DISSERTATION THE EFFECTS OF DOCOSAHEXAENOIC ACID INTAKE DURING PREGNANCY AND LACTATION ON INFANT GROWTH AND NEUROCOGNITIVE DEVELOPMENT AND THE ASSOCIATED EFFECTS OF GENETIC VARIANTS OF THE FADS1 FADS2 GENE CLUSTER Submitted by Stacy Marie Miller Department of Food Science and Human Nutrition In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2014 Doctoral Committee: Advisor: Mary Harris Adam Chicco Susan Baker Melinda Frye Copyright by Stacy Marie Miller 2014 All Rights Reserved ABSTRACT THE EFFECTS OF DOCOSAHEXAENOIC ACID INTAKE DURING PREGNANCY AND LACTATION ON INFANT GROWTH AND NEUROCOGNITIVE DEVELOPMENT AND THE ASSOCIATED EFFECTS OF GENETIC VARIANTS OF THE FADS1 FADS2 GENE CLUSTER Maternal docosahexaenoic acid (DHA) intake during pregnancy and/or lactation has been positively associated with infant growth and neurocognitive development. However, randomized controlled clinical trials (RCT) report mixed results. Several RCT that failed to demonstrate an effect of DHA supplementation have found correlations between DHA status and cognitive benefits, possibly due to a failure to account for total maternal DHA intake. The majority of studies to date investigating neurocognitive development have not examined the effect of supplementing women with DHA during both pregnancy and lactation and fail to determine the effects of maternal genetic variation on infant neurocognitive development. Single nucleotide polymorphisms (SNPs) within the fatty acid desaturase (FADS)1 FADS2 gene cluster encoding for Δ5- and Δ6-desaturase enzymes were previously reported to be associated with altered omega-3 (n-3) and omega-6 (n-6) fatty acid proportions of erythrocyte, plasma phospholipids and breastmilk, possibly effecting DHA transfer to the infant. This study was conducted to determine the relationship between DHA intake in women obtaining varying amounts of DHA daily during pregnancy and lactation and infant neurocognitive development in the first year of life and the association of maternal SNPs in the FADS1 FADS2 gene cluster. One hundred and fifteen pregnant women were randomized to ii receive purified tuna oil supplement containing 300 mg of DHA and 67 mg EPA per day or an identical placebo (Sunola oil) for the last trimester of pregnancy through the first 3 months of lactation in a double-blinded placebo controlled clinical trial. Two SNPs in the FADS1 FADS2 gene cluster, rs174575 and rs174561, were genotyped from maternal DNA and erythrocyte, plasma phospholipid and breastmilk fatty acids and daily DHA intake from food frequency questionnaires (FFQ) were analyzed. Neurocognitive development of the infants was measured using the Mental Development Index (MDI) of the Bayley Scales of Infant Development III (BSID-III) at 4 and 12 months of age. Gestational length in days was calculated based upon last menstrual period and birth date. Infant birth weights and lengths were obtained from pediatric medical records at delivery and at 2 months of age. Total daily DHA intake was estimated to range from 18 mg to 1.374 g per day calculated from all sources of DHA, including food and supplementation. Data was analyzed based on treatment group, placebo versus DHA, and by total daily DHA intake broken into three groups: low = 0-299 mg per day DHA, medium = 300-599 mg per day DHA, high = ≥600 mg per day DHA. DHA portion of 2 month breastmilk fatty acids directly correlated with daily DHA intake (r=0.37, p=0.0002). Erythrocyte and breastmilk DHA proportions significantly increased in women homozygous for the major allele (SNP rs174575, p=0.0002 and p=0.030, respectively; SNP rs174561, p=0.003 and p=0.040, respectively) in the high daily DHA intake compared to the low intake group. However, daily DHA intake had no effect significantly increasing DHA proportions in women homozygous for the minor alleles of both SNPs studied. Infants born to mothers in the high DHA intake group showed significantly higher scores on the 12 month cognitive scale of the MDI of the BSID-III (p=0.018), compared to the low intake group. No significant differences were seen between treatment groups or DHA intake groups on any of the iii 4 month infant cognitive testing. Genotype had no direct effect on BSID-III scores, however, ANCOVA for 12 month cognitive MDI subtest showed a statistically significant interaction between SNP rs174575 genotype and daily DHA intake group (p=0.023). Additionally, infants born to mothers in the DHA treatment group had an increase of 4.5 days in gestational age (p=0.048) and significantly lower incidence of preterm birth (5%; n=3) compared to infants born to mothers in the control group (18%; n=10; χ2=4.97, p=0.026). No significant differences were seen between treatment groups or DHA intake groups in infant growth measurements at birth or at 2 months of age, although 2 month breastmilk DHA proportion of fatty acids was negatively correlated with 2 month weight (r= -0.22, p=0.048). An intake of 600 mg of DHA per day or greater during the third trimester of gestation throughout the first three months of breastfeeding was associated with improved infant neurocognitive development. Genetic variants of the FADS1 FADS2 gene cluster influence erythrocyte and breastmilk fatty acids, and increased DHA intake does not effectively increase DHA proportions in minor allele carriers. Additionally, DHA supplementation increased gestational length and decreased preterm birth risk, however, did not affect infant birth weights. DHA supplementation during pregnancy and lactation could be beneficial for improving the neurocognitive development of infants, however, genetic variation may affect DHA transfer to the infant. iv ACKNOWLEDGEMENTS I have received a great deal of support, encouragement and guidance from a great number of individuals. I would like to thank my entire advisory committee for the time, patience and flexibility they have had in completing this project. To my advisor, Mary Harris, I would like to extend heartfelt gratitude. Her expertise and guidance throughout the study, analysis and writing process was invaluable and I am grateful to have had her assistance. My committee member, Adam Chicco, provided crucial advice, support and direction for which I am grateful. I would also like to thank Susan Baker and Melinda Frye for their time and expertise throughout this project. I am very grateful for so many other individuals who were instrumental in making this project a success. I would like to thank Deana Davalos in the CSU Department of Psychology for her advisement, expertise, and for collecting and managing the cognitive data essential to the success of this project. Kathryn McGirr was an integral part of organizing the project, maintaining data files and providing expert assistance throughout the project. Thank you to Sherry Nash for managing the study supplements and aiding the breastfeeding coaches. Thank you to Alena Clark for your help with the study design, breastfeeding expertise and training of our coaches. To our breastfeeding coaches, Annaliese Zieler and Janice Gillespie, thank you for all of your help and continued dedication to the study. Without you, this project would not have been a success. And thank you Nicole Booker for your excitement and recruiting efforts and Ruth Inglis-Widrick for your ongoing project assistance. During my data collection, analysis, and writing, Chris Mulligan provided extensive laboratory expertise, guidance and support. Without Chris and his GC wisdom, I feel that I v would not have been able to successfully complete all aspects of this project. I am grateful for my previous employer, Aftab Ahmad, at the University of Colorado, for his support and allowing me to use the real-time PCR equipment and laboratory space that was necessary for genetic analysis. Additionally, I would like to thank John Wilson for his computer expertise and help with the CDC z-scores, and Jim zumBrunnen and the graduate students in the Colorado State University Statistical Laboratory for their unending and vital statistics guidance. Finally, I owe a great deal to my friends and family who supported me in this journey. First and foremost, I want to acknowledge my husband, Kyle. His unwavering support, perspective and encouragement was and is vital to my continued success. To my baby girl, Hadley, I appreciate your support and understanding while I wrote and was at “work”. I want to acknowledge my parents, Paul and Vicki, for their never-ending, immense support and encouragement. If it were not for them, I never would have learned to believe in myself, push myself and reach for my dreams. To my in-laws, Bruce and Debbie, your continued love and support (and Starbucks) helped me immensely, thank you. For all of my friends and family that listened to me, supported and encouraged me throughout this process, I am in debt to your assistance. And to my dogs, Marley and Dylan, thank you for your love and therapeutic runs and walks. Without the immense support from my family and friends, achieving my dreams would never have been possible. Whatever you do, do it well. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1: INTRODUCTION ....................................................................................................1