1 Spontaneous Preterm Birth Decreasing the Disparities Nutrigenomic Profiling for Early Risk Stratification and Preventive Intervention John S Finnell, ND, MPH, LAc AOMA Graduate School of Integrative Medicine

2 Outline The Rising Syndemic Seasonality of Risk A Culture of DisparityNourishing Relationships Twilight of Risk Assessment Spinning the Thread of Life Shining the Light on Microparticles Dawn of a Culture of Health Outline

3 “Never lose an opportunity of seeing anything beautiful, for beauty is God’s handwriting.” Ralph Waldo Emerson ( )

4 Suboptimal Vitamin D a Rising SyndemicIndoor living Climate control Artificial lighting Sedentary lifestyle Poor nutrition Sunscreen and cosmetics Self-image and biological racism Hygiene Suboptimal Vitamin D a Rising Syndemic

5 Seasonality of SPTB Spontaneous Preterm BirthSPTB is associated with the season of conception Bodnar LM, Simhan HN. The prevalence of preterm birth and season of conception. Paediatr Perinat Epidemiol 2008;22: Vitamin D insufficiency Small for gestational age Sablok A, Batra A, Thariani K, et al. Supplementation of vitamin D in pregnancy and its correlation with feto-maternal outcome. Clin Endocrinol (Oxf) 2015;83: Preeclampsia Baca KM, Simhan HN, Platt RW, Bodnar LM. Low maternal 25-hydroxyvitamin D concentration increases the risk of severe and mild preeclampsia. Ann Epidemiol 2016;26:853-7 e1. 13,14 Vitamin D Levels Optimal level of >=40 ng/mL, esp. 3rd trimester Wagner CL, Baggerly C, McDonnell SL, et al. Post-hoc comparison of vitamin D status at three timepoints during pregnancy demonstrates lower risk of preterm birth with higher vitamin D closer to delivery. J Steroid Biochem Mol Biol 2015;148: Seasonality of SPTB

6 A Culture of Disparity Across the Spectrum Health outcomesSpontaneous preterm birth (SPTB) Vitamin deficiencies Sociocultural determinants of health Burden of chronic disease A Culture of Disparity

7 Nourishing RelationshipsMaternal-Placental Risk Infection and inflammation, Boyle AK, Rinaldi SF, Norman JE, Stock SJ. Preterm birth: Inflammation, fetal injury and treatment strategies. J Reprod Immunol 2017;119:62-6. Maternal stress, Fry-Johnson YW, Rowley DL. The enigma of spontaneous preterm birth. N Engl J Med 2010;362:2032. Smoking, Braillon A, Bewley S. The enigma of spontaneous preterm birth. N Engl J Med 2010;362:2032. Nutritional status, and Antony AC. The enigma of spontaneous preterm birth. N Engl J Med 2010;362:2033. Advanced maternal age. Takayama JI, Matsuo N. The enigma of spontaneous preterm birth. N Engl J Med 2010;362: Nourishing Relationships

8 Twilight of a Syndemic Overcoming Barriers to Parity Early detectionRisk stratification Assessment and diagnosis Current level of care, education and beliefs Intervention mapping Implementation of preventative strategies Twilight of a Syndemic

9 Spinning the Thread of LifeKlotho named after the Greek Fate who spins the thread of life Makoto Kuro-o, et. al., Nature, 1997 the klotho mouse exhibited shortened life span multiple age-like phenotypes dysregulation of VitD Axis Klotho is purported to have pleiotropic effects that slow the aging process. Spinning the Thread of Life M. Kuro-o, et. al., “Mutation of the mouse klotho gene leads to a syndrome resembling ageing,” Nature. 1997; 390(6655) Kurosu, Rosenblatt, Kuro-o, et. al., “Suppression of Aging in Mice by the Hormone Klotho,” Science. 2005; 309(5742)

10 Perinatal Endocrine FactorsEndocrine Fibroblastic Growth Factors and Klothos FGFR1-4 involved in embryological development via paracrine FGFs FGF19 subfamily bind to FGFR1-4 Klotho and beta-Klotho as cognate co-receptors Respond to nutritional and metabolic factors. Klotho – cognate co-receptor for fibroblastic growth factor 23 (FGF23) and is a regulator of vitamin D homeostasis and mineral metabolism beta-Klotho – acts as a cognate co-receptor for FGF19 and FGF21 binding to FGFRs, which are regulators of bile acid synthesis, and glucose and lipid metabolism, respectively. Perinatal Endocrine Factors M. Kuro-o, et. al., “Mutation of the mouse klotho gene leads to a syndrome resembling ageing,” Nature. 1997; 390(6655) Kurosu, Rosenblatt, Kuro-o, et. al., “Suppression of Aging in Mice by the Hormone Klotho,” Science. 2005; 309(5742)

11 Pre/Postnatal DevelopmentPrenatal Development Klotho expressed in placenta & involved in fetal development Klotho/FGF23 - bone mineral homeostasis and skeletal formation Beta-Klotho/FGF21 - lipid, glucose metabolism Beta-Klotho/FGF21 - bile-acid homeostasis Neurological development Postnatal Development Bone mineral homeostasis Prevents thymic involution Promotes normal reproductive development and function Increases Endothelia Function Reduces oxidative stress and insulin-like signaling Protective of stem cells Decreases over the lifespan Pre/Postnatal Development M. Kuro-o, et. al., Nature. 1997; 390(6655); Kurosu, Rosenblatt, Kuro-o, et. al., Science. 2005; 309(5742)

12 where is Klotho expressed?main expression: kidney distal convoluted tubules choroid plexus of the brain also expressed in: hypothalamus and pituitary gland parathyroid gland pancreas gonads placenta inner ear breast tissues Placenta Gonads Kidney Brain Breast where is Klotho expressed? M. Kuro-o, et. al., Nature. 1997; 390(6655); Kurosu, Rosenblatt, Kuro-o, et. al., Science. 2005; 309(5742)

13 Klotho in Normal MaternityMiranda, J., et al. (2014). "The anti-aging factor alpha-klotho during human pregnancy and its expression in pregnancies complicated by small-for-gestational-age neonates and/or preeclampsia." J Matern Fetal Neonatal Med 27(5):

14 Klotho in-vivo Stress Resilience in PregnancyMaternal Stress and Depression associated with decreased Klotho Prather, A. A., et al. (2015). "Longevity factor klotho and chronic psychological stress." Transl Psychiatry 5: e585. Klotho attenuates stem cell senescence Bian, A., et al. (2015). "Klotho, stem cells, and aging." Clin Interv Aging 10: Endothelial function Klotho’s potential role in preeclampsia Loichinger, M. H., et al. (2016). "Systemic and placental alpha-klotho: Effects of preeclampsia in the last trimester of gestation." Placenta 41: Fan, C., et al. (2016). "Clinic significance of markedly decreased alpha-klothoin women with preeclampsia." Am J Transl Res 8(5): Cecati, M., et al. (2016). "Potential Role of Placental Klotho in the Pathogenesis of Preeclampsia." Cell Biochem Biophys 74(1): Klotho in-vivo

15 Klotho in-vivo MetabolismCalcium (Ca), phosphate (Pi) and VitD homeostasis Tomiyama K, Maeda R, Urakawa I, et al. Relevant use of Klotho in FGF19 subfamily signaling system in vivo. Proceedings of the National Academy of Sciences of the United States of America 2010;107: Yoshida T, Fujimori T, Nabeshima Y. Mediation of unusually high concentrations of 1,25-dihydroxyvitamin D in homozygous klotho mutant mice by increased expression of renal 1alpha-hydroxylase gene. Endocrinology 2002;143:683-9. Kurosu H, Yamamoto M, Clark JD, et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309: Endothelial function Fukino K, Suzuki T, Saito Y, et al. Regulation of angiogenesis by the aging suppressor gene klotho. Biochemical and biophysical research communications 2002;293:332-7. Saito Y, Nakamura T, Ohyama Y, et al. In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome. Biochemical and biophysical research communications 2000;276: Normal energy metabolism Mori K, Yahata K, Mukoyama M, et al. Disruption of klotho gene causes an abnormal energy homeostasis in mice. Biochemical and biophysical research communications 2000;278: PTH, FGF23, Klotho, and Phosphate Excretion Prasad, N. and D. Bhadauria (2013). "Renal phosphate handling: Physiology." Indian J Endocrinol Metab 17(4): Klotho in-vivo

16 Klotho in-vivo Oxidative Stress and InflammationGlutathione production Xu Y, Dutcher L, Sun Z. Anti-aging gene klotho regulates cellular antioxidant capacity via binding to S-fromylglutathione hydrolase through the N285 glycosylation site (1095.3). FASEB J 2014;28:Supplement Regulation of oxidative stress Yamamoto M, Clark JD, Pastor JV, et al. Regulation of oxidative stress by the anti-aging hormone klotho. The Journal of biological chemistry 2005;280: Mitobe M, Yoshida T, Sugiura H, Shirota S, Tsuchiya K, Nihei H. Oxidative stress decreases klotho expression in a mouse kidney cell line. Nephron Experimental nephrology 2005;101:e67-74. Hsieh CC, Kuro-o M, Rosenblatt KP, Brobey R, Papaconstantinou J. The ASK1-Signalosome regulates p38 MAPK activity in response to levels of endogenous oxidative stress in the Klotho mouse models of aging. Aging (Albany NY) 2010;2: Inflammation Zhao Y, Banerjee S, Dey N, et al. Klotho depletion contributes to increased inflammation in kidney of the db/db mouse model of diabetes via RelA (serine)536 phosphorylation. Diabetes 2011;60: Suppression of the insulin/IGF-1 signaling Kurosu H, Yamamoto M, Clark JD, et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309: Klotho in-vivo

17 Achieving vitamin D SufficiencyBiomarker changes upon correction of vitamin D insufficiency and deficiency (10K IU/d for 12 weeks) Vitamin D increase p< Klotho increase p=0.019 FGF23 increase p=0.0570 IGF-1 increase p=0.0188 Insulin increase p=0.0088 HOMA insulin resistance increase p=0.0208 Total cholesterol decrease p=0.0177 Heart rate decrease p=0.0488 No significant differences in 1,25(OH)D, FGF-23, PTH, Calcium, Phosphate, SBP, DBP Achieving vitamin D Sufficiency Ancilary study (not published) to Traub, M. L., et al. (2014). "Impact of Vitamin D3 Dietary Supplement Matrix on Clinical Response." The Journal of clinical endocrinology and metabolism: jc

18 Change in Klotho with AgeChange in Klotho after Correction of Vitamin D Deficiency and Insufficiency, by age Change in Klotho with Age Ancilary study (not published) to Traub, M. L., et al. (2014). "Impact of Vitamin D3 Dietary Supplement Matrix on Clinical Response." The Journal of clinical endocrinology and metabolism: jc

19 vitamin D/ Klotho proposed modelFGF23 VDRE~~KL kidney KL mKL KL KLOTHO KL FGFR KL KL KL KL Pre-Vitamin D3 KL KL KL sKL KL KL Cholecalciferol KL KL KL sKL hepatocyte KL KL CYP27B1 CYP24A1 25(OH)D 1,25(OH)2D ≈1,25(OH)2D 24,25(OH)2D

20 Shining Light on MicroparticlesEarly Identification of Risk for SPTB is Paramount Preterm Risk Associated Biomarker Function SPTB ≤ 34 weeks (10-12 weeks gestation) SPTB (n=25) vs. Control (n=50) Biomarkers of SPTB Coagulation and wound healing Inflammation Oxidative stress Coagulation and complement interplay Compliment and adaptive immunity Fibrinolysis and anticoagulation Lipid metabolism Thyroid function Shining Light on Microparticles Differential dependency network analysis Cantonwine DE, Zhang Z, Rosenblatt K, et al. Evaluation of proteomic biomarkers associated with circulating microparticles as an effective means to stratify the risk of spontaneous preterm birth. Am J Obstet Gynecol 2016;214:631 e1- e11.

21 The proposed study will develop and standardize nutrigenomic diagnostic patterns for the prevention of SPTB in high risk populations, via: early diagnosis of SPTB, identification of nutrient biomarker patterns characterization of CMPs nutrient deficient versus sufficient status attenuation of pathophysiology of SPTB personalized nutritional strategies intervention mapping Research Goals

22 Nutrient Biomarker PatternsNutrient biomarker acquisition and analysis. ascorbic acid carotenoids, tocopherol, and retinol thiamin, riboflavin, niacin, and pyridoxal-5-phoshate folate and vitamin B12 25-OH vitamin D fatty acid concentrations calcium, magnesium, iron, iodine and zinc Nutrient Biomarker Patterns Marangoni, F., et al. (2016). "Maternal Diet and Nutrient Requirements in Pregnancy and Breastfeeding. An Italian Consensus Document." Nutrients 8(10). Bowman, G. L., et al. (2011). "Reliability and validity of food frequency questionnaire and nutrient biomarkers in elders with and without mild cognitive impairment." Alzheimer disease and associated disorders 25(1):

23 Aim1: To examine the cross-sectional relationship of nutrient status to: 1) proteomic and CMP biomarkers associated with elevated risk for SPTB, 2) gestational benchmarks, and 3) SPTB outcomes. Aim2: To use a Placenta-on-a-Chip micro-device to simulate and analyze CMP and epigenomic patterns under differential nutritional conditions to attenuate oxidative stress and inflammation, while promoting endothelial function at the utero-placental interface. Aim3: To develop nutrient biomarker pattern (NBP) for the determination of personalized nutritional status and intervention recommendation for the prevention of high-risk SPTB. Specific Aims

24 Collaborations K23 Career Development Award ApplicationWell characterized study population Banked samples – EDTA plasma (-80F) Microparticle risk stratification Nutrient biomarker acquisition and analysis Future Research Placenta-on-Chip Micro-particles – nutrient sensing and crosstalk Nutrient exchange at maternal-placental interface Epigenomics of normal maternity Collaborations

25 Dawn of a Culture of HealthMore research is needed! Dawn of a Culture of Health

26 Thank you – Grassroots Health and MUSCCollaborators Ryan Bradley ND, MPH Kevin Rosenblatt MD, PhD Nataliya Bulayeva, PhD Bradley Saul, PhD Funding and Analysis AOMA Graduate School of Integrative Medicine Bastyr University Research Institute University of Texas Health Science Center Proteomics Core Facility Diabetes Action Research and Education Foundation National Institutes of Health, National Center for Complementary and Alternative Medicine (5T32AT000815) Thank you – Grassroots Health and MUSC Acknowledgements