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Title:Dietary intervention with whole flaxseed or vitamin D has beneficial effects on leiomyomas in the oviduct of the laying hen
Author(s):Sizemore, Courtni Renee
Advisor(s):Nowak, Romana
Contributor(s):Miller, David; Hales, Dale B
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Vitamin D
Abstract:Uterine leiomyomas, or fibroids, have a symptomatic incidence of around 40% and are a leading cause of reproductive morbidity in reproductively-aged women. Symptoms include menorrhagia, pelvic pain, and infertility. They are characterized by excessive production of extracellular matrix components, such as collagen. Leiomyomas are responsive to estrogen and progesterone and occur more frequently as women age. Risk factors for fibroids include obesity, race, localized injury, and stress, while parity has been shown to be protective against fibroids. Inflammation has been hypothesized to play a key role in their development. Traditionally, hysterectomy or myomectomy has been the treatment for fibroids. However, hysterectomy does not preserve a woman’s fertility. Currently, there are no effective long-term, non-surgical treatments available for fibroids. Hormone therapies, such as GnRH analogs cannot be used for long-term treatment because they induce a state of hypoestrogenism which leads to post-menopausal symptoms. Researchers have turned to dietary intervention as a possible therapy for fibroids because it is non-invasive, safe for long-term use, and relatively inexpensive. Two potential candidates for dietary intervention are flaxseed and vitamin D3. Flaxseed consists of two parts: lignan and oil. The lignan portion of flaxseed contains phytoestrogens which may inhibit fibroid growth through the antagonism of estrogen. Flaxseed oil is rich in α-linolenic acid, which has known anti-inflammatory properties. Vitamin D3 is a hormone involved in calcium and bone homeostasis that also possesses anti-inflammatory and anti-fibrotic properties. Because the laying hen is the only animal to develop leiomyomas spontaneously as they age, they are an ideal animal model for the study of fibroids. Hen leiomyomas occur as polyps on the smooth muscle layer of the oviduct with increasing abundance as hens age and have the same histological and molecular markers as human uterine fibroids including desmin, smooth muscle actin, increased collagen, and steroid hormone receptors. Two studies were conducted to assess the efficacy of dietary intervention with whole flaxseed and vitamin D3. The first study involved feeding White Leghorn hens an isocaloric control (basic layer) or 15% whole flaxseed diet ad libitum from the end of their second year of lay to the completion of their third. At the end of the study, hens were euthanized and fibroids were counted, measured, and collected. Immunohistochemical analyses of fibrosis (Gomori’s Trichrome Stain), proliferation (Proliferating Cell Nuclear Antigen), angiogenesis (von Willebrand Factor), NF-κB p65, oxidative stress (nitrotyrosine), cellular senescence (Sudan Black B), and prevalence of mast cells (Toluidine Blue) were performed on fibroid tissue sections. Quantitative RT-PCR was performed to determine changes in expression levels of mRNA for collagen I, transforming growth factor-β3 (TGF-β3), and prostaglandin-endoperoxide synthase 2 (PTGS2). Western blots were carried out to determine changes in protein levels for COX-2. There was a slight decrease in the incidence of leiomyomas in the flaxseed group versus the controls. There was no significant difference in the volume of leiomyomas (p=0.3), although previous work by our group demonstrated a significant decrease in the size of leiomyomas from hens fed a similar diet of 10% whole flaxseed diet for 24 months. Fibroids from hens fed the flaxseed-supplemented diet for 12 months showed a modest decrease in proliferation when compared to controls and a significant decrease in numbers of blood vessels per unit area of fibroid. There were no differences in p65 translocation to the nucleus, a measure of Nuclear Factor kappa B (NF-κB) activation, or tissue levels of nitrotyrosine, a measure of oxidative stress, between treatment groups. Sudan Black B staining showed that leiomyomas from both treatment groups were not undergoing cellular senescence while Toluidine Blue staining did show mast cell infiltration into fibroids from both groups. Fibroids from hens fed the whole flaxseed diet had slightly decreased collagen I (4-fold change, p=0.06) and TGFβ-3 expression (6-fold change, p=0.06), while PTGS2 (6-fold change, p=0.04) expression was significantly downregulated when compared to fibroids from hens fed the control diet. COX-2 analysis through western blot showed a non-significant decrease in relative COX-2 expression in leiomyomas from flaxseed-fed hens compared to those from control hens. In the second study, White Leghorn hens having completed their second year of lay were fed either an isocaloric control (basic layer), 10% whole flaxseed, or vitamin D3 (69 µg/kg/day) diet for 15 months. Twenty-five hens were sacrificed at the start of the trial to determine the baseline incidence of leiomyomas. Blood was drawn from the same subset of hens every three months until the end of the trial. After 15 months on their respective diets, hens were euthanized. Oviducts were collected, and fibroids were counted, measured, and either flash frozen in liquid nitrogen or fixed in 10% neutral buffered formalin for further analysis. Leiomyoma incidence increased from 44% at the start of the trial to 78%, 74%, and 71% in the control, whole flaxseed, and vitamin D groups, respectively after 15 months. The number of leiomyomas per hen in all three groups increased significantly after fifteen months when compared to the baseline; there were no differences between groups after fifteen months. Plasma carbonylated protein levels decreased significantly in the whole flaxseed and vitamin D3-treated hens after 6 months on their respective diets; these levels were significantly lower than those of the control hens after 9 months in both groups. qRT-PCR analysis showed a downregulation of collagen I (50-fold change, p=0.02), TGF-β3 (50-fold change, p=0.003), and PTGS2 (100-fold change, p=0.001) gene expression in leiomyomas from hens fed the vitamin D3-supplemented diet after 15 months when compared to those from control hens; there were no differences between the whole flaxseed group when compared to the control. We conclude that dietary intervention with whole flaxseed or vitamin D3 may have beneficial, therapeutic effects on leiomyomas found in the oviduct of the laying hen. Our results support further investigation into the use of these supplements in women with fibroids as they could provide a safe, effective, long-term, and non-invasive treatment option that currently is not available.
Issue Date:2018-12-20
Rights Information:Copyright 2018 Courtni Renee Sizemore
Date Available in IDEALS:2019-02-07
Date Deposited:2018-12

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