(OH)D concentrations above 20 ng/ml were associated with\nreduced fibroid growth. Results for analyses examining 25(OH)D concentrations above\n30 ng/ml provided suggestive evidence for increased likelihood of fibroid loss and a\npossible reduction in fibroid incidence. Intervention trials with vitamin D\nsupplementation to attain and maintain sufficient 25(OH)D will be needed to further\nassess the impact of vitamin D on fibroid development.","source_license":"CC0","license_restricted":false}