Source: AUBURN UNIVERSITY submitted to
POSTNATAL DIET, AGE AT SEXUAL MATURITY, AND RISKS FOR UTERINE FIBROIDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1002091
Grant No.
(N/A)
Project No.
ALA018-1-13007
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 2, 2014
Project End Date
Apr 30, 2018
Grant Year
(N/A)
Project Director
Berry, WA, DE.
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Poultry Science
Non Technical Summary
Background: Over 70% of women develop uterine fibroid tumors by the time they reach 50 years old. Uterine fibroids have a significant effect on fertility and quality-of-life, costing the US $34.4 billion every year. To date, little progress has been made in determine causes of the disease, in part due to a lack of experimental models for study of the disease. In particular, virtually no study has examined how developmental environment, such as childhood diet, affects the disease risk. Egg-type chicken hens provide an excellent model for studying developmental origin and mechanisms of uterine fibroids as hens spontaneously develop fibroid tumors at a high rate. In addition, our team was the first to discover that hen oviductal fibroids share key characteristics with human uterine fibroids, such as molecular markers. Our goals of the proposed research are to: 1) determine the effect of low- and high-protein diet during postnatal period and timing of sexual maturity on risks of developing uterine fibroid, 2) use the obtained data to apply for PAR-13-204 by NIH and USDA, and 3) help establish guidelines for optimal nutrition during development to alleviate incidence and severity of fibroid tumors. Significance: Childhood diet as a potential origin of uterine fibroids has been practically untested. This proposal pioneers the field because not only does it examine the mechanistic link between postnatal diet and fibroid risk, but also disentangle diet from timing of sexual maturity in relation to fibroid risk. We hypothesize that high-protein postnatal nutrition and early onset of sexual maturity increase risks of uterine fibroids through alteration in the insulin-like growth factor (IGF) system and cell proliferation signals. Approach: To test the hypothesis, egg-type female chicks will be exposed to 1) various levels of protein in the diet and 2) alterations of photoperiod to manipulate onset of sexual maturity. Size and number of fibroid tumors will be determined biyearly along with plasma IGF and IGF binding protein (IGFBP) levels, as well as IGF receptor levels, cell proliferation signals including Bcl-2, PCNA, and an apoptosis signal, caspase-3 in fibroid tissues. The incidence of fibroid tumors is expected to rise with increasing proportion of protein during rearing and decreasing age at sexual maturity, accompanied by high levels of IGF, Bcl-2, PCNA, and low levels of IGFBP and caspase-3. The long-term goal of the proposed research is to lower prevalence of uterine fibroids to improve quality of life for millions of women. The results from this research will inform recommended consumption guidelines for protein during infancy and prepubertal period to prevent or lessen the severity of fibroid tumors.
Animal Health Component
100%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30532101020100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3210 - Egg-type chicken, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
1. Gather preliminary data to be included in a PAR-13-204 proposal (a NIH-USDA grant titled "Dual purpose with dual benefit: research in biomedicine and agriculture using agriculturally important domestic animal species (R01)")2. Understand the causal relationship among postnatal diet, timing of onset of sexual maturity and uterine fibroid3. Help establish a guideline for optimal nutrition during development to alleviate incidence and severity of fibroid tumors
Project Methods
Experimental Design 270 one-day old female chicks will be randomly allocated into 6 groups following a 3x2 full factorial design (n=45): three diets containing 10%, 18% or 25% crude protein, and two levels (early or late) of onset of sexual maturity. All diets will be isocaloric and fall within the range of protein content given to hens in a commercial setting (North and Bell, 1990). The hens will be raised on the assigned diet from 1 day of age to until euthanasia. All chickens will have ad lib access to food. Onset of sexual maturity will be controlled by photoperiod manipulation: day length will be switched from 8L:16D to 16L:8D, on 16 or 24 weeks. 10 Data collection and analyses Hens will be bled to obtain serum levels of IGF and IGFBP at 18 weeks (pre-sexual maturity), 26 weeks (post-sexual maturity), and every 6 months thereafter. Fifteen hens per treatment will be euthanized at 6 months, 1 year, and 1.5 years of age by CO2 narcosis. Size, number, and location of fibroid tumors will be recorded. Fibroid tissues will be collected from each hen and half will be snap-frozen using dry ice and the other half will be fixed in 10% formalin. Normal oviduct tissue from the same bird will serve as a control for comparison. Frozen tissue will be later homogenized, centrifuged, and supernatant will be used to quantify IGF-I, IGF-IR, and IGFBP-1 protein levels using a commercial ELISA kits (RayBiotech, Inc., Norcross, GA). The fixed tissue will be embedded in paraffin blocks and sliced into 5 μm sections for immunohistochemistry. Three immunohistochemical staining will be performed on 3 consecutive sections using primary antibodies against Bcl-2 (R&D Systems Inc., Minneapolis, MN), proliferating cell nuclear antigen (Lab Vision Corp., Fremont, CA), and caspase-3, a marker of cell apoptosis (Abcam, Cambridge, MA). Statistical analyses The effects of protein content of rearing diet and onset of sexual maturity on size, number of fibroid tumors will be analyzed using two-way ANCOVA with body weight as a covariate. The effects of protein content of rearing diet and onset of sexual maturity on IGF system, Bcl-2, PCNA, and caspase-3 staining will be analyzed using independent repeated measures ANCOVAs with respective levels in the normal tissues for each bird as a covariate. The alpha level will be set to 0.05.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Biomedical research professionals working in reproductive cancer research and developmental biologists are the primary target audiences. Status of this work is briefed to gynecologic oncologists at University of South Alabama Medical School. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Wada and Dr. Berry have both gained expertise in various new laboratory techniques related to data gathered in this study. How have the results been disseminated to communities of interest?Conversations regarding the progress of this study have been had with gynecologic oncologists at University of South Alabama Medical School. These conversations led to a collaboration with colleagues at USA resulting in submission of a proposal to the DOD reproductive cancer program. The proposal was funded and research will be underway in February 2018. What do you plan to do during the next reporting period to accomplish the goals?Complete sample analysis, draw conclusions from results, publish manuscripts in peer reviewed journals.

Impacts
What was accomplished under these goals? Hens in this study were found to develop significant numbers of fibroid tumors of the oviduct from two years of age. From initiation of the experiment at 1 day of age up to 24 months of age, blood and tissue samples were obtained from laying hens. All blood sampling has been completed with plasma harvested from the blood samples and stored at -80C for later analysis. Similarly, periodic tissue samples, from control and treated hens, including normal and fibroid tumor samples were collected. All tissue sampling is now complete. Samples of all tissues were collected in liquid nitrogen and stored frozen at -80C. Duplicate samples were also collected and stored in an RNA preservation solution. Hen body weight and age, and fibroid number and weight were recorded. The results obtained will be used as the basis for a proposal to NIH (PAR-13-204 NIH-USDA or similar program) or other appropriate agency. 2. Analysis of the collected samples is ongoing. Initial results indicate that earlier onset of sexual maturity is associated with an increased incidence of oviduct fibroid tumors in laying hens. As analysis proceeds, conclusions will be drawn regarding the relationships between diet, sexual maturation, and development of reproductive tract fibroids. 3. Completion of assay procedures and analysis will allow conclusions to be drawn, and potentially, recommendations made regarding diet.

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:Biomedical research professionals working in reproductive cancer research and developmental biologists are the primary target audiences. Status of this work is briefed to gynecologic oncologists at University of South Alabama Medical School. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Wada and Dr. Berry have both gained expertise in various new laboratory techniques related to data gathered in this study. How have the results been disseminated to communities of interest?Conversations regarding the progress of this study have been had with gynecologic oncologists at University of South Alabama Medical School. These physician scientists are potential collaborators on any oviduct fibroid studies going forward. An abstract is in preparation and will be submitted for the 2017 Poultry Science Meeting. Manuscript(s) will be submitted to appropriate journals upon completion of data analysis. What do you plan to do during the next reporting period to accomplish the goals?Continue with data gathering and analysis. Samples are being analyzed for changes in components of the IGF system.

    Impacts
    What was accomplished under these goals? 1. Data is still being gathered and analyzed. Oviduct fibroids typically appear in laying hens at around 24 months of age. This occurred as expected in the current study. From inititation of the experiment at 1 day of age up to 24 months of age, blood and tissue samples were obtained from laying hens. To date, periodic blood sampling has been completed with plasma harvested from the blood samples and stored at -80C for later analysis. Periodic tissue samples, from control and treated hens, including normal and fibroid tumor samples were collected. Samples of all tissues were collected in liquid nitrogen and stored frozen at -80C. Duplicate samples were also collected and stored in an RNA preservation solution. Hen body weight and age, and fibroid number and weight were recorded. The results obtained will be used as the basis for a proposal to NIH (PAR-13-204 NIH-USDA or similar program) or other appropriate agency. 2. Analysis is ongoing, however, to date, it appears that earlier onset of sexual maturity is associated with an increased incidence of oviduct fibroid tumors in laying hens. Analysis of samples collected is proceeding and will allow conclusions to be drawn regarding the relationships between diet, sexual maturation, and development of reproductive tract fibroids. 3. Completion of assay procedures and analysis will allow conclusions to be drawn, and potentially, recommendations made regarding diet.

    Publications


      Progress 10/01/14 to 09/30/15

      Outputs
      Target Audience:Gynecologic Oncologists at University of South Alabama Medical School and University of Alabama Birmingham Medical School. Changes/Problems:No major changes. The study is proceeding as planned. What opportunities for training and professional development has the project provided?Dr. Berry and Dr. Wada have been training in techniques for molecular analysis of markers for tumor development. How have the results been disseminated to communities of interest?Results from this study will be forthcoming following the final tissue sampling when the hens are 24 months old. What do you plan to do during the next reporting period to accomplish the goals?Analysis of all samples, data collection and reduction, and preparation of manuscripts will occur during the next reporting period.

      Impacts
      What was accomplished under these goals? This study is entirely contingent upon the development of reproductive tract fibroid tumors. Development of these tumors generally occurs during the second to third year of life in high producing lines of laying hens. The hens are now approaching 24 months of age. The second of three sets of tissue samples have been obtained. Collection of the final set of samples and beginning of molecular analysis of samples is imminent.

      Publications


        Progress 05/02/14 to 09/30/14

        Outputs
        Target Audience:Readers of College of Agriculture and Poultry Science Department publications have read news articles outlining reproductive tract tumor studies within this Hatch project. Dr. Berry has briefed gynecologic oncologists at the University of South Alabama School of Medicine on this work. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?At least two articles have been published in College of Agriculture and Poultry Science Department literature. This literature has gone out to students, faculty, alumni, state legislators, and stakeholders. What do you plan to do during the next reporting period to accomplish the goals?The study will come to completion during the next reporting period. All samples will be collected and analyzed.

        Impacts
        What was accomplished under these goals? 1. Initial samples have been gathered and are being analyzed. 2-3. Progress towards goals 2-3 must await analysis of all samples at the end of the study.

        Publications