THE ROLE OF THE GUT MICROBIOTA IN ESTROGEN METABOLISM AND DIETARY FLAX AS A POTENTIAL MODULATOR

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1013253
• Project Start Date: Jul 26, 2017
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Food Science & Human Nutrition

Non Technical Summary
It is now known that the human microbiota, particularly the trillions of organisms that reside in the gastrointestinal tract, influence host metabolic status and overall health. While the effects of diet and other external influences on gut microbial composition and metabolism have been well studied, little direct evidence exists about the interaction between endogenous hormones, such as estrogen, and the gut microbiota. Estrogen modulation of metabolic homeostasis is a fundamental component of women's health. Systemic exposure to estrogen is a major factor in determining risk of developing chronic diseases such as non-alcoholic fatty liver disease (NAFLD), type-2 diabetes (T2D), cardiovascular disease (CVD) and cancer. The goal of the current proposal is to determine the effects of endogenous estrogen on the profile of the gut microbiota. Specifically, we will determine if estrogen suppression in premenopausal women results in changes in gut microbial community composition and how these changes might alter relative levels of systemic parent estrogens (estradiol and estrone) and estrogen metabolites (i.e. 2OH-, 4-OH, 16-OH pathways). We will also determine the effect of dietary flaxseed as a possible modifiable source of beneficial phyotestrogens on the gut microbiota and estrogen profile in the absence of ovarian estrogen.

Animal Health Component

10%

Research Effort Categories

Basic

70%

Applied

10%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
724	4010	1010	50%
702	1842	1010	50%

Knowledge Area
724 - Healthy Lifestyle; 702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
1842 - Flax; 4010 - Bacteria;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

menopause

gut microbiota

estrogen metabolism

flaxseed phytoestrogens

Goals / Objectives
The goal of this project is to determine the role of estrogen on the composition of the gut microbiota in premenopausal women treated with GnRH agonist to suppress endogenous estrogenand whether 30 days of ground flaxseed consumption will restore the microbiota structure to pre-supression status. The main objectives are to:1. Establish a model of estrogen suppression in premenopausal women for the study of the effect of endogenous estrogen on the composition of gut microbiota--suppression will beconfirmed byestrogen levels in plasma2. Determine the effect of endogenous estrogen suppression on gut microbiota as measured by sequencing the v3-v4 region of the 16s rRNA3. Measure the effect of ingestion of 25g/day ground flaxseed (high in lignans--microbiota metabolism produces phyotestrogens)on the gut microbiota in premenopausal women treated with GnRH agonist to suppress endogenous estrogen3. Collect urine, feces and blood for future measurement of systemic estrogens4. Assess associations between estrogensuppression andflaxseed intervention on gut microbiotastructure andestrogen profiles associated with disease.

Project Methods
Overview. Upon completion of informed consent and fulfillment of inclusion criteria, baseline samples of blood, urine and feces will be collected. Each participant will then receive an intramuscular dose of the estrogen suppressing drug, GnRHAG. They will return in 4 weeks for one month collection of specimens, a second shot of GnRHAG and to pick up a 4-week supply of ground flaxseed. After 4-weeks of 40-45g dietary flaxseed supplementation per day, participants will return for final specimen collection. Sequencing of gut microbiota, plasma estrogen, metabolic panels and short chain fatty acids will be measured at each timepoint.Study participants. We will study 15 healthy, normal to overweight (BMI 22-28 kg/m2), non- smoking, premenopausal (normally menstruating) women(20-40y). They must not have contra-indications to GnRHAG therapy (i.e. low bone mineral density, history of breast cancer etc.). They must be sedentary to moderately active (< 120 min exercise/week), not have used estrogen-based contraception for > 6 months, antibiotic or probiotics for>2mo nor taking phytoestrogenic supplements, or lipid- or glucose-lowering medications.Screening. Prior to beginning the study, volunteers will undergo an extensive medical history (including menstrual cycle history), physical examination, and a clinical laboratory evaluation including creatinine, liver function tests, thyroid function tests, and a complete blood count overseen by Dr. Hoenig (see letter of support). Other screening measures will include body composition (DEXA), food frequency and physical activity questionnaires and asked about use of probiotics or other supplements. Participants will also begin tracking their menstrual cycle, so that baseline samples will be collected in the early follicular phase of the menstrual cycle when estrogen levels are lowest.Examinations, Laboratory Tests, Procedures, and Follow-up VisitsBlood and serum measures - At each time point, fasted (12hr) blood samples will be obtained for the measurement of total estrogen and sex hormone binding globulin (SHBG). Samples will be stored at -80º C prior to processing.GnRHAG therapy.After baseline samples are collected, 2 mo of GnRHAG therapy(monthlyintramuscular injection of leuprolide acetate 3.75 mg for depot suspension; Lupron Depot (Abbvie, Inc., Chicago, IL) will be initiated to chronically suppress ovarian hormones.; injections and oversight by Dr. Hoenig. GnRHAG therapy has been used clinically for the management of endometriosis and for pre-operative management of anemia caused by uterine fibroids for up to 1 year without serious adverse events, although there is a decrease in BMD that recovers following cessation of treatment.42,43. Side effects are those typical of hypoestrogenemia. In studiesof women withuterinefibroids,GnRHAG 3.75mg/mo.induced amenorrheain61%,86%, and 90% of women after 1, 2, and 3 mo of treatment, respectively.44,45Return of menses typically occurs within 1 to 3 mo of cessation of therapy.Absence of pregnancy (urine test) will be confirmed before each dosing. Participants will be observed for 30 min after drug injection to monitor for hypersensitivity reactions. Women whodo not tolerate GnRHAG therapy can refuse further treatment after each monthly dosing. In 94 women treated with GnRHAG in studies done by colleagues at UC AMC (R01 AG018198, P50 HD073063), this occurred only once. Participants will also meet with the study physician at each visit to discuss adverse responses via a health status questionnaire, the Menopausal Symptom List (MSL) to monitor the frequency and severity of menopause-related symptoms (e.g., hot flashes, depressed feelings);46the Eating Behavior Questionnaire (EBQ) to monitor dietary restraint, disinhibition, and hunger; and the Pittsburgh Sleep Quality Index (PSQI) to monitor sleeping behavior.47Flaxseed supplementation. Organic flaxseed will be stored at 4C and ground fresh the day before it is given to the participants. Each participant will be given a 1 pound bag of ground flaxseed at the second study visit (start of month 2) and instructed to store it in the refrigerator for the duration of the study. Starting the morning following the 2-month visit, participants will be asked to consume 3 tablespoons (40-45g) per day. They can sprinkle the powder over foods such as hot or cold cereals, cottage cheese, applesauce etc., or mix it into soups or smoothies.Sample collection. Subjects will visit the Human Performance Clinical Research Lab (HPCRL) after an overnight fast (12hr). They will have 10cc of venous blood drawn into lithium heparin and EDTA-coated vacutainers. Wholeblood will be used for a comprehensive metabolic panel and the remaining blood will be centrifuged to obtain plasma which will be frozen at -80C for the analysis of systemic inflammation and total estrogen. Fecal samples will be collected according to study provided instructions in participants' homes in specified containers and immediately frozen. Participants will be given ice packs and coolers to return their samples to CSU. On arrival, samples will be maintained at -80C until processing.Gut microbe sequencing. DNA will be extracted from all samples using the Microbiome DNA extraction kit (Invitrogen) according to the manufacturer's instructions. All samples will be quantified with the Quanti-iT PicoGreen dsDNA assay (Life Technologies, Grand Island, NY) and stored at −20°C prior to sequencing the V3-V4 region of the16s rRNA gene. Library preparation will be conducted by tagging amplicons using Illumina indices for multiplexing and sequenced as paired-end reads on an Illumina MiSeq.Short-chain fatty acids (SCFA). Weighed, frozen feces will be mixed with acidified water (pH 2.5) containing 1 mmol/L of ethylbutyric acid as an internal standard, sonicated, centrifuged, filtered through 0.45 μmol/L nylon filters and stored at −80°C prior to analysis. The samples will be analyzed using an Agilent 6890 Series Gas Chromatographer (Agilent Inc, Santa Clara, CA). SCFA's will be quantified using standards of commercially purchased compounds and samples adjusted for extraction efficiency differences by normalizing to the internal standard.Inflammatory profile. Systemic (plasma) inflammation will be assayed at each time point using a multi-plex assay (Luminex platform) per manufacturer protocol. Fecal lipocalin, a measure of sub- clinical intestinal inflammation, will be measured via elisa.Data AnalysisPower determination. Inour animal studies, we were able to detect differences in bacterialdiversity as well as between bacterial phyla using n= 4 animals per group, and differences in gut bacterial outcomes with dietary interventions in 3-4 human subjects per group 48. Recent human studies have found significant correlations between bacterial diversity and estrogen metabolites in as few as 9 postmenopausal women 17. We therefore propose that 15 will be a sufficient number of participants to detect differences in bacterial diversity, specific bacterial populations, SCFA and inflammatory markers at each time-point with a within subject design.Statistical analyses. Sequence data will be processed in mothur 49 following the published SOP for Illumina data. Multivariate statistical analysis will be conducted to determine differences in overall bacterial community composition across treatment groups. Specific pairwise tests will be performed to detect differences in bacterial communities between baseline and estrogen suppression time points. Bacterial diversity indices and relative abundance of phyla will be compared via the nonparametric Kruskal- Wallis one-way analysis of variance by ranks test.Group differences in SCFA and inflammatory marker concentrations will be analyzed via independent samples t-test with p=0.05. Statistics will be done using SPSS v. 24.

Progress 07/26/17 to 09/30/19

Outputs
Target Audience:The target audience reached by the project in this reporting period include the participants in the study and collaborators who are processing the samples. In the case of the study participants, the hypothesis, biology/physiology,and practical implicationswerecommunicated throughout. Communications with collaborators included potential outcomes, methodology and data analysis. The study was also discussed with graduate students and community members in casual settings when the topic arose. These communications ranged from lay terminology to technical discussions about women's health, the gut microbiome and nutrition. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided valuable training opportunities for the investigators involved. The graduate student (Evan Stoecker) who worked on the project learned several valuable skills including study management (recruiting, IRB approval, the consent process), database set-up and management (REDCap), sample collection/storage, wet lab methods (DNA isolation, enzyme kinetics assay development), and writing (contributed to a book chapter (Microbial Metabolites in Cancer Promotion or Prevention). Evan also presented the study design at several poster sessions on the CSU campus and based partly on his work with the projectwas accepted to and attended theJohn Milner Nutrition and Cancer Prevention Research Practicum. The primary investigators were asked to contribute a chapter to the above-mentioned book and used the current project as a baseline. Dr. Cox-York presented the study in several spaces including the CCTSI Summit and invited guest lectures at CSU and UC AMC. This project was also the catalyst for developing a collaboration with Dr. Nilesh Gaikwad at the University of California Davis. Dr. Gaikwad has published in respected journals in the area of estrogen metabolites and health. Depending on the outcomes of the study, Dr. Cox-York is in conversations with collaborators at UC AMC to continue the work. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? It is now known that the human microbiota, particularly the trillions of organisms that reside in the gastrointestinal tract (gut), influence host metabolic status and overall health. While the effects of diet and other external influences on gut microbial composition and metabolism have been well studied, little direct evidence exists about the interaction between endogenous (produced by the body) hormones, such as estrogen, and the gut microbiota. Estrogen modulation of metabolic homeostasis or balance is a fundamental component of women's health. Systemic exposure to estrogen is a major factor in determining risk of developing chronic diseases such as nonalcoholic fatty liver disease (NAFLD), type-2 diabetes (T2D), cardiovascular disease (CVD) and cancer. The goal of the current proposal is to determine the effects of endogenous estrogen on the profile of the gut microbiota. Specifically, we will determine if estrogen suppression in premenopausal women results in changes in gut microbial community composition and how these changes might alter relative levels of systemic parent estrogens (estradiol and estrone) and estrogen metabolites (i.e. 2OH-, 4- OH, 16-OH pathways). We will also determine the effect of dietary flaxseed as a possible modifiable source of beneficial fiber and phytoestrogens on the gut microbiota and estrogen profile in the absence of ovarian estrogen. The results of this study have the potential to impact women's health significantly. Understanding the impact of estrogen loss on the gut microbiota will help elucidatethe systems that are affected by menopause and identify potential targets for reducing the negative health effects of estrogen loss. If there are dietary means by which women can modulate their gut microbiota and ultimately their estrogen levels and profile, the impact on quality and span of life could be significant. In the broader picture, using dietary, rather than pharmacological means of maintaining the estrogen profile helps reorient people to the importance of food for health. Flaxseed was chosen for the current project because it is not only an excellent source of fiber, but contains a high concentration of phytoestrogen precursors that are transformed by the gut microbiota. However, it is possible that multiple fiber types could elicit beneficial effects.Therefore, while flaxseed is grown almost exclusively in North Dakota and Minnesota, it is possible that similar effects could result from consuming high-fiber crops grown in Colorado. We are just in the process of analyzing the samples collected from the study, so we don't yet know the impact of our work. Objectives: 1. Establish a model of estrogen suppression in premenopausal women for the study of the effect of endogenous estrogen on the composition of gut microbiota--suppression will beconfirmed byestrogen levels in plasma 1) Major activities completed / experiments conducted: estrogen suppression (about one month) was induced by a single administration of a gonadal hormone agonist (Lupron) in 12 premenopausal women. All women received the shot and tolerated it well. We will have confirmation of estrogen suppression when we receive the analysis of plasma hormones. 2) Data collected: the only data collected at this point is survey data to monitor the tolerance of the suppression. We monitored mood, eating/shopping behaviors and menopausal symptoms (e.g. hot flashes). 3) Summary statistics and discussion of results: NA - awaiting results of estrogen levels/profile 4) Key outcomes or other accomplishments realized: While collaborators at the University of Colorado Anschutz Medical Campus have used Lupron extensively, this was the first use of the model at CSU. It was administered and monitored by a physician and was well-tolerated. 2. Determine the effect of endogenous estrogen suppression on gut microbiota as measured by sequencing the v3-v4 region of the 16s rRNA 1) Major activities completed / experiments conducted: fecal samples (x6) were collected from 12 women under estrogen suppression with and without dietary flaxseed (2T per day for 60d). DNA has been isolated from the fecal samples, a sequencing library has been constructed and the samples have been sent for sequencing to determine changes in gut microbiota - type, number, number of different types (diversity). 2) Data collected: awaiting results 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: we collected fecal samples in two ways, one typically used by investigators in our department and one suggested by a scientific article comparing different methods. We were successful in isolating DNA from both sample collection methods. 3. Measure the effect of ingestion of 25g/day ground flaxseed (high in lignans--microbiota metabolism produces phytoestrogens)on the gut microbiota in premenopausal women treated with GnRH agonist to suppress endogenous estrogen 1) Major activities completed / experiments conducted: 12 women completed all components of the study. Flaxseed intake was well-tolerated. Estrogen suppression was also well-tolerated. Fecal, blood and urine samples were collected at all timepoints for each subject. 2) Data collected: Awaiting results 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: daily intake of 2T of flaxseed was well-tolerated. If the results indicate that dietary flaxseed has the potential to modulate estrogen profiles favorably, our study suggests that it is a reasonable intervention. 4. Collect urine, feces and blood for future measurement of systemic estrogens - see above 5. Assess associations between estrogensuppression andflaxseed intervention on gut microbiotastructure andestrogen profiles associated with disease. - Awaiting results of sample analysis. Once we have this data, we will be able to assess associations between estrogen, dietary flaxseed and the gut microbiota.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Women and scientists researching women's health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Mr. Stoecker sucessfully established the protocols for measuring two of the major estrogen-metabolizing enzymes produced by the gut microbiota. This was the basis for his thesis, which he wrote and successfully defended. While working as my graduate research assistant, Mr. Stoecker was selected to attendthe competative John Milner Nutrition and Cancer Prevention Practicum at the National Cancer Institute and helped author a book chapter on gut metabolites and cancer. He also presented his work at the CSU Graduate Showcase. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to finish recruiting participants and collecting their samples. We will then isolate DNA from the fecal samples and complete sequencing and analysis of the gut microbes. We will send the plasma samples to our collaborator, Nilesh Gaikwad, PhD at UC Davis for analysis of circulating estrogen metabolites. We anticipate having data analysis completed by the next reporting date and a manuscript in preparation.

Impacts
What was accomplished under these goals? With the onset of menopause, the risk for several chronic diseases, including cardiovascular disease, type 2 diabetes,bone loss, and some cancersincreases.Itisbelieved that estrogen metabolism plays a significant role in these increases, asmenopause is marked by a significant decrease in total estrogen and changes in the types of circulating estrogen. As estrogens that are produced in the ovaries and adipose tissue are metabolized, they eventually end up in the gut, where products (enzymes)of the microbiota residing there further metabolize them. This step determines if the estrogen compounds will be excreted or taken back up into circulation. This step is therefore critical in determining the amount and types of estrogen that are available in the body, and play a role in disease risk. It is important to understand which bacteria are associated with estrogen metabolism and what enzymes may be active in metabolising estrogen. While there have been reported changesin the gut microbiota with menopause, these are only associations. There are many metabolic changes occuring in menopause, so it is difficult to know if the microbiota are responding to estrogen per se, or someother factor.We are therefore using a drug to supress estrogen in premenopausal women and studing the effects on the gut microbiota, the activity of their enzymes and the impact on circulating estrogens. We are studing these effects in a group of control women (no dietary changes) and an experimental group consuming daily dietary flaxseed. My graduate student, Evan Stockerand I have worked together to establish the secure database for collecting study visit and survey data in RedCap, recruit participants and manage sample collection and storage. Mr. Stoeckerworked out the methods for the enzyme activities. Mark Hoenig, MD is the study physician and has administered the GnRH agonist injections and provided medical oversight to the study. 1. Establish a model of estrogen suppression in premenopausal women for the study of the effect of endogenous estrogen on the composition of gut microbiota--suppression will beconfirmed byestrogen levels in plasma. We have successfully recruited and taken 12 participants through the 3-month experimental protocol. 3. Collect urine, feces and blood for future measurement of systemic estrogensWe have collected and stored fecal, urine and blood samples for 6 timepoints for each participant. The protocol has been well-tolerated. We will analyze samples for gut microbiota and estrogen levels when all participants have completed the study.

Publications

Progress 07/26/17 to 09/30/17

Outputs
Target Audience:The target audience of this research is women in general, with specific emphasis on peri- and postmenopausal women. Changes/Problems:We have changed the protocol to include a control group. Rather than studying the women pre- and post-flaxseed intake with 2 months of estrogen suppression, we have shortened the suppression periodto one month:10 women will consume flaxseed 30 days prior to, and during the 30 day suppressionperiod, and 10 women will maintain theirnormal diet for the entire study period. What opportunities for training and professional development has the project provided?This project is supporting the training of a master's student in database design and use, the clinical research process and assay development. It has also been the basis of authorship of a book chapter for this student and contributed to the acceptance of the student to a National Cancer Institute practicum in Cancer Nutrition. For the project director, the project has given the opportunity to act as primary investigator on a clinical trial, for increased skills base in database design and management,for primary mentorship, for expanding collaborations with investigators at UC Denver AMC, for partnering with the flaxseed industry, and the potential for additional funding based on the results. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue to enroll participants, process samples and establish laboratory assays.

Impacts
What was accomplished under these goals? a. Establish a model of estrogen suppression in premenopausal womento study the effect of endogenous estrogen on the composition of the gut microbiota' b. Circulating estrogen concentrations and types change as women enter the menopause, with implications for metabolic health outcomes e.g. bone health, cancer risk, obesity etc.The gutmicrobiota metabolizeestrogen and influence circulating levels and types of estrogen. Given that microbial profilesare modifiable with diet, it may be possible to modulate the gut microbiota to helpmaintainestrogenprofilesassociated withdiseaseprevention. Researchto date hasbeen in postmenopausal women only, which is confounded bytime-since-menopause, changes inmetabolic rate and body composition. A modelthatcircumvents theseconfounding factorswillhelpidentify theeffects ofestrogenper se on gut microbiotaprofiles. c. We have obtained IRB and IBC approval, set up the study database and electronic surveys in RedCap, and successfully recruited 5 participants via CSU email listservs and social media (NextDoor). The project director (Dr. Cox-York) and graduate research assistant Evan Stoecker have conducted all of the work thus far. Blood samples have been taken andprocessed bytheHumanPerformance and ClinicalResearchLabat CSU. d.Three of the 5 participants are 90% through the protocol and the other two are in the beginning stages.The samples from these subjects have been processed and stored to be analyzed in batch. e. We expect this work to result in science-based knowledge about the reciprocal regulation of estrogen and the gut microbiota. This knowledge has the potential to influence women's health across the lifespan.

Publications