Changes in thyroid hormone concentrations over time in dogs with autoimmune thyroiditis.

OBJECTIVE: The objective of this study was to follow long-term changes in the concentration of thyroid hormones in dogs with subclinical thyroiditis. SAMPLES: Samples were obtained from 125 dogs with subclinical thyroiditis. The study population included 70 female and 55 male dogs. The mean testing interval was 3.9 years from initial testing (SD, 2.3 years; range, 1 to 9 years). METHODS: Dogs with subclinical thyroiditis were identified retrospectively using results from the Orthopedic Foundation for Animals Canine Thyroid Profile performed by the Endocrinology Section of the Michigan State University Veterinary Diagnostic Lab. Owners were invited to submit follow-up serum samples with their veterinarian along with a medical history form, including subsequent treatments. RESULTS: At the time of retesting, 30% of the dogs had progressed to hypothyroidism and/or were treated with thyroxine. Fifty percent maintained positive or equivocal thyroglobulin autoantibody (TgAA) results while remaining euthyroid. Fourteen percent of the dogs became TgAA negative and remained euthyroid. In 6% of the cases tested, proper medical histories were not available, and a final classification could not be determined. CLINICAL RELEVANCE: These results indicate that most dogs with elevated thyroglobulin autoantibodies either exhibit persistent autoimmune thyroiditis with continued risk of hypothyroidism or progress to hypothyroidism when monitored for more than 1 year. Thyroid function in dogs with subclinical thyroiditis should be monitored every 12 months or if there is change in the clinical presentation.

Serum gastrin concentrations in dogs with primary hyperparathyroidism.

BACKGROUND: Hypercalcemia has been associated with hypergastrinemia in humans. Hypergastrinemia could be responsible for gastrointestinal (GI) signs in dogs with primary hyperparathyroidism (PHPT). HYPOTHESIS/OBJECTIVES: (a) Determine whether hypergastrinemia occurs in dogs with PHPT, (b) assess for potential correlations among ionized calcium (iCa), parathyroid hormone (PTH), and serum gastrin concentrations, and (c) determine whether gastrin concentrations decrease after management of PHPT. ANIMALS: Phase 1: 151 client-owned dogs at the time of PHPT diagnosis, Phase 2: 24 dogs that underwent treatment for PHPT. METHODS: Dogs with azotemia, concurrent disease, or those receiving acid suppressants were excluded. Twenty-four treated dogs had baseline and repeat quantification of serum gastrin, PTH, and iCa concentrations 4 weeks after treatment. The effect of treatment on gastrin, iCa, and PTH concentrations was assessed using Wilcoxon signed rank sum tests. Fisher exact testing was used to compare the proportion of dogs with hypergastrinemia in dogs with and without GI signs. RESULTS: Twenty-seven of 151 PHPT dogs (17.9%) had increased pre-treatment serum gastrin concentrations (median, 45.0 ng/L; interquartile range [IQR], 20.0 ng/L). Gastrin concentrations were not correlated with iCa (P = .92) or PTH (P = .60). Treatment of PHPT decreased PTH (P < .001) and iCa concentrations (P < .001), but not gastrin concentrations (P = .15). The proportion of dogs with hypergastrinemia with and without GI signs did not differ (P = 1.00). CONCLUSIONS AND CLINICAL IMPORTANCE: Mild increases in serum gastrin concentrations may be seen in dogs with PHPT, but this finding is independent of the presence of GI signs.

Molecular Action of Tamoxifen in the Ovaries of Rats with Mammary Neoplasia.

Tamoxifen (TAM) is a drug commonly used in patients with breast cancer. The anticancer effect of TAM occurs via its ability to antagonize estrogen-dependent growth of mammary epithelial cells. Previously, we demonstrated that TAM prevented the chemotherapy-induced loss of ovarian follicular reserves in both cancer-free rats and rats with cancer. Such follicular loss is a main cause of infertility in young women treated for cancer. The current study was undertaken to discover the molecules and intracellular pathways involved in the action of TAM in the ovaries of rats with mammary tumors. To meet this goal we used transcriptomic (RNA-Seq) and proteomic (2D-DIGE/MS) approaches. TAM inhibited the expression of genes and lncRNAs involved in ovarian steroidogenesis. Moreover, TAM altered the expression of genes related to primordial follicle activation or arrest. In addition, proteomic screening indicated the importance of basic metabolic processes in the ovarian actions of TAM. Although simple extrapolation of these data to humans is not possible, the results of this study emphasize the need to explore the ability of TAM to affect ovarian function in women undergoing cancer treatment.

Establishment of a reference interval for 12-hour fasted serum gastrin concentration in adult dogs.

BACKGROUND: Adherence to traditional 24-h fasting periods for serum gastrin concentration in dogs can be challenging and may delay the institution of therapies for suspected hypergastrinemia. Peer-reviewed publications regarding serum gastrin reference intervals (RI) are lacking. Hypercalcemia is associated with hypergastrinemia in people; limited data exist in dogs. OBJECTIVE: The objective of the study was to generate a RI for a 12-h fasted serum gastrin concentration in dogs and to investigate whether correlations exist with age, weight, sex, and total calcium concentration. METHODS: Fifty-five healthy adult dogs (>1 year of age). The screening included: medical history, physical examination, CBC (15 dogs), and serum chemistry (55 dogs). Gastrin was measured via a commercial radioimmunoassay. The RI for 12-h fasted serum gastrin concentration was calculated according to the recommendations of the American Society for Veterinary Clinical Pathology. Additionally, data were evaluated for correlation with selected variables. RESULTS: The RI for serum gastrin following a 12-h fasting period was 15.1-78.9 ng/L with 90% confidence intervals for the lower and upper limits of 14.0-22.9 and 68.3-83.0 ng/L, respectively. A generalized linear model did not detect significant relationships between gastrin and age (P = 0.48), sex (P = 0.30), weight (P = 0.93), or total calcium concentration (P = 0.84). CONCLUSIONS: A 12-h fasted serum gastrin concentration RI has been established. Given the limited range of serum calcium concentrations in our healthy study population, additional investigations are needed to determine the effects of hypercalcemia on serum gastrin concentrations in dogs and for any potential clinical consequences thereof.

Tamoxifen decreases ovarian toxicity without compromising cancer treatment in a rat model of mammary cancer.

BACKGROUND: Premenopausal women diagnosed with breast cancer often face aggressive chemotherapy resulting in infertility. Tamoxifen (TAM) is a selective estrogen receptor modulator that was previously suggested as a protective agent against chemotherapy-induced ovarian failure. In the current study, we examined mechanisms of the protective action of TAM in the ovaries of tumor-bearing rats treated with the chemotherapy drug cyclophosphamide (CPA). RESULTS: TAM prevented CPA-induced loss of ovarian follicular reserves. The protective TAM effect in the rat ovary partially resulted from decreased apoptosis. In addition, transcriptomic and proteomic screening also implicated the importance of DNA repair pathways as well as cell adhesion and extracellular matrix remodeling in the protective ovarian actions of TAM. CONCLUSIONS: Tamoxifen shielded the ovary from the side effects of chemotherapy without lessening the tumoricidal actions of mammary cancer treatment.

Relationship between serum magnesium, calcium, and parathyroid concentrations in dogs with abnormally low serum 25-hydroxyvitamin D concentration and chronic or protein-losing enteropathy.

BACKGROUND: The relationship between the development of SHPT and ionized magnesium (iMg) concentrations in blood of dogs with chronic gastrointestinal (GI) disease and abnormally low 25(OH)D is undefined. OBJECTIVES: Evaluate relationships between ionized magnesium (iMg), PTH, ionized calcium (iCa), and 25(OH)D in dogs with chronic enteropathy (CE) with or without protein-losing enteropathy (PLE) and abnormal 25(OH)D. Determine whether dogs with CE or PLE, decreased 25(OH)D and SHPT have differences in iMg, iCa, or 25(OH)D when compared to dogs that do not have SHPT. ANIMALS: Fifty dogs with CE +/- PLE and abnormally low serum 25(OH)D. METHODS: Retrospective search of submissions database at a veterinary diagnostic laboratory for vitamin D profiles submitted in years 2017 to 2020. Cases were excluded if supplemented with Ca, Mg, or vitamin D. Spearman correlation was performed to evaluate relationships between iMg, PTH, 25(OH)D, and iCa. Ionized Mg, iCa, and 25(OH)D concentrations were compared between dogs with SHPT and those with normal PTH concentrations. RESULTS: Concentrations of iMg were weakly negatively correlated with PTH (rho, -.31; P = .03), and weakly positively correlated with serum 25(OH)D (rho, .34, P = .02) and iCa (rho, .42, P = .003). Ionized magnesium concentrations were lower in dogs with abnormally low 25(OH)D and SHPT compared to dogs with abnormally low 25(OH)D and normal parathyroid hormone concentrations (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Hypomagnesemia might contribute to alterations in iCa and parathyroid hormone in dogs with CE +/- PLE and abnormally low 25(OH)D.

PRESUMPTIVE CONGENITAL HYPOTHYROIDISM IN RED PANDAS (AILURUS FULGENS FULGENS) FROM FOUR SUCCESSIVE LITTERS.

High neonatal mortality among red pandas (Ailurus fulgens) challenges the long-term sustainability of the Species Survival Plan population. Congenital hypothyroidism (CH) is a rare condition in domestic animals, typically due to an inherited genetic defect. Nongoitrous CH was presumptively diagnosed in 75% (n = 6/8) of red panda neonates from four successive litters, with a common sire and two closely related dams. Antemortem diagnosis of CH was made in three cubs (n = 3/6) based on elevated thyroid stimulating hormone and decreased free thyroxine and total thyroxine levels. Affected cubs also had suggestive clinical signs, which included delayed growth with cretinous dwarf appearance, atonic bladder, delayed gastrointestinal motility, hypercholesterolemia, and hypocalcemia. With sodium levothyroxine therapy, two of the three cubs developed into normal adult red pandas in terms of body size, appearance, and behavior. On necropsy cubs (n = 4) were small with varying degrees of cretin dwarf appearance and hypoplastic thyroids with reduced to no colloid in follicles. These cases demonstrate the importance of collecting thyroid tissue, (or proximal trachea/larynx if gross visualization not possible), in neonates for histopathology. Further investigation into the role of thyroid disease in neonatal red panda mortality is warranted.

Multiple Lesions Contribute to Infertility in Males Lacking Autoimmune Regulator.

Male factors, including those of autoimmune origin, contribute to approximately 50% of infertility cases in humans. However, the mechanisms underlying autoimmune male infertility are poorly understood. Deficiency in autoimmune regulator (AIRE) impairs central immune tolerance because of diminished expression of self-antigens in the thymus. Humans with AIRE mutations and mice with engineered ablation of Aire develop multiorgan autoimmunity and infertility. To determine the immune targets contributing to infertility in male Aire-deficient (-/-) mice, Aire-/- or wild-type (WT) males were paired with WT females. Aire-/- males exhibited dramatically reduced mating frequency and fertility, hypogonadism, and reduced serum testosterone. Approximately 15% of mice exhibited lymphocytic infiltration into the testis, accompanied by atrophy, azoospermia, and reduced numbers of mitotically active germ cells; the remaining mice showed normal testicular morphology, sperm counts, and motility. However, spermatozoa from all Aire-/- mice were defective in their ability to fertilize WT oocytes in vitro. Lymphocytic infiltration into the epididymis, seminal vesicle, and prostate gland was evident. Aire-/- male mice generated autoreactive antibodies in an age-dependent manner against sperm, testis, epididymis, prostate gland, and seminal vesicle. Finally, expression of Aire was evident in the seminiferous epithelium in an age-dependent manner, as well as in the prostate gland. These findings suggest that Aire-dependent central tolerance plays a critical role in maintaining male fertility by stemming autoimmunity against multiple reproductive targets.

VALIDATION OF CHEMILUMINESCENT ASSAY FOR CANINE THYROID STIMULATING HORMONE IN RED PANDAS (AILURUS FULGENS FULGENS).

Thyroid abnormalities have been anecdotally reported in red pandas (Ailurus fulgens fulgens); however, definitive diagnosis is hampered by a lack of established reference ranges and validated diagnostic tests. The chemiluminescent assay for canine thyroid stimulating hormone (cTSH) has been validated for use in domestic canids and felids. This study aims to validate the cTSH assay for use in red pandas. Validation was performed via serial dilutions of banked serum samples (n = 15) and both inter- and intra-assay testing. High estimated recoveries and low coefficients of variability indicate that the cTSH assay accurately and consistently measures TSH concentrations in red panda serum. Further studies to generate red panda age and sex TSH reference ranges are indicated.

Randomized Phase IIB Trial of the Lignan Secoisolariciresinol Diglucoside in Premenopausal Women at Increased Risk for Development of Breast Cancer.

We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was -1.8% in the SDG arm (P = 0.001) and -1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = -2.2%; P = 0.002) but not placebo (median = -1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (<0.5 or >2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.

Autoimmune Regulator is required in female mice for optimal embryonic development and implantation†.

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis.

Radioactive iodine uptake in hyperthyroid cats after administration of recombinant human thyroid stimulating hormone.

BACKGROUND: Radioactive iodine therapy is considered the treatment of choice for hyperthyroidism in cats, but the availability of this modality is limited by costs and hospitalization requirements. Administration of recombinant human thyroid stimulating hormone (rh-TSH) to humans with thyroid neoplasia or nodular goiter can increase thyroidal iodine uptake, thereby allowing the use of lower radioactive iodine doses for treatment. Veterinary studies of this subject are limited, and results are conflicting. OBJECTIVE: To investigate the effects of rh-TSH administration on thyroidal iodine uptake in hyperthyroid cats. ANIMALS: Ten client-owned hyperthyroid cats. METHODS: In this prospective clinical study, cats were administered saline (placebo), 50 µg rh-TSH (low-dose), and 100 µg rh-TSH (high-dose) in randomized crossover design with treatments separated by 7-10 days. After each treatment, thyroid scintigraphy was performed by administering 300 µCi 123 I and assessing radionuclide uptake 8 and 24 hours later. Serum thyroid hormone concentrations were measured at each visit. RESULTS: Thyroidal percent iodine uptakes (mean ± SD at 8 and 24 hours) in cats treated with placebo (25.2 ± 13.4%, 30.0 ± 12.8%), low-dose (24.1 ± 12.5%, 29.4 ± 13.7%), and high-dose rh-TSH (24.2 ± 16.3%, 30.8 ± 15.3%) were not different (P = .76). Independent of rh-TSH administration, percent iodine uptakes were positively correlated with serum thyroid hormone concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: One-time administration of rh-TSH, even at high doses, would not be expected to lower radioactive iodine doses needed for treatment of hyperthyroidism in cats. Investigations of alternate strategies to increase thyroidal uptake of radioactive iodine are warranted.

Immune response to a model shared placenta/tumor-associated antigen reduces cancer risk in parous mice.

During human pregnancy, paternally inherited antigens expressed by the fetal-placental unit can elicit expansion of antigen-specific CD8+ T cells. These cells can persist for years as memory T cells, but their effects on long-term maternal health are unknown. Shared placenta/tumor-associated antigens are expressed by placenta and tumors, but are minimally expressed or absent in normal adult tissues. We hypothesized that maternal T cells elicited against these antigens can alter risk of cancers expressing the same antigen after pregnancy, and tested this in mice using chicken ovalbumin (OVA) as a surrogate shared placenta/tumor antigen. Hemizygous OVA transgenic males were bred to wild-type C57BL/6 females (H2b haplotype) such that the fetuses inherited and expressed OVA. Maternal OVA/H2Kb-specific CD8+ T cells became detectable during gestation, and persisted in some animals for up to 24 weeks. To determine whether these cells might influence growth of OVA-expressing tumors in OVA-bred females, E.G7-OVA thymoma cells were inoculated subcutaneously in OVA-bred, wild-type bred, and virgin females, and monitored for growth. OVA-bred mice had prolonged survival as compared to virgin mice and the progression of tumors was delayed in comparison to wild-type bred and virgin females. Thus, paternally inherited OVA antigen elicited a CD8+ T cell response during pregnancy that was associated with delayed growth of OVA-expressing tumors following pregnancy. These data suggest a possible role of antigen-specific T cells in protecting parous females against tumors bearing shared placenta/tumor antigens.

The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERß expression and the incidence of apoptosis in granulosa cells of medium porcine follicles.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor ß (ERß) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERß and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

Modulation of Breast Cancer Risk Biomarkers by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Premenopausal Women.

Higher intakes of the omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) have been variably associated with reduced risk of premenopausal breast cancer. The purpose of this pilot trial was to assess feasibility and explore the effects of high-dose EPA and DHA on blood and benign breast tissue risk biomarkers before design of a placebo-controlled phase IIB trial. Premenopausal women with evidence of hyperplasia ± atypia by baseline random periareolar fine needle aspiration were given 1860 mg of EPA + 1500 mg of DHA ethyl esters daily for 6 months. Blood and benign breast tissue were sampled during the same menstrual cycle phase prestudy and a median of 3 weeks after last dose. Additional blood was obtained within 24 hours of last dose. Feasibility, which was predefined as 50% uptake, 85% retention, and 70% compliance, was demonstrated with 46% uptake, 94% completion, and 85% compliance. Cytologic atypia decreased from 77% to 38% (P = 0.002), and Ki-67 from a median of 2.1% to 1.0% (P = 0.021) with an increase in the ratio of EPA + DHA to AA in erythrocyte phospholipids but no change in blood hormones, adipokines, or cytokines. Exploratory breast proteomics assessment showed decreases in several proteins involved in hormone and cytokine signaling with mixed effects on those in the AKT/mTOR pathways. Further investigation of EPA plus DHA for breast cancer prevention in a placebo-controlled trial in premenopausal women is warranted.

Clinical Trial of Acolbifene in Premenopausal Women at High Risk for Breast Cancer.

The purpose of this study was to assess the feasibility of using the selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women. To do so, we assessed change in proliferation in benign breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) as a primary endpoint, along with changes in other risk biomarkers and objective and subjective side effects as secondary endpoints. Twenty-five women with cytologic hyperplasia ± atypia and ≥2% of breast epithelial cells staining positive for Ki-67, received 20 mg acolbifene daily for 6-8 months, and then had benign breast tissue and blood risk biomarkers reassessed. Ki-67 decreased from a median of 4.6% [interquartile range (IQR), 3.1%-8.5%] at baseline to 1.4% (IQR, 0.6%-3.5%) after acolbifene (P < 0.001; Wilcoxon signed-rank test), despite increases in bioavailable estradiol. There were also significant decreases in expression (RT-qPCR) of estrogen-inducible genes that code for pS2, ERα, and progesterone receptor (P ≤ 0.026). There was no significant change in serum IGF1, IGFBP3, IGF1:IGFBP3 ratio, or mammographic breast density. Subjective side effects were minimal with no significant increase in hot flashes, muscle cramps, arthralgias, or fatigue. Objective measures showed a clinically insignificant decrease in lumbar spine bone density (DEXA) and an increase in ovarian cysts but no change in endometrial thickness (sonography). In summary, acolbifene was associated with favorable changes in benign breast epithelial cell proliferation and estrogen-inducible gene expression but minimal side effects, suggesting a phase IIB placebo-controlled trial evaluating it further for breast cancer prevention.

Modulation of Breast Cancer Risk Biomarkers by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Postmenopausal Women.

Associational studies suggest higher intakes/blood levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) are associated with reduced breast cancer risk. We performed a pilot study of high-dose EPA + DHA in postmenopausal women to assess feasibility before initiating a phase IIB prevention trial. Postmenopausal women with cytologic evidence of hyperplasia in their baseline random periareolar fine needle aspiration (RPFNA) took 1,860 mg EPA +1500 mg DHA ethyl esters daily for 6 months. Blood and breast tissue were sampled at baseline and study conclusion for exploratory biomarker assessment, with P values uncorrected for multiple comparisons. Feasibility was predefined as 50% uptake, 80% completion, and 70% compliance. Trial uptake by 35 study entrants from 54 eligible women was 65%, with 97% completion and 97% compliance. Favorable modulation was suggested for serum adiponectin (P = 0.0027), TNFα (P = 0.016), HOMA 2B measure of pancreatic ß cell function (P = 0.0048), and bioavailable estradiol (P = 0.039). Benign breast tissue Ki-67 (P = 0.036), macrophage chemoattractant protein-1 (P = 0.033), cytomorphology index score (P = 0.014), and percent mammographic density (P = 0.036) were decreased with favorable effects in a proteomics array for several proteins associated with mitogen signaling and cell-cycle arrest; but no obvious overall effect on proteins downstream of mTOR. Although favorable risk biomarker modulation will need to be confirmed in a placebo-controlled trial, we have demonstrated feasibility for development of high-dose EPA and DHA ethyl esters for primary prevention of breast cancer.

Effects of Flaxseed Lignan Secoisolariciresinol Diglucosideon Preneoplastic Biomarkers of Cancer Progression in a Model of Simultaneous Breast and Ovarian Cancer Development.

Breast cancer prevention efforts are focused increasingly on potentially beneficial dietary modifications due to their ease of implementation and wide acceptance. Secoisolariciresinol diglucoside (SDG) is a lignan found in high concentration in flaxseed that may have selective estrogen receptor modulator-like effects resulting in antiestrogenic activity in a high estrogen environment. In parallel with a human phase II prevention trial, female ACI rats (n = 8-10/group) received 0, 10, or 100 ppm SDG in the feed. The 100 ppm SDG treatment produced similar blood lignan levels as those observed in our human pilot study. Mammary and ovarian cancer progression were induced using local ovarian DMBA treatment and subcutaneous sustained release 17ß-estradiol administered starting at 7 weeks of age. Mammary gland and ovarian tissues were collected at 3 mo after initiation of treatment and examined for changes in epithelial cell proliferation (Ki-67, cell counts), histopathology, and dysplasia scores, as well as expression of selected genes involved in proliferation, estrogen signaling, and cell adhesion. Treatment with SDG normalized several biomarkers in mammary gland tissue (dysplasia, cell number, and expression of several genes) that had been altered by carcinogen. There is no indication that SDG promotes preneoplastic progression in the ovarian epithelium.

Tamoxifen prevents apoptosis and follicle loss from cyclophosphamide in cultured rat ovaries.

Recent studies documented that the selective estrogen receptor modulator tamoxifen prevents follicle loss and promotes fertility following in vivo exposure of rodents to irradiation or ovotoxic cancer drugs, cyclophosphamide and doxorubicin. In an effort to characterize the ovarian-sparing mechanisms of tamoxifen in preantral follicle classes, cultured neonatal rat ovaries (Day 4, Sprague Dawley) were treated for 1-7 days with active metabolites of cyclophosphamide (i.e., 4-hydroxycyclophosphamide; CTX) (0, 1, and 10 µM) and tamoxifen (i.e., 4-hydroxytamoxifen; TAM) (0 and 10 µM) in vitro, and both apoptosis and follicle numbers were measured. CTX caused marked follicular apoptosis and follicular loss. TAM treatment decreased follicular loss and apoptosis from CTX in vitro. TAM alone had no effect on these parameters. IGF-1 and IGF-1 receptor were assessed in ovarian tissue showing no impact of TAM or CTX on these endpoints. Targeted mRNA analysis during follicular rescue by TAM revealed decreased expression of multiple genes related to inflammation, including mediators of lipoxygenase and prostaglandin production and signaling (Alox5, Pla2g1b, Ptgfr), cytokine binding (Il1r1, Il2rg ), apoptosis (Tnfrsf1a), second messenger signaling (Mapk1, Mapk14, Plcg1), as well as tissue remodeling and vasodilation (Bdkrb2, Klk15). The results suggest that TAM protects the ovary from CTX-mediated toxicity through direct ovarian actions that oppose follicular loss.

Challenges and Potential for Ovarian Preservation with SERMs.

Tamoxifen (TAM) is a selective estrogen receptor modulator with tissue-specific effects on estrogen signaling used predominantly for treatment and chemoprevention of breast cancers. Recent studies have shown that TAM prevents infertility and decreases follicular loss from common cancer chemotherapy and radiation therapy in preclinical models. Here we review current and novel uses of selective estrogen receptor modulator s and advantages and challenges for translation of TAM for human fertility preservation.