Exploring the Impact of 3,3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women

Exploring the Impact of 3,3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring the Impact of 3,3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women Mark Newman, Jaclyn Smeaton This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3944853/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background 3, 3’-diindolylmethane (DIM) is a phytonutrient derived from cruciferous vegetables that is an often used supplement in the complementary and alternative medicine space. The most common goal for providers when recommending DIM to their patients is to alter estrogen metabolism, yet research into DIM’s effect on the estrogen profile is lacking in the published literature. The objective of this study was to comprehensively evaluate DIM’s effect on the urinary estrogen profile. Methods In this retrospective cohort study, we analyzed data from a clinical laboratory, including urinary estrogen and estrogen metabolite concentrations. Analyte concentrations were determined from dried urine samples using a gas chromatography-tandem mass spectrometry assay. Individuals were separated into 2 groups, either reporting taking DIM (N = 909) or reporting not taking DIM (N = 18,385). Comparisons between individuals in these two groups were made using the Wilcoxon rank sum test. Additionally, we were also able to explore a subset of women who had laboratory results in the database before and after initiating DIM treatment (N = 53). In this subset, differences were assessed with Wilcoxon signed rank tests. Results In the larger group, significant differences were observed in the concentrations of almost every urinary estrogen and estrogen metabolite (with the only exception being 2-methoxyestrone) in the urinary estrogen profiles of those taking DIM compared to those not taking DIM (all P values < 0.001). In the smaller subset of individuals with before and after measurements, differences were only seen in 4 of the urinary estrogens and estrogen metabolites (P < 0.001 for estrone, estradiol, estriol, and 16-hydroxyestrone). Differences in total estrogens were significant in both the larger group and the smaller subset (both with P < 0.001). Additionally, observed differences in the ratios of metabolites followed a similar trend with more significant differences observed in the larger group. Notably, the 2-hydroxyestrone:16-hydroxyestrone ratio increased significantly in both the larger and the before and after group. Conclusions The results of this study provide the most comprehensive evaluation to date of DIM’s effect on the urinary estrogen profile. Additionally, the results demonstrate that the dried urine collection and accompanying assay used captures changes that are similar in direction, but not necessarily magnitude, to previous reports in the literature. Considered together, these two things highlight the clinical validity and utility of this approach to the evaluation of DIM supplementation and suggest the need for additional studies using this approach to fully understand the potential clinical utility of DIM. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As part of the burgeoning interest in integrative and functional medicine approaches to women’s health, dietary supplements such as indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) have gained attention for their potential impact on estrogen metabolism. I3C and DIM are derived from cruciferous vegetables including broccoli, Brussels sprouts, bok choy, cabbage, cauliflower, collards, and kale. 1 Once ingested, I3C is rapidly converted to multiple oligomers of which DIM is the predominant active agent, and the molecule responsible for the majority of the observed effects. 1–3 However, although considerable experimental and theoretical effort has been aimed at understanding the mechanism of action (MOA) and effects of DIM in other contexts, 4–10 little focus has been directed at understanding it’s MOA and effects in applications common to functional and integrative medicine. The MOA of DIM is complex and not completely understood. When recommending DIM supplements to patients, the goal of integrative and functional medicine providers is usually to alter estrogen metabolism or lower total estrogen levels. In this setting, the most relevant component of DIM’s MOA is the modulation of estrogen metabolism via the induction and inhibition of cytochrome P450 (CYP) enzymes. In previous studies, DIM was shown to be an inducer of CYP1A1, CYP1A2, and, to a lesser extent, CYP3A4. 11,12 Importantly, DIM’s MOA has many other proposed components that aren’t directly relevant to the current study. In order to understand the potential impact of DIM on estrogen metabolism, it is necessary to understand the role of CYP enzymes in estrogen metabolism. In phase 1 of estrogen metabolism estrone (E1) and estradiol (E2) undergo hydroxylation reactions which are mediated by the same CYP enzymes that DIM induces, along with CYP1B1, and to a lesser extent CYP3A5 and CYP3A7. 13 CYP1A1 and CYP1A2 catalyze the hydroxylation of E1 and E2, leading to the formation of 2-hydroxyestrone (2-OHE1) and 2-hydroxyestradiol (2-OHE2) which are considered to be less proliferative compared to other phase 1 metabolites. 12,14 In contrast, CYP1B1 catalyzes the the 4-hydroxylation of E1 and E2 producing 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2), which are considered to be more proliferative metabolites due to the reactive quinones they can yield via redox cycling. 12 The 4-hydroxylation reaction is also catalyzed, to a lesser extent, by CYP3A4 and CYP3A5. 13 In addition to there being little investigation of the MOA of DIM as it relates to integrative and functional medicine indications, there is similarly little research into understanding the effects of DIM on the entire estrogen profile. The studies that have been published generally only look at one component of the estrogen profile that is of interest, such as the 2-OHE1/16-OHE1 ratio. 15,16 This fact has presented functional and integrative medicine practitioners with a dilemma. Despite compelling anecdotal evidence of the effectiveness of DIM in integrative and functional medicine applications, there are no published studies that provide definitive evidence of the expected changes in the estrogen profile. Compounding this issue is the fact that the mechanism of action of DIM is not completely understood. The dearth of direct evidence in the published literature urged us to explore potential mechanisms that may be illuminated in the urinary estrogen profile. Previous studies have validated the collection and assay method used in the current study for exploring estrogen metabolism in other contexts. 17–21 The primary aim of this study was to use this validated method to explore and present a detailed analysis of DIM’s effects on estrogen metabolism in humans by evaluating the complete urinary estrogen profile of premenopausal women in the luteal phase of their cycle. The complete urinary estrogen profile we sought to explore includes parent estrogens, estrogen metabolites, and relevant ratios. Material and Methods Data Source In this retrospective data mining study registered as Precision Analytical Retrospective Data Correlation (Clinical Trials ID, NCT04305093), data were available for 144,561 laboratory accessions from 129,883 individuals between January 1, 2016 and December 9, 2019. The study was approved by the National University of Natural Medicine Institutional Review Board, and since all data analyzed in this data mining study were deidentified, the institutional review board determined that written informed consent could be waived. Deidentified data were extracted from the database and included the following variables: age, sex, menstrual status (regular, irregular, none), last menstrual period, body mass index (BMI) calculated from self-reported height and weight, use of DIM, comorbidities, and urinary measures of estrone, estradiol, estriol, 𝛼 and 𝛽-pregnanediol, 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, 4-hydroxyestradiol, 16-hydroxyestrone, and 2-methoxyestrone. Data on race/ethnicity were not available. Inclusion criteria for this study included being a premenopausal woman reporting regular menses and having collected samples in the luteal phase of the menstrual cycle. Exclusion criteria included the following: a BMI less than 16 or greater than 60 kg/m 2 ; pregnancy; any reported kidney disease; reported adrenal insufficiency or excess; suspected or diagnosed PCOS; missing data for urinary estrogens or estrogen metabolites (with the exception of 4-hydroxyestradiol and 2-methoxyestradiol); use of any estrogen supplementation (oral, sublingual pelleted, injected, or other transdermal formulation); use of 𝛽-human chorionic gonadotropin; use of anastrazole; use of tamoxifen; evidence of overly dilute urine (any spot urine creatinine < 0.1 mg/ml). Sample Collection and Laboratory Methods The sample collection and assay methods used to produce the data analyzed in this study, including assay performance characteristics, have been described previously. 17,18 Briefly, participants collected a total of four urine samples on filter paper over 24 hours. These samples were allowed to dry and then shipped to the laboratory for analysis. Once samples were received in the laboratory, urine was extracted from the filter paper and the four samples were combined into a single representative sample. This combination was done by using creatinine concentrations from each individual sample to determine the volume of sample that should be added to the combined sample to best represent measured concentrations over a 24-hour period. The combined sample was then analyzed via gas chromatography-tandem mass spectrometry on an Agilent 7890/7000B (Agilent Technologies, Santa Clara, CA). Estrogen and estrogen metabolite concentrations were normalized to creatinine to account for variation in both urine concentration and filter paper saturation. Statistical Methods All values are presented as medians and interquartile ranges as they were not normally distributed. In the larger group, we compared characteristics of premenopausal women taking DIM and premenopausal women not taking DIM. These comparisons were done using the Wilcoxon rank-sum test. In the smaller subset of individuals who had laboratory results available both before and after initiating DIM, comparisons were made with the Wilcoxon signed rank test. Because this was a data mining study using a convenience sample, no sample size or power calculation was performed a priori, and all comparisons should be considered exploratory and hypothesis generating in nature. All analyses were conducted using R version 4.3.1 (R Version for Statistical Computing, Vienna, Austria). Results Comparison of Women Not Taking DIM to Women Taking DIM After applying inclusion and exclusion criteria, there were 18,385 women who did not report taking DIM and 909 women who reported taking DIM. Patient characteristics and urinary estrogen and estrogen metabolite concentrations for these two groups of women are reported and compared in Table 1 . The urinary estrogen metabolite ratios for these two groups are reported and compared in Table 2 . Significant differences were observed in both age and BMI along with all urinary estrogen metabolites with the exception of 2-methoxyestrone. Urinary E1, E2, and E3 concentrations were all lower in the group taking DIM, as were the concentrations of 16-hydroxyestrone and total urinary estrogens. Concentrations of 2-hydroxyestrone, 4-hydroxyestrone, 2 hydroxyestradiol, and 4-hydroxyestradiol were all higher in the women taking DIM. These differences in metabolite concentrations translated to significant differences in all of the metabolite ratios that were calculated. Before and After Initiating DIM There were a total of 53 women who had laboratory results available both before and after starting DIM. Age, BMI, and urinary estrogen and estrogen metabolite concentrations for these women are reported and compared in Table 3 . Urinary estrogen metabolite ratios for these women before and after initiating DIM are compared in Table 4 . The before and after values for some of the metabolites and some of the ratios can be seen in the context of the estrogen metabolic pathway in Fig. 1 and Fig. 2 . With the exception of 2-methoxyestrone and 2OHE2, all parent estrogens and metabolites changed in the same direction as in the larger group. However, only the changes in E1, E2, E3, and 16-OHE1 reached the threshold for significance. All of the ratios changed in the same direction as in the larger group; however, the only ratio changes that reached significance were the 2-OHE1/16-OHE1, 2-OHE1/E1, 2-OHE1/E2, and the 2-methoxyE1/2-OHE1 ratios. The percent changes for E2, 2-OHE1, 4-OHE1, 16-OHE1, and the 2/16 ratio are presented in Table 5 . Table 1 Patient Characteristics and Urinary Estrogen Concentrations Characteristic No DIM, N = 18,385 1 DIM, N = 909 1 p-value 2 Age at Collection 38 (33, 43) 40 (35, 44) < 0.001 BMI 23.8 (21.3, 26.6) 23.4 (21.0, 25.8) 0.003 Urinary Estrone (ng/mg-Cr) 18.00 (12.70, 24.90) 14.70 (10.30, 20.70) < 0.001 Urinary Estradiol (ng/mg-Cr) 3.10 (2.22, 4.34) 2.62 (1.89, 3.86) < 0.001 Urinary Estriol (ng/mg-Cr) 9.60 (5.90, 15.10) 5.20 (3.00, 9.50) < 0.001 Urinary 2-hydroxyestrone (ng/mg-Cr) 8.24 (5.40, 11.50) 11.55 (7.90, 15.81) < 0.001 Urinary 4-hydroxyestrone (ng/mg-Cr) 1.09 (0.75, 1.50) 1.37 (0.97, 2.00) < 0.001 Urinary 16-hydroxyestrone (ng/mg-Cr) 1.21 (0.73, 1.99) 0.82 (0.45, 1.44) < 0.001 Urinary 2-methoxyestrone (ng/mg-Cr) 4.20 (2.79, 5.91) 4.05 (2.70, 5.97) 0.7 Urinary 2-hydroxyestradiol (ng/mg-Cr) 0.66 (0.36, 1.09) 0.99 (0.55, 1.58) < 0.001 Urinary 4-hydroxyestradiol (ng/mg-Cr) 0.30 (0.20, 0.40) 0.40 (0.20, 0.50) < 0.001 Missing Values 5,560 260 Total Urinary Estrogens (ng/mg-Cr) 48.50 (36.50, 63.30) 44.20 (33.00, 58.10) < 0.001 1 Median (IQR) 2 Wilcoxon rank sum test Table 2 Patient Characteristics and Urinary Estrogen Metabolite Ratios Characteristic No DIM, N = 18,385 1 DIM, N = 909 1 p-value 2 Age at Collection 38 (33, 43) 40 (35, 44) < 0.001 BMI 23.8 (21.3, 26.6) 23.4 (21.0, 25.8) 0.003 2OHE1/16OHE1 Ratio 6.89 (3.72, 12.10) 15.36 (7.49, 25.65) < 0.001 Missing Values 1 0 2OHE1/E1 Ratio 0.44 (0.31, 0.61) 0.79 (0.54, 1.10) < 0.001 Missing Values 1 0 2OHE1/E2 Ratio 2.56 (1.69, 3.63) 4.30 (2.87, 5.99) < 0.001 2OHE1/4OHE1 Ratio 7.27 (5.36, 9.46) 8.15 (6.00, 10.43) < 0.001 Missing Values 2 0 2-methoxyE1/2OHE1 Ratio 0.51 (0.40, 0.66) 0.35 (0.27, 0.46) < 0.001 2OHE2/2OHE1 Ratio 0.08 (0.06, 0.11) 0.08 (0.06, 0.12) 0.004 1 Median (IQR) 2 Wilcoxon rank sum test Table 3 Before and After Estrogen & Estrogen Metabolite Concentrations Variable Before DIM 1 After DIM 1 p-value 2 Age at Collection 39 (32, 42) 40 (34, 44) 0.2 BMI 24.1 (21.3, 26.6) 23.4 (21.3, 25.7) 0.8 Urinary a-pregnanediol (ng/mg-Cr) 417 (284, 843) 427 (308, 681) 0.6 Urinary b-pregnanediol (ng/mg-Cr) 1,350 (1,144, 1,657) 1,263 (973, 1,964) 0.7 Urinary Estrone (ng/mg-Cr) 23.40 (18.40, 32.10) 15.10 (11.10, 22.30) < 0.001 Urinary Estradiol (ng/mg-Cr) 4.10 (3.00, 5.55) 2.84 (2.17, 4.03) < 0.001 Urinary Estriol (ng/mg-Cr) 10.60 (6.50, 19.90) 4.70 (2.90, 8.00) < 0.001 Urinary 2-hydroxyestrone (ng/mg-Cr) 10.50 (7.70, 14.50) 11.70 (8.46, 16.64) 0.4 Urinary 4-hydroxyestrone (ng/mg-Cr) 1.37 (1.10, 2.10) 1.57 (1.12, 2.07) 0.5 Urinary 16-hydroxyestrone (ng/mg-Cr) 1.80 (1.00, 3.62) 0.70 (0.39, 1.24) < 0.001 Urinary 2-methoxyestrone (ng/mg-Cr) 4.70 (3.70, 6.20) 4.86 (3.20, 6.12) 0.8 Urinary 2-hydroxyestradiol (ng/mg-Cr) 1.10 (0.51, 1.86) 1.01 (0.58, 1.68) 0.8 Urinary 4-hydroxyestradiol (ng/mg-Cr) 0.35 (0.30, 0.50) 0.40 (0.30, 0.60) 0.6 Missing Values 37 5 Urinary 2-methoxyestradiol (ng/mg-Cr) 0.55 (0.48, 0.83) 0.50 (0.30, 0.73) 0.4 Missing Values 37 5 Total Urinary Estrogens (ng/mg-Cr) 64.0 (50.3, 77.0) 47.2 (35.3, 60.7) < 0.001 1 Median (IQR) 2 Wilcoxon rank sum test Table 4 Before and After Estrogen Metabolite Ratio Values Variable Before DIM 1 After DIM 1 p-value 2 Age at Collection 39 (32, 42) 40 (34, 44) 0.2 BMI 24.1 (21.3, 26.6) 23.4 (21.3, 25.7) 0.8 2OHE1/16OHE1 Ratio 5.67 (2.79, 13.66) 18.20 (9.64, 31.58) < 0.001 2OHE1/E1 Ratio 0.45 (0.30, 0.61) 0.88 (0.56, 1.14) < 0.001 2OHE1/E2 Ratio 2.40 (1.59, 3.81) 4.27 (3.05, 5.80) < 0.001 2OHE1/4OHE1 Ratio 6.92 (5.41, 9.33) 7.51 (6.21, 9.69) 0.3 2-methoxyE1/2OHE1 Ratio 0.41 (0.32, 0.57) 0.35 (0.27, 0.51) 0.027 2OHE2/2OHE1 Ratio 0.09 (0.07, 0.12) 0.08 (0.07, 0.11) 0.6 1 Median (IQR) 2 Wilcoxon rank sum test Table 5 Percent Change in Key Metabolites and the 2/16 Ratio (In women with results before and after initiating DIM) Variable Percent Change Estradiol -23.13% 2-Hydroxyestrone 19.24% 4-Hydroxyestrone 8.95% 16-Hydroxyestrone -55.56% 2OHE1/16OHE1 Ratio 188.31% Discussion In this study, we have examined retrospective data in an attempt to more completely understand the effect of DIM on estrogen metabolism in premenopausal women. We found significant differences in almost all estrogens, estrogen metabolites, and estrogen metabolite ratios when we compared the larger groups of women who were either taking DIM or not taking DIM. In the smaller group of women who had laboratory results available before and after starting DIM, although we saw differences in the same directions as in the larger group, fewer of these differences reached significance. This study has several limitations. The data used in this analysis is from a convenience sample and thus there is limited external generalizability to populations that are different from the population studied. However, given the large sample size, valuable insights can be garnered to inform the design of future, more targeted studies. We also had no access to data on participants’ diet which could have a meaningful impact if a participant’s diet was particularly high in cruciferous vegetables (or particularly low). In addition to not having access to data on diet, although we did collect some data regarding prescription medication and supplement use, we were unable to collect and exhaustive list of concurrent prescription medications and over the counter supplements. In this patient population there is sometimes concurrent use of other estrogen-lowering or estrogen metabolism altering agents such as calcium-d-glucarate. 22 These agents may have also contributed to some of the lower estrogen and estrogen metabolites concentrations seen, but these agents would be less likely to affect the ratios of estrogen metabolites. We did not collect data regarding dose or formulation of DIM/I3C, frequency of administration, or adherence. Although this is certainly a limitation, it also reflects the real-world nature of the data and represents what is likely seen in actual clinical practice. There are DIM products which claim higher bioavailability, 23 so it possible that the use of different products may have significantly different impacts on the urinary estrogen profile. The results of this study highlight that this collection method and assay would be a useful tool to investigate potential differing impacts between DIM products. Because of differing bioavailability and half-life, some products may recommend more or less frequent dosing, however dosing is usually once or twice daily. Finally, we do not have data on the rationale employed for initiating DIM. The reasons for using DIM vary widely in the integrative and functional medicine space, and capturing the potential “indications” for DIM use concisely is beyond the scope of this manuscript. Notwithstanding these limitations, this study provides both a comparative evaluation of premenopausal women taking vs. not taking DIM and a repeated measures evaluation of a subset of women using DIM. These evaluations shed light on the impact of DIM on the entire urinary estrogen profile which, to the best of our knowledge, has not previously been explored this comprehensively. Many of the previous studies that evaluated the effects of DIM focused on changes in 2-OHE1, 16-OHE1, and the 2/16 ratio. 6,15,16,24 In addition to the focus on the 2-OHE1 and 16-OHE1, Dalessandri and colleagues also reported urinary E1, E2, and E3 concentrations before and after DIM use, along with urinary 6𝛽-hydroxycortisol (6𝛽-OHC), cortisol, and the 6𝛽-OHC/cortisol ratio, which has shown to be an indication of CYP3A4 enzyme activity. 25 As discussed earlier, regarding DIM, the activity of CYP3A4 influences production of 16-OHE1 and 4-OHE2, the non-favored estrogen metabolites. One of the only studies in the literature that is similar to the present study is one done by Green and colleagues which used the same assay as the present study. 16 However, participants in that study were given a dietary supplement that contained I3C and at least 8 additional ingredients. Moreover, the women participating in that study may not have been adequately fluid restricted and samples were collected during the day. The combination of these 2 factors may have led to low creatinine values to which the estrogen metabolite concentrations are indexed. Furthermore, the overall estrogen and estrogen metabolite concentrations for the study participants were low compared to established reference ranges. The primary aim of that study was simply to determine if the nutraceutical product used, Estrosense, increased the 2/16 ratio. Results in this study are similar in direction to the four previously mentioned studies conducted by Rajoria and colleagues, Thomson and colleagues, Godinez-Martinez and colleagues, and Green and colleagues. Here, just as in all of those studies, 2-OHE1 increased and 16-OHE1 decreased with DIM treatment, which translated to an increase in the 2/16 ratio. One interesting observation concerns the change in 4-OHE1. In the present study, although 4-OHE1 increased in the before and after group, the change did not reach significance. Similar results were seen in the study by Thomson and colleagues. They reported an initial small median decrease at 6 months, followed by an increase at 12 months; however median change from baseline to 12 months was 0.0, and the P-value comparing DIM treated participants with the placebo arm was not significant (P = 0.534) indicating no difference in effect on 4-OHE1 between DIM and placebo. This echoes the conclusion that we have drawn from our data. This conclusion may be surprising to some integrative and functional medicine providers who expect DIM to reduce 4-OHE1. The currently available evidence does not support DIM use for this purpose. The 2OHE1/4OHE1 ratio did increase in both groups, although this change did not reach the threshold for significance in the before and after group. Interestingly, in the study conducted by Green and colleagues, the only outcome measures with changes from baseline that reached statistical significance were 2-OHE1 (P = 0.01) and the 2/16 ratio (P < 0.001). This indicates that the change in the primary outcome was driven by the observed increased in 2-OHE1 which was only 0.43 ng/mg-Cr. In the present study we saw a somewhat opposite result in that even though the median 2-OHE1 concentration increased by a larger margin (1.20 ng/mg-Cr), the difference from baseline was not significant (P = 0.4). However, the difference in 16-OHE1 was significant (P < 0.001). This suggests that although the outcome measure of increase in the 2/16 ratio was the same in both studies, the underlying change in estrogen metabolism driving the increase in the ratio was different in these two studies. This has important implications for clinical decision making depending on the goals of the provider. Future randomized, placebo-controlled studies with multiple DIM products are needed to fully understand the potential clinical utility of DIM supplementation. The tools presented here for assessing the effects of DIM seem to be the most comprehensive approach to gaining actionable clarity. Conclusion Although a complete understanding of the mechanism of action of DIM continues to elude researchers, this and previous studies serve to illuminate some of the expected effects in premenopausal women. The results of this study demonstrate that the dried urinary sampling method and corresponding assay used to measure estrogen metabolites is a useful approach to monitoring the effects of DIM therapy in real world clinical practice. To conclude, we propose that the changes seen here in parent estrogen concentrations, estrogen metabolite concentrations, and estrogen metabolite ratios represent a key component to understanding the complex and somewhat convoluted mechanism of action of DIM. The specific changes that we have either confirmed or uncovered provide a molecular explanation for some of the observed effects of DIM. The results of this study, taken together with the theoretical and observed partial mechanism of action of DIM, provide moderate evidence supporting the potential clinical utility of DIM and strong evidence supporting the definite clinical utility of urinary estrogen and estrogen metabolite monitoring in the setting of DIM supplementation. Additional work is needed to completely reveal how the complex mechanism of action of DIM is reflected in the complex estrogen metabolism pathway. Disentangling the complexity of estrogen metabolism remains a formidable challenge, which may well demand the use of novel methods. We posit that the use of urinary estrogen and estrogen metabolite concentration changes to explore theoretical and actual consequences of altered estrogen metabolism will prove to be a drama of refound opportunity in the coming years. Declarations Ethics approval and consent to participate The study was approved by the National University of Natural Medicine Institutional Review Board, and since all data analyzed in this data mining study were deidentified, the institutional review board determined that written informed consent could be waived. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests M.N. is the founder and CEO of Precision Analytical, Inc. J.S. is an employee of Precision Analytical, Inc. Funding None Authors’ contributions MN conceived and designed the study and acquired the data. Both MN and JS analyzed and interpreted the data. Both MN and JS wrote and revised the manuscript. The final version of the manuscript was read and approved by both authors. References Thomson CA, Ho E, Strom MB. Chemopreventive properties of 3,30-diindolylmethane in breast cancer: Evidence from experimental and human studies. Nutrition Reviews . 2016;74(7):432-443. doi:10.1093/nutrit/nuw010 Bradlow HL, Zeligs MA. Diindolylmethane (DIM) Spontaneously Forms from Indole-3-carbinol (I3C) During Cell Culture Experiments. in vivo . Published online 2010. 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Indoles Derived From Glucobrassicin: Cancer Chemoprevention by Indole-3-Carbinol and 3,3’-Diindolylmethane. Frontiers in Nutrition . 2021;8:734334. doi:10.3389/fnut.2021.734334 Yang L, Zheng C, Xia YF, Dai Y, Wei ZF. 3, 3’-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology . 2023;116:154874. doi:10.1016/j.phymed.2023.154874 Li CS, Nguyen TV, Chai OH, Park BH, Lee JS, Kim SM. 3,3’-Diindolylmethane Augments 5-Fluorouracil-InducedGrowth Suppression in Gastric Cancer Cells through Suppression of the Akt/GSK-3β and WNT/Beta-Catenin. Journal of Oncology . 2023;2023:8268955. doi:10.1155/2023/8268955 Tucci P, Brown I, Bewick GS, Pertwee RG, Marini P. The Plant Derived 3-3’-Diindolylmethane (DIM) Behaves as CB2 Receptor Agonist in Prostate Cancer Cellular Models. International Journal of Molecular Sciences . 2023;24(4):3620. doi:10.3390/ijms24043620 Sowers MR, Wilson AL, Kardia SR, Chu J, McConnell DS. CYP1A1 and CYP1B1 Polymorphisms and Their Association with Estradiol and Estrogen Metabolites in Women Who Are Premenopausal and Perimenopausal. The American Journal of Medicine . 2006;119(9):S44-S51. doi:10.1016/j.amjmed.2006.07.006 Szaefer H, Licznerska B, Krajka-Kuźniak V, Bartoszek A, Baer-Dubowska W. Modulation of CYP1A1, CYP1A2 and CYP1B1 Expression by Cabbage Juices and Indoles in Human Breast Cell Lines. Nutrition and Cancer . 2012;64(6):879-888. doi:10.1080/01635581.2012.690928 Sood D, Johnson N, Jain P, et al. CYP3A7*1C allele is associated with reduced levels of 2-hydroxylation pathway oestrogen metabolites. British Journal of Cancer . 2017;116(3):382-388. doi:10.1038/bjc.2016.432 Tsuchiya Y, Nakajima M, Yokoi T. Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Letters . 2005;227(2):115-124. doi:10.1016/j.canlet.2004.10.007 Godínez-Martínez E, Santillán R, Sámano R, Chico-Barba G, Tolentino MC, Hernández-Pineda J. Effectiveness of 3,3’-Diindolylmethane Supplements on Favoring the Benign Estrogen Metabolism Pathway and Decreasing Body Fat in Premenopausal Women. Nutrition and Cancer . 2023;75(2):510-519. doi:10.1080/01635581.2022.2123535 Green T, See J, Schauch M, et al. A randomized, double-blind, placebo-controlled, cross-over trial to evaluate the effect of EstroSense ® on 2-hydroxyestrone:16α-hydroxyestrone ratio in premenopausal women. Journal of Complementary and Integrative Medicine . 2023;20(1):199-206. doi:10.1515/jcim-2022-0301 Newman M, Pratt SM, Curran DA, Stanczyk FZ. Evaluating urinary estrogen and progesterone metabolites using dried filter paper samples and gas chromatography with tandem mass spectrometry (GCMS/MS). BMC Chemistry . 2019;13(1):1-12. doi:10.1186/s13065-019-0539-1 Newman M, Curran DA. Reliability of a dried urine test for comprehensive assessment of urine hormones and metabolites. BMC Chemistry . 2021;15(1):18. doi:10.1186/s13065-021-00744-3 Newman MS, Curran DA, Mayfield BP, Saltiel D, Stanczyk FZ. Assessment of estrogen exposure from transdermal estradiol gel therapy with a dried urine assay. Steroids . 2022;184:109038. doi:10.1016/j.steroids.2022.109038 Newman MS, Mayfield BP, Saltiel D, Stanczyk FZ. Assessing estrogen exposure from transdermal estradiol patch therapy using a dried urine collection and a GCMS/MS assay. Steroids . 2023;189:109149. doi:10.1016/j.steroids.2022.109149 Newman MS, Saltiel D, Smeaton J, Stanczyk FZ. Comparative estrogen exposure from compounded transdermal estradiol creams and Food and Drug Administration-approved transdermal estradiol gels and patches. Menopause . Published online October 18, 2023. doi:10.1097/GME.0000000000002266 Oredipe OA, Barth RF, Dwivedi C, Webb TE. Dietary glucarate-mediated inhibition of initiation of diethylnitrosamine-induced hepatocarcinogenesis. Toxicology . 1992;74(2-3):209-222. doi:10.1016/0300-483X(92)90140-A Anderton MJ, Manson MM, Verschoyle R, et al. PHYSIOLOGICAL MODELING OF FORMULATED AND CRYSTALLINE 3,3’-DIINDOLYLMETHANE PHARMACOKINETICS FOLLOWING ORAL ADMINISTRATION IN MICE. Drug Metabolism and Disposition . 2004;32(6):632-638. doi:10.1124/dmd.32.6.632 Thomson C, Chow S, Roe D, et al. Effect of diindolylmethane on estrogen-related hormones, metabolites and tamoxifen metabolism: Results of a randomized, placebo-controlled trial. Cancer Epidemiology, Biomarkers & Prevention . 2017;26(3):435. doi:10.1158/1055-9965.EPI-17-0027 Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF. Pilot study: Effect of 3,3’-diindolylmethane supplements on urinary hormone metabolites in postmenopausal women with a history of early-stage breast cancer. Nutrition and Cancer . 2004;50(2):161-167. doi:10.1207/s15327914nc5002_5 Additional Declarations Competing interest reported. M.N. is the founder and CEO of Precision Analytical, Inc. J.S. is an employee of Precision Analytical, Inc. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 16 Oct, 2024 Reviews received at journal 28 Jun, 2024 Reviews received at journal 23 Jun, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviewers invited by journal 04 May, 2024 Editor assigned by journal 03 May, 2024 Editor invited by journal 18 Apr, 2024 Submission checks completed at journal 18 Apr, 2024 First submitted to journal 09 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3944853","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293762167,"identity":"15b9fe3f-fefc-4edd-bdcd-85504cfd0132","order_by":0,"name":"Mark Newman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYHACZhiD8QEPkDQgRQuzAQ9QgwEbkHmASC1sEkRp0W0/+9iAcYdN4tr2s8cq3rb9kTeXbz78+QODnZxuA3YtZmfSjRMYz6QlbjuTl3ZzbpuB4c42tjSJAwzJxmY4rDI7kMZ8gLHtcO62Azlmt3nbDBIMjvGYAR12IHEbLi3nn4G0/M/ddv6NWTFEC//nD3i13EhjTmBsO5C77UaOGTPUFgYJ/FqeMRsktiXXb7vxxlhyzjljww3H0swkzhjg8cv5NGaJj212xmbncww/vCmTkzc4fPjxh4oKOzlcWsAgAVOImDQwCkbBKBgFowAnAADyg2CHDKsfswAAAABJRU5ErkJggg==","orcid":"","institution":"Precision Analytical","correspondingAuthor":true,"prefix":"","firstName":"Mark","middleName":"","lastName":"Newman","suffix":""},{"id":293762169,"identity":"e4ab5587-96ab-48a5-8f23-ab610da9bf51","order_by":1,"name":"Jaclyn Smeaton","email":"","orcid":"","institution":"Precision Analytical","correspondingAuthor":false,"prefix":"","firstName":"Jaclyn","middleName":"","lastName":"Smeaton","suffix":""}],"badges":[],"createdAt":"2024-02-10 03:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3944853/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3944853/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55514616,"identity":"29a0cfce-e0d6-451a-9c8d-cf3a5fc748e0","added_by":"auto","created_at":"2024-04-29 13:03:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":310150,"visible":true,"origin":"","legend":"\u003cp\u003eUrinary Estrogen Metabolite Before and After Values\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/549776613da10ef339e302a0.jpg"},{"id":55513606,"identity":"fcb81ecc-8e00-4fa3-956a-44ebc2e5ff92","added_by":"auto","created_at":"2024-04-29 12:55:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":125088,"visible":true,"origin":"","legend":"\u003cp\u003eUrinary Estrogen Metabolite Ratio Before and After Values\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/30debd782f12d36ed5d81d86.jpg"},{"id":55515588,"identity":"7b3c0226-8585-4f87-873f-94834aae3cd3","added_by":"auto","created_at":"2024-04-29 13:11:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47508,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/0a320a3310e8cc0ce21e8a59.jpg"},{"id":55515589,"identity":"014aca52-f630-44ff-a658-20f7dc09a7cb","added_by":"auto","created_at":"2024-04-29 13:11:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49965,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/feb2b4d920877146891a7ee8.jpg"},{"id":55513602,"identity":"8f560d2d-fa56-4e04-ac75-c153507d42ba","added_by":"auto","created_at":"2024-04-29 12:55:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43322,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/4e5199e309c111d35139eb7b.jpg"},{"id":55513600,"identity":"2884fa3d-38c3-477e-a9a6-4e59804594c8","added_by":"auto","created_at":"2024-04-29 12:55:24","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46810,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/93fdbdfc3af8149ae3da5a90.jpg"},{"id":55516796,"identity":"a603b733-6e62-42e5-9cf0-e1be0570ab61","added_by":"auto","created_at":"2024-04-29 13:19:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":662972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944853/v1/eca4b8de-30b0-404a-9c0a-905fee872e04.pdf"}],"financialInterests":"Competing interest reported. M.N. is the founder and CEO of Precision Analytical, Inc. J.S. is an employee of Precision Analytical, Inc.","formattedTitle":"Exploring the Impact of 3,3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs part of the burgeoning interest in integrative and functional medicine approaches to women\u0026rsquo;s health, dietary supplements such as indole-3-carbinol (I3C) and 3,3\u0026rsquo;-diindolylmethane (DIM) have gained attention for their potential impact on estrogen metabolism. I3C and DIM are derived from cruciferous vegetables including broccoli, Brussels sprouts, bok choy, cabbage, cauliflower, collards, and kale.\u003csup\u003e1\u003c/sup\u003e Once ingested, I3C is rapidly converted to multiple oligomers of which DIM is the predominant active agent, and the molecule responsible for the majority of the observed effects.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e However, although considerable experimental and theoretical effort has been aimed at understanding the mechanism of action (MOA) and effects of DIM in other contexts,\u003csup\u003e4\u0026ndash;10\u003c/sup\u003e little focus has been directed at understanding it\u0026rsquo;s MOA and effects in applications common to functional and integrative medicine.\u003c/p\u003e \u003cp\u003eThe MOA of DIM is complex and not completely understood. When recommending DIM supplements to patients, the goal of integrative and functional medicine providers is usually to alter estrogen metabolism or lower total estrogen levels. In this setting, the most relevant component of DIM\u0026rsquo;s MOA is the modulation of estrogen metabolism via the induction and inhibition of cytochrome P450 (CYP) enzymes. In previous studies, DIM was shown to be an inducer of CYP1A1, CYP1A2, and, to a lesser extent, CYP3A4.\u003csup\u003e11,12\u003c/sup\u003e Importantly, DIM\u0026rsquo;s MOA has many other proposed components that aren\u0026rsquo;t directly relevant to the current study.\u003c/p\u003e \u003cp\u003eIn order to understand the potential impact of DIM on estrogen metabolism, it is necessary to understand the role of CYP enzymes in estrogen metabolism. In phase 1 of estrogen metabolism estrone (E1) and estradiol (E2) undergo hydroxylation reactions which are mediated by the same CYP enzymes that DIM induces, along with CYP1B1, and to a lesser extent CYP3A5 and CYP3A7.\u003csup\u003e13\u003c/sup\u003e CYP1A1 and CYP1A2 catalyze the hydroxylation of E1 and E2, leading to the formation of 2-hydroxyestrone (2-OHE1) and 2-hydroxyestradiol (2-OHE2) which are considered to be less proliferative compared to other phase 1 metabolites.\u003csup\u003e12,14\u003c/sup\u003e In contrast, CYP1B1 catalyzes the the 4-hydroxylation of E1 and E2 producing 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2), which are considered to be more proliferative metabolites due to the reactive quinones they can yield via redox cycling.\u003csup\u003e12\u003c/sup\u003e The 4-hydroxylation reaction is also catalyzed, to a lesser extent, by CYP3A4 and CYP3A5.\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn addition to there being little investigation of the MOA of DIM as it relates to integrative and functional medicine indications, there is similarly little research into understanding the effects of DIM on the entire estrogen profile. The studies that have been published generally only look at one component of the estrogen profile that is of interest, such as the 2-OHE1/16-OHE1 ratio.\u003csup\u003e15,16\u003c/sup\u003e This fact has presented functional and integrative medicine practitioners with a dilemma. Despite compelling anecdotal evidence of the effectiveness of DIM in integrative and functional medicine applications, there are no published studies that provide definitive evidence of the expected changes in the estrogen profile. Compounding this issue is the fact that the mechanism of action of DIM is not completely understood.\u003c/p\u003e \u003cp\u003eThe dearth of direct evidence in the published literature urged us to explore potential mechanisms that may be illuminated in the urinary estrogen profile. Previous studies have validated the collection and assay method used in the current study for exploring estrogen metabolism in other contexts.\u003csup\u003e17\u0026ndash;21\u003c/sup\u003e The primary aim of this study was to use this validated method to explore and present a detailed analysis of DIM\u0026rsquo;s effects on estrogen metabolism in humans by evaluating the complete urinary estrogen profile of premenopausal women in the luteal phase of their cycle. The complete urinary estrogen profile we sought to explore includes parent estrogens, estrogen metabolites, and relevant ratios.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Source\u003c/h2\u003e \u003cp\u003eIn this retrospective data mining study registered as Precision Analytical Retrospective Data Correlation (Clinical Trials ID, NCT04305093), data were available for 144,561 laboratory accessions from 129,883 individuals between January 1, 2016 and December 9, 2019. The study was approved by the National University of Natural Medicine Institutional Review Board, and since all data analyzed in this data mining study were deidentified, the institutional review board determined that written informed consent could be waived. Deidentified data were extracted from the database and included the following variables: age, sex, menstrual status (regular, irregular, none), last menstrual period, body mass index (BMI) calculated from self-reported height and weight, use of DIM, comorbidities, and urinary measures of estrone, estradiol, estriol, \u0026#120572; and \u0026#120573;-pregnanediol, 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, 4-hydroxyestradiol, 16-hydroxyestrone, and 2-methoxyestrone. Data on race/ethnicity were not available. Inclusion criteria for this study included being a premenopausal woman reporting regular menses and having collected samples in the luteal phase of the menstrual cycle. Exclusion criteria included the following: a BMI less than 16 or greater than 60 kg/m\u003csup\u003e2\u003c/sup\u003e; pregnancy; any reported kidney disease; reported adrenal insufficiency or excess; suspected or diagnosed PCOS; missing data for urinary estrogens or estrogen metabolites (with the exception of 4-hydroxyestradiol and 2-methoxyestradiol); use of any estrogen supplementation (oral, sublingual pelleted, injected, or other transdermal formulation); use of \u0026#120573;-human chorionic gonadotropin; use of anastrazole; use of tamoxifen; evidence of overly dilute urine (any spot urine creatinine\u0026thinsp;\u0026lt;\u0026thinsp;0.1 mg/ml).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection and Laboratory Methods\u003c/h2\u003e \u003cp\u003eThe sample collection and assay methods used to produce the data analyzed in this study, including assay performance characteristics, have been described previously.\u003csup\u003e17,18\u003c/sup\u003e Briefly, participants collected a total of four urine samples on filter paper over 24 hours. These samples were allowed to dry and then shipped to the laboratory for analysis. Once samples were received in the laboratory, urine was extracted from the filter paper and the four samples were combined into a single representative sample. This combination was done by using creatinine concentrations from each individual sample to determine the volume of sample that should be added to the combined sample to best represent measured concentrations over a 24-hour period. The combined sample was then analyzed via gas chromatography-tandem mass spectrometry on an Agilent 7890/7000B (Agilent Technologies, Santa Clara, CA). Estrogen and estrogen metabolite concentrations were normalized to creatinine to account for variation in both urine concentration and filter paper saturation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Methods\u003c/h2\u003e \u003cp\u003eAll values are presented as medians and interquartile ranges as they were not normally distributed. In the larger group, we compared characteristics of premenopausal women taking DIM and premenopausal women not taking DIM. These comparisons were done using the Wilcoxon rank-sum test. In the smaller subset of individuals who had laboratory results available both before and after initiating DIM, comparisons were made with the Wilcoxon signed rank test. Because this was a data mining study using a convenience sample, no sample size or power calculation was performed a priori, and all comparisons should be considered exploratory and hypothesis generating in nature. All analyses were conducted using R version 4.3.1 (R Version for Statistical Computing, Vienna, Austria).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eComparison of Women Not Taking DIM to Women Taking DIM\u003c/h2\u003e \u003cp\u003eAfter applying inclusion and exclusion criteria, there were 18,385 women who did not report taking DIM and 909 women who reported taking DIM. Patient characteristics and urinary estrogen and estrogen metabolite concentrations for these two groups of women are reported and compared in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The urinary estrogen metabolite ratios for these two groups are reported and compared in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Significant differences were observed in both age and BMI along with all urinary estrogen metabolites with the exception of 2-methoxyestrone. Urinary E1, E2, and E3 concentrations were all lower in the group taking DIM, as were the concentrations of 16-hydroxyestrone and total urinary estrogens. Concentrations of 2-hydroxyestrone, 4-hydroxyestrone, 2 hydroxyestradiol, and 4-hydroxyestradiol were all higher in the women taking DIM. These differences in metabolite concentrations translated to significant differences in all of the metabolite ratios that were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBefore and After Initiating DIM\u003c/h2\u003e \u003cp\u003eThere were a total of 53 women who had laboratory results available both before and after starting DIM. Age, BMI, and urinary estrogen and estrogen metabolite concentrations for these women are reported and compared in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Urinary estrogen metabolite ratios for these women before and after initiating DIM are compared in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The before and after values for some of the metabolites and some of the ratios can be seen in the context of the estrogen metabolic pathway in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. With the exception of 2-methoxyestrone and 2OHE2, all parent estrogens and metabolites changed in the same direction as in the larger group. However, only the changes in E1, E2, E3, and 16-OHE1 reached the threshold for significance. All of the ratios changed in the same direction as in the larger group; however, the only ratio changes that reached significance were the 2-OHE1/16-OHE1, 2-OHE1/E1, 2-OHE1/E2, and the 2-methoxyE1/2-OHE1 ratios. The percent changes for E2, 2-OHE1, 4-OHE1, 16-OHE1, and the 2/16 ratio are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Characteristics and Urinary Estrogen Concentrations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo DIM, N\u0026thinsp;=\u0026thinsp;18,385\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDIM, N\u0026thinsp;=\u0026thinsp;909\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at Collection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 (33, 43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (35, 44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.8 (21.3, 26.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.4 (21.0, 25.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.00 (12.70, 24.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.70 (10.30, 20.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.10 (2.22, 4.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.62 (1.89, 3.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estriol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.60 (5.90, 15.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.20 (3.00, 9.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.24 (5.40, 11.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.55 (7.90, 15.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 4-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.09 (0.75, 1.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.37 (0.97, 2.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 16-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.21 (0.73, 1.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.82 (0.45, 1.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-methoxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.20 (2.79, 5.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.05 (2.70, 5.97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-hydroxyestradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66 (0.36, 1.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.99 (0.55, 1.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 4-hydroxyestradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.30 (0.20, 0.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40 (0.20, 0.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Urinary Estrogens (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.50 (36.50, 63.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.20 (33.00, 58.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eWilcoxon rank sum test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Characteristics and Urinary Estrogen Metabolite Ratios\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo DIM, N\u0026thinsp;=\u0026thinsp;18,385\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDIM, N\u0026thinsp;=\u0026thinsp;909\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at Collection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38 (33, 43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (35, 44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.8 (21.3, 26.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.4 (21.0, 25.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/16OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.89 (3.72, 12.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.36 (7.49, 25.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/E1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.44 (0.31, 0.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.79 (0.54, 1.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/E2 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.56 (1.69, 3.63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.30 (2.87, 5.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/4OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.27 (5.36, 9.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.15 (6.00, 10.43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-methoxyE1/2OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51 (0.40, 0.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35 (0.27, 0.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE2/2OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08 (0.06, 0.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08 (0.06, 0.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eWilcoxon rank sum test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBefore and After Estrogen \u0026amp; Estrogen Metabolite Concentrations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore DIM\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAfter DIM\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at Collection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 (32, 42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (34, 44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.1 (21.3, 26.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.4 (21.3, 25.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary a-pregnanediol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e417 (284, 843)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e427 (308, 681)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary b-pregnanediol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,350 (1,144, 1,657)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,263 (973, 1,964)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.40 (18.40, 32.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.10 (11.10, 22.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.10 (3.00, 5.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.84 (2.17, 4.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary Estriol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.60 (6.50, 19.90)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.70 (2.90, 8.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.50 (7.70, 14.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.70 (8.46, 16.64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 4-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.37 (1.10, 2.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.57 (1.12, 2.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 16-hydroxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.80 (1.00, 3.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70 (0.39, 1.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-methoxyestrone (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.70 (3.70, 6.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.86 (3.20, 6.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-hydroxyestradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.10 (0.51, 1.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01 (0.58, 1.68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 4-hydroxyestradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.35 (0.30, 0.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40 (0.30, 0.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrinary 2-methoxyestradiol (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.55 (0.48, 0.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50 (0.30, 0.73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMissing Values\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Urinary Estrogens (ng/mg-Cr)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.0 (50.3, 77.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.2 (35.3, 60.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eWilcoxon rank sum test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBefore and After Estrogen Metabolite Ratio Values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore DIM\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAfter DIM\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at Collection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 (32, 42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (34, 44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.1 (21.3, 26.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.4 (21.3, 25.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/16OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.67 (2.79, 13.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.20 (9.64, 31.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/E1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.45 (0.30, 0.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.88 (0.56, 1.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/E2 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.40 (1.59, 3.81)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.27 (3.05, 5.80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/4OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.92 (5.41, 9.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.51 (6.21, 9.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-methoxyE1/2OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.41 (0.32, 0.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35 (0.27, 0.51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE2/2OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.09 (0.07, 0.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08 (0.07, 0.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e1\u003c/sup\u003eMedian (IQR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003e2\u003c/sup\u003eWilcoxon rank sum test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003ePercent Change in Key Metabolites and the 2/16 Ratio\u003c/b\u003e (In women with results before and after initiating DIM)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePercent Change\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstradiol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-23.13%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-Hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.24%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-Hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.95%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16-Hydroxyestrone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-55.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2OHE1/16OHE1 Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e188.31%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have examined retrospective data in an attempt to more completely understand the effect of DIM on estrogen metabolism in premenopausal women. We found significant differences in almost all estrogens, estrogen metabolites, and estrogen metabolite ratios when we compared the larger groups of women who were either taking DIM or not taking DIM. In the smaller group of women who had laboratory results available before and after starting DIM, although we saw differences in the same directions as in the larger group, fewer of these differences reached significance.\u003c/p\u003e \u003cp\u003eThis study has several limitations. The data used in this analysis is from a convenience sample and thus there is limited external generalizability to populations that are different from the population studied. However, given the large sample size, valuable insights can be garnered to inform the design of future, more targeted studies. We also had no access to data on participants\u0026rsquo; diet which could have a meaningful impact if a participant\u0026rsquo;s diet was particularly high in cruciferous vegetables (or particularly low). In addition to not having access to data on diet, although we did collect some data regarding prescription medication and supplement use, we were unable to collect and exhaustive list of concurrent prescription medications and over the counter supplements. In this patient population there is sometimes concurrent use of other estrogen-lowering or estrogen metabolism altering agents such as calcium-d-glucarate.\u003csup\u003e22\u003c/sup\u003e These agents may have also contributed to some of the lower estrogen and estrogen metabolites concentrations seen, but these agents would be less likely to affect the ratios of estrogen metabolites. We did not collect data regarding dose or formulation of DIM/I3C, frequency of administration, or adherence. Although this is certainly a limitation, it also reflects the real-world nature of the data and represents what is likely seen in actual clinical practice. There are DIM products which claim higher bioavailability,\u003csup\u003e23\u003c/sup\u003e so it possible that the use of different products may have significantly different impacts on the urinary estrogen profile. The results of this study highlight that this collection method and assay would be a useful tool to investigate potential differing impacts between DIM products. Because of differing bioavailability and half-life, some products may recommend more or less frequent dosing, however dosing is usually once or twice daily. Finally, we do not have data on the rationale employed for initiating DIM. The reasons for using DIM vary widely in the integrative and functional medicine space, and capturing the potential \u0026ldquo;indications\u0026rdquo; for DIM use concisely is beyond the scope of this manuscript. Notwithstanding these limitations, this study provides both a comparative evaluation of premenopausal women taking vs. not taking DIM and a repeated measures evaluation of a subset of women using DIM. These evaluations shed light on the impact of DIM on the entire urinary estrogen profile which, to the best of our knowledge, has not previously been explored this comprehensively.\u003c/p\u003e \u003cp\u003eMany of the previous studies that evaluated the effects of DIM focused on changes in 2-OHE1, 16-OHE1, and the 2/16 ratio.\u003csup\u003e6,15,16,24\u003c/sup\u003e In addition to the focus on the 2-OHE1 and 16-OHE1, Dalessandri and colleagues also reported urinary E1, E2, and E3 concentrations before and after DIM use, along with urinary 6\u0026#120573;-hydroxycortisol (6\u0026#120573;-OHC), cortisol, and the 6\u0026#120573;-OHC/cortisol ratio, which has shown to be an indication of CYP3A4 enzyme activity.\u003csup\u003e25\u003c/sup\u003e As discussed earlier, regarding DIM, the activity of CYP3A4 influences production of 16-OHE1 and 4-OHE2, the non-favored estrogen metabolites. One of the only studies in the literature that is similar to the present study is one done by Green and colleagues which used the same assay as the present study.\u003csup\u003e16\u003c/sup\u003e However, participants in that study were given a dietary supplement that contained I3C and at least 8 additional ingredients. Moreover, the women participating in that study may not have been adequately fluid restricted and samples were collected during the day. The combination of these 2 factors may have led to low creatinine values to which the estrogen metabolite concentrations are indexed. Furthermore, the overall estrogen and estrogen metabolite concentrations for the study participants were low compared to established reference ranges. The primary aim of that study was simply to determine if the nutraceutical product used, Estrosense, increased the 2/16 ratio.\u003c/p\u003e \u003cp\u003eResults in this study are similar in direction to the four previously mentioned studies conducted by Rajoria and colleagues, Thomson and colleagues, Godinez-Martinez and colleagues, and Green and colleagues. Here, just as in all of those studies, 2-OHE1 increased and 16-OHE1 decreased with DIM treatment, which translated to an increase in the 2/16 ratio. One interesting observation concerns the change in 4-OHE1. In the present study, although 4-OHE1 increased in the before and after group, the change did not reach significance. Similar results were seen in the study by Thomson and colleagues. They reported an initial small median decrease at 6 months, followed by an increase at 12 months; however median change from baseline to 12 months was 0.0, and the P-value comparing DIM treated participants with the placebo arm was not significant (P\u0026thinsp;=\u0026thinsp;0.534) indicating no difference in effect on 4-OHE1 between DIM and placebo. This echoes the conclusion that we have drawn from our data. This conclusion may be surprising to some integrative and functional medicine providers who expect DIM to reduce 4-OHE1. The currently available evidence does not support DIM use for this purpose. The 2OHE1/4OHE1 ratio did increase in both groups, although this change did not reach the threshold for significance in the before and after group.\u003c/p\u003e \u003cp\u003eInterestingly, in the study conducted by Green and colleagues, the only outcome measures with changes from baseline that reached statistical significance were 2-OHE1 (P\u0026thinsp;=\u0026thinsp;0.01) and the 2/16 ratio (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This indicates that the change in the primary outcome was driven by the observed increased in 2-OHE1 which was only 0.43 ng/mg-Cr. In the present study we saw a somewhat opposite result in that even though the median 2-OHE1 concentration increased by a larger margin (1.20 ng/mg-Cr), the difference from baseline was not significant (P\u0026thinsp;=\u0026thinsp;0.4). However, the difference in 16-OHE1 was significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This suggests that although the outcome measure of increase in the 2/16 ratio was the same in both studies, the underlying change in estrogen metabolism driving the increase in the ratio was different in these two studies. This has important implications for clinical decision making depending on the goals of the provider.\u003c/p\u003e \u003cp\u003eFuture randomized, placebo-controlled studies with multiple DIM products are needed to fully understand the potential clinical utility of DIM supplementation. The tools presented here for assessing the effects of DIM seem to be the most comprehensive approach to gaining actionable clarity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAlthough a complete understanding of the mechanism of action of DIM continues to elude researchers, this and previous studies serve to illuminate some of the expected effects in premenopausal women. The results of this study demonstrate that the dried urinary sampling method and corresponding assay used to measure estrogen metabolites is a useful approach to monitoring the effects of DIM therapy in real world clinical practice. To conclude, we propose that the changes seen here in parent estrogen concentrations, estrogen metabolite concentrations, and estrogen metabolite ratios represent a key component to understanding the complex and somewhat convoluted mechanism of action of DIM. The specific changes that we have either confirmed or uncovered provide a molecular explanation for some of the observed effects of DIM. The results of this study, taken together with the theoretical and observed partial mechanism of action of DIM, provide moderate evidence supporting the potential clinical utility of DIM and strong evidence supporting the definite clinical utility of urinary estrogen and estrogen metabolite monitoring in the setting of DIM supplementation. Additional work is needed to completely reveal how the complex mechanism of action of DIM is reflected in the complex estrogen metabolism pathway. Disentangling the complexity of estrogen metabolism remains a formidable challenge, which may well demand the use of novel methods. We posit that the use of urinary estrogen and estrogen metabolite concentration changes to explore theoretical and actual consequences of altered estrogen metabolism will prove to be a drama of refound opportunity in the coming years.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the National University of Natural Medicine Institutional Review Board, and since all data analyzed in this data mining study were deidentified, the institutional review board determined that written informed consent could be waived.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eM.N. is the founder and CEO of Precision Analytical, Inc. J.S. is an employee of Precision Analytical, Inc.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eMN conceived and designed the study and acquired the data. Both MN and JS analyzed and interpreted the data. Both MN and JS wrote and revised the manuscript. The final version of the manuscript was read and approved by both authors.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThomson CA, Ho E, Strom MB. Chemopreventive properties of 3,30-diindolylmethane in breast cancer: Evidence from experimental and human studies. \u003cem\u003eNutrition Reviews\u003c/em\u003e. 2016;74(7):432-443. doi:10.1093/nutrit/nuw010\u003c/li\u003e\n\u003cli\u003eBradlow HL, Zeligs MA. Diindolylmethane (DIM) Spontaneously Forms from Indole-3-carbinol (I3C) During Cell Culture Experiments. \u003cem\u003ein vivo\u003c/em\u003e. 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Indoles Derived From Glucobrassicin: Cancer Chemoprevention by Indole-3-Carbinol and 3,3\u0026rsquo;-Diindolylmethane. \u003cem\u003eFrontiers in Nutrition\u003c/em\u003e. 2021;8:734334. doi:10.3389/fnut.2021.734334\u003c/li\u003e\n\u003cli\u003eYang L, Zheng C, Xia YF, Dai Y, Wei ZF. 3, 3\u0026rsquo;-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway. \u003cem\u003ePhytomedicine: International Journal of Phytotherapy and Phytopharmacology\u003c/em\u003e. 2023;116:154874. doi:10.1016/j.phymed.2023.154874\u003c/li\u003e\n\u003cli\u003eLi CS, Nguyen TV, Chai OH, Park BH, Lee JS, Kim SM. 3,3\u0026rsquo;-Diindolylmethane Augments 5-Fluorouracil-InducedGrowth Suppression in Gastric Cancer Cells through Suppression of the Akt/GSK-3\u0026beta; and WNT/Beta-Catenin. \u003cem\u003eJournal of Oncology\u003c/em\u003e. 2023;2023:8268955. doi:10.1155/2023/8268955\u003c/li\u003e\n\u003cli\u003eTucci P, Brown I, Bewick GS, Pertwee RG, Marini P. 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A randomized, double-blind, placebo-controlled, cross-over trial to evaluate the effect of EstroSense \u003csup\u003e\u0026reg;\u003c/sup\u003e on 2-hydroxyestrone:16\u0026alpha;-hydroxyestrone ratio in premenopausal women. \u003cem\u003eJournal of Complementary and Integrative Medicine\u003c/em\u003e. 2023;20(1):199-206. doi:10.1515/jcim-2022-0301\u003c/li\u003e\n\u003cli\u003eNewman M, Pratt SM, Curran DA, Stanczyk FZ. Evaluating urinary estrogen and progesterone metabolites using dried filter paper samples and gas chromatography with tandem mass spectrometry (GCMS/MS). \u003cem\u003eBMC Chemistry\u003c/em\u003e. 2019;13(1):1-12. doi:10.1186/s13065-019-0539-1\u003c/li\u003e\n\u003cli\u003eNewman M, Curran DA. Reliability of a dried urine test for comprehensive assessment of urine hormones and metabolites. \u003cem\u003eBMC Chemistry\u003c/em\u003e. 2021;15(1):18. doi:10.1186/s13065-021-00744-3\u003c/li\u003e\n\u003cli\u003eNewman MS, Curran DA, Mayfield BP, Saltiel D, Stanczyk FZ. Assessment of estrogen exposure from transdermal estradiol gel therapy with a dried urine assay. \u003cem\u003eSteroids\u003c/em\u003e. 2022;184:109038. doi:10.1016/j.steroids.2022.109038\u003c/li\u003e\n\u003cli\u003eNewman MS, Mayfield BP, Saltiel D, Stanczyk FZ. Assessing estrogen exposure from transdermal estradiol patch therapy using a dried urine collection and a GCMS/MS assay. \u003cem\u003eSteroids\u003c/em\u003e. 2023;189:109149. doi:10.1016/j.steroids.2022.109149\u003c/li\u003e\n\u003cli\u003eNewman MS, Saltiel D, Smeaton J, Stanczyk FZ. Comparative estrogen exposure from compounded transdermal estradiol creams and Food and Drug Administration-approved transdermal estradiol gels and patches. \u003cem\u003eMenopause\u003c/em\u003e. Published online October 18, 2023. doi:10.1097/GME.0000000000002266\u003c/li\u003e\n\u003cli\u003eOredipe OA, Barth RF, Dwivedi C, Webb TE. Dietary glucarate-mediated inhibition of initiation of diethylnitrosamine-induced hepatocarcinogenesis. \u003cem\u003eToxicology\u003c/em\u003e. 1992;74(2-3):209-222. doi:10.1016/0300-483X(92)90140-A\u003c/li\u003e\n\u003cli\u003eAnderton MJ, Manson MM, Verschoyle R, et al. PHYSIOLOGICAL MODELING OF FORMULATED AND CRYSTALLINE 3,3\u0026rsquo;-DIINDOLYLMETHANE PHARMACOKINETICS FOLLOWING ORAL ADMINISTRATION IN MICE. \u003cem\u003eDrug Metabolism and Disposition\u003c/em\u003e. 2004;32(6):632-638. doi:10.1124/dmd.32.6.632\u003c/li\u003e\n\u003cli\u003eThomson C, Chow S, Roe D, et al. Effect of diindolylmethane on estrogen-related hormones, metabolites and tamoxifen metabolism: Results of a randomized, placebo-controlled trial. \u003cem\u003eCancer Epidemiology, Biomarkers \u0026amp; Prevention\u003c/em\u003e. 2017;26(3):435. doi:10.1158/1055-9965.EPI-17-0027\u003c/li\u003e\n\u003cli\u003eDalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF. Pilot study: Effect of 3,3\u0026rsquo;-diindolylmethane supplements on urinary hormone metabolites in postmenopausal women with a history of early-stage breast cancer. \u003cem\u003eNutrition and Cancer\u003c/em\u003e. 2004;50(2):161-167. doi:10.1207/s15327914nc5002_5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3944853/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3944853/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e3, 3\u0026rsquo;-diindolylmethane (DIM) is a phytonutrient derived from cruciferous vegetables that is an often used supplement in the complementary and alternative medicine space. The most common goal for providers when recommending DIM to their patients is to alter estrogen metabolism, yet research into DIM\u0026rsquo;s effect on the estrogen profile is lacking in the published literature. The objective of this study was to comprehensively evaluate DIM\u0026rsquo;s effect on the urinary estrogen profile.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this retrospective cohort study, we analyzed data from a clinical laboratory, including urinary estrogen and estrogen metabolite concentrations. Analyte concentrations were determined from dried urine samples using a gas chromatography-tandem mass spectrometry assay. Individuals were separated into 2 groups, either reporting taking DIM (N\u0026thinsp;=\u0026thinsp;909) or reporting not taking DIM (N\u0026thinsp;=\u0026thinsp;18,385). Comparisons between individuals in these two groups were made using the Wilcoxon rank sum test. Additionally, we were also able to explore a subset of women who had laboratory results in the database before and after initiating DIM treatment (N\u0026thinsp;=\u0026thinsp;53). In this subset, differences were assessed with Wilcoxon signed rank tests.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the larger group, significant differences were observed in the concentrations of almost every urinary estrogen and estrogen metabolite (with the only exception being 2-methoxyestrone) in the urinary estrogen profiles of those taking DIM compared to those not taking DIM (all P values\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the smaller subset of individuals with before and after measurements, differences were only seen in 4 of the urinary estrogens and estrogen metabolites (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for estrone, estradiol, estriol, and 16-hydroxyestrone). Differences in total estrogens were significant in both the larger group and the smaller subset (both with P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, observed differences in the ratios of metabolites followed a similar trend with more significant differences observed in the larger group. Notably, the 2-hydroxyestrone:16-hydroxyestrone ratio increased significantly in both the larger and the before and after group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe results of this study provide the most comprehensive evaluation to date of DIM\u0026rsquo;s effect on the urinary estrogen profile. Additionally, the results demonstrate that the dried urine collection and accompanying assay used captures changes that are similar in direction, but not necessarily magnitude, to previous reports in the literature. Considered together, these two things highlight the clinical validity and utility of this approach to the evaluation of DIM supplementation and suggest the need for additional studies using this approach to fully understand the potential clinical utility of DIM.\u003c/p\u003e","manuscriptTitle":"Exploring the Impact of 3,3’-Diindolylmethane on the Urinary Estrogen Profile of Premenopausal Women","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 12:55:20","doi":"10.21203/rs.3.rs-3944853/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-16T14:20:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-28T05:32:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T13:41:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164321213499871152696534304709414846423","date":"2024-06-14T05:36:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59122159841981308183358071504532753019","date":"2024-06-13T18:35:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-04T22:31:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-03T11:32:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-18T20:06:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-18T20:01:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2024-02-10T03:34:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6fe0d7f5-21c1-4394-80ad-fcf076515088","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-12T12:08:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-29 12:55:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3944853","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3944853","identity":"rs-3944853","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}