Maternal plasma n-3 and n-6 polyunsaturated fatty acids during pregnancy and features of fetal health: Fetal growth velocity, birth weight and duration of pregnancy

Accepted Manuscript Maternal plasma N-3 and N-6 polyunsaturated fatty acids during pregnancy and features of fetal health: Fetal growth velocity, birth weight and duration of pregnancy Nina H. Grootendorst-Van Mil, MD PhD, Henning Tiemeier, MD PhD, Jolien Steenweg-De Graaff, PhD, Berthold Koletzko, MD PhD, Hans Demmelmair, PhD, Vincent W.V. Jaddoe, MD PhD, Eric A.P. Steegers, MD PhD, Régine P.M. SteegersTheunissen, MD PhD PII:

S0261-5614(17)30221-2

DOI:

10.1016/j.clnu.2017.06.010

Reference:

YCLNU 3167

To appear in:

Clinical Nutrition

Received Date: 23 February 2017 Revised Date:

12 May 2017

Accepted Date: 5 June 2017

Please cite this article as: Grootendorst-Van Mil NH, Tiemeier H, Steenweg-De Graaff J, Koletzko B, Demmelmair H, Jaddoe VWV, Steegers EAP, Steegers-Theunissen RPM, Maternal plasma N-3 and N-6 polyunsaturated fatty acids during pregnancy and features of fetal health: Fetal growth velocity, birth weight and duration of pregnancy, Clinical Nutrition (2017), doi: 10.1016/j.clnu.2017.06.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

MATERNAL PLASMA N-3 AND N-6 POLYUNSATURATED FATTY ACIDS DURING PREGNANCY AND FEATURES OF FETAL HEALTH: FETAL GROWTH VELOCITY, BIRTH

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WEIGHT AND DURATION OF PREGNANCY

Nina H. GROOTENDORST-VAN MIL, MD PhD1,2,3, Henning TIEMEIER, MD PhD2,4,5, Jolien

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STEENWEG-DE GRAAFF, PhD1,2, Berthold KOLETZKO, MD PhD6, Hans DEMMELMAIR,

STEEGERS-THEUNISSEN, MD PhD3

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PhD6, Vincent W.V. JADDOE, MD PhD1,7, Eric A.P. STEEGERS, MD PhD3, Régine P.M.

The Generation R Study Group, 2Department of Child and Adolescent Psychiatry, 3Department

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Department of Paediatrics

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of Obstetrics and Gynaecology, 4Department of Epidemiology, 5Department of Psychiatry

Erasmus MC, University Medical Center Rotterdam Dr. Molewaterplein 50

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3015 GE Rotterdam, The Netherlands

Div. Metabolic and Nutritional Medicine, Dr. Von Hauner Children's Hospital, Ludwig-

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Maximilians-University, Munich, Germany.

ACCEPTED MANUSCRIPT 2 Corresponding Author Régine Steegers-Theunissen, MD, PhD Professor in Periconception Epidemiology

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Erasmus MC, University Medical Center Department of Obstetrics and Gynaecology P.O. Box 2040, 3000 CA Rotterdam

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Tel. +31 10 70 43598

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E-mail: [email protected]

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Short Title: pregnancy n-3 and n-6 fatty acids and birth outcomes

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ABSTRACT

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Background & Aims: Maternal fatty acids are essential for fetal growth and development. Here,

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we examine associations between maternal mid-pregnancy plasma n-3 and n-6 polyunsaturated

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fatty acids (PUFAs) and fetal health determined by fetal growth velocity, birth weight and

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duration of pregnancy.

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Methods: Participants were 6,974 pregnant women and their infants from a population-based

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birth cohort, the Generation R Study. Maternal plasma n-3:n-6 PUFA ratio and n-3 and n-6

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PUFA percentage in glycerolphospholipids in mid-pregnancy were related to fetal growth

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velocity calculated from repeatedly measured weight, length and head circumference, birth

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weight, and duration of pregnancy.

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Results: A higher maternal mid-pregnancy n-3:n-6 PUFA ratio was associated with a higher

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growth velocity of the fetal weight (β=0.082 SD-score/week, 95%CI 0.055; 0.108, P<0.001),

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length (β=0.085 SD-score/week, 95%CI 0.052; 0.119, P<0.001); and head (β=0.055 SD-

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score/week, 95%CI 0.019; 0.091, P=0.003).

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We also observed positive associations between n-3:n-6 PUFA ratio and birth weight (β=0.76

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SD-score, 95%CI 0.22; 1.29, P=0.006), and duration of pregnancy (β =1.32 weeks, 95%CI 0.24;

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2.40, P=0.02).

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Conclusions: These results are consistent with the hypothesis that a higher n-3:n-6 PUFA ratio is

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important for fetal health.

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• Keywords birth weight, fatty acids, fetal growth, population-based cohort, pregnancy

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INTRODUCTION

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Derangements in fetal growth, low birth weight and preterm birth are features of fetal health and

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the leading causes of infant death and long-term metabolic and neurological disabilities (1). Low

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birth weight can be the result of preterm birth or fetal growth restriction or both. A better

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understanding of the factors that underlie preterm or small-for gestational age (SGA) births can

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help to improve fetal health and reduce mortality, morbidity and associated health care costs. Even in well-nourished populations, the quality of dietary patterns is associated with the

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course and outcome of pregnancy (2, 3). As humans are incapable of synthesizing

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polyunsaturated fatty acids (PUFAs) they depend on dietary intake. As these PUFAs have

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implications for many different biological properties including energy storage, oxygen transport,

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conformation of cell membranes, and regulation of cell proliferation and inflammation, they are

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considered as potential biomarkers and determinants of preterm and SGA births (4, 5).

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These studies, that mostly relied on estimated intake of fatty acids, suggest that in

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particular excessive intake of n-6 PUFAs relative to n-3 PUFAs may increase the probability of

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adverse pregnancy outcomes (6), although results are not consistent. Observational studies have

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associated higher intakes of n-3 PUFA during pregnancy with a longer duration of pregnancy (7).

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Different meta-analyses confirmed the results on higher birth weight and length of gestation after

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n-3 PUFA supplementation during pregnancy (8, 9). The use of n-3 PUFA supplements to reduce

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the risk of adverse birth outcomes, however, is not generally accepted (10). In addition, no

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consensus about the type, dose, timing and duration of the used n-3 PUFA supplements have

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been reached.

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Against this background, we aimed to explore the association between the percentage of maternal plasma n-3 and n-6 PUFAs in glycerolphospholipids and features of fetal health. In a

ACCEPTED MANUSCRIPT 5 large population-based cohort study, percentage of n-3 and n-6 PUFAs in glycerolphospholipids

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were measured in mid-pregnancy and related to duration of pregnancy, birth weight and

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parameters of fetal growth (weight, length and head circumference) based on three repeated

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ultrasounds in early, mid and late pregnancy. As a higher maternal n-3:n-6 PUFA ratio has been

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reported to predict the infant size at birth, we hypothesised that a higher maternal n-3:n-6 PUFA

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ratio is positively associated with the fetal growth velocity from mid-pregnancy onward.

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ACCEPTED MANUSCRIPT 6

METHODS

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Participants

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This study was embedded in the Generation R Study, an ongoing population-based birth cohort

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from early fetal life onward. The Generation R Study, designed to identify early environmental

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and genetic determinants of growth, development and health, has been previously described in

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detail (11). Eligible mothers were those who were resident in the city of Rotterdam, The

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Netherlands, at the moment of delivery and had an (expected) delivery date from April 2002 until

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January 2006. Enrolment of mothers was aimed in early pregnancy (gestational age until 17

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weeks), but was possible until birth of their child. Midwifes and obstetricians gave eligible

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mothers at their first prenatal visit during routine antenatal care verbal information about the

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study, handed out an information package and asked these mothers to make an appointment for

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the first ultrasound examination. During the study programme, assessments with physical

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examinations, biological samples and detailed questionnaires are repeatedly performed.

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Of 8,663 mothers enrolled in the study before < 25 weeks pregnancy, percentage of n-3 and n-6

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PUFA in glycerolphospholipids were available for 7,186 mothers (82.9%). Since growth

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potentials for the individual fetus in multiple pregnancies are not comparable to singleton

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pregnancies, we excluded twin pregnancies (n=146). In total, information on duration of

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pregnancy and birth weight was available for 6,974 mother-infant pairs in singleton pregnancies.

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The study has been approved by the Medical Ethics Committee of the Erasmus MC, University

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Medical Center, in Rotterdam, The Netherlands. Written informed consent was obtained from all

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participants.

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ACCEPTED MANUSCRIPT 7 Maternal plasma PUFAs during pregnancy

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Our study aimed to explore opportunities of earlier intervention during pregnancy and therefore

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focused on mid-pregnancy PUFA status. The second trimester is of relevance for PUFA

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metabolism because during that period, maternal DHA status begins to decline when DHA stores

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are mobilized and transferred to the fetus (12). In mid-pregnancy (median: 20.5 weeks of

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pregnancy; 90% range: 18.8–22.9 weeks), venous samples were drawn and stored at room

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temperature before being transported to the regional laboratory for processing and storage for

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future studies. Processing was aimed to finish within a maximum of 3 hours after venipuncture.

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The samples were centrifuged and thereafter stored at –80ºC (without anti-oxidant). To analyse

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fatty acid concentrations, EDTA plasma samples were picked and transported to the Division of

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Metabolic Diseases and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of

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Munich Medical Centre, in 2010. After being thawed, the analysis of plasma glycerophospholipid

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fatty acid composition performed using gas chromatography by a sensitive and precise high-

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throughput method described recently by Glaser et al. (13). This study describes the PUFA

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content of plasma glycerophosphocholine, which was by far the most abundant plasma

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glycerophospholipid, to be similar to red blood cell glycerophosphocholine PUFA content.

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Therefore, it can be expected that glycerophospholipids PUFA composition is representative of

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the FA status in a variety of physiological and pathological conditions, including pregnancy (13)

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and can be seen as a valid measure of PUFA status in the current study population.

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PUFAs are expressed as percentage by weight (% wt/wt) of all glycerophospholipid fatty acids

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detected with a chain length between 14 and 22 carbon atoms.

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The available essential PUFAs and their desaturated/ elongated deratives were used for

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calculation of the n-3:n-6 PUFA ratio by dividing ∑ n3 PUFAs (C18:3n3, C20:3n3, C20:5n3,

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C22:5n3, and C22:6n3) by ∑ n6 PUFAs (C18:2n6, C18:3n6, C20:2n6, C20:3n6, C20:4n6,

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C22:4n6, and C22:5n6) (Table S1).

100 Fetal growth

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Fetal ultrasound examinations were carried out in two dedicated research centers in early (median

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12.4 weeks of pregnancy, 95% range 10.7–13.9), mid (median 20.5 weeks of pregnancy, 95%

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range 18.5–23.5) and late pregnancy (median 30.3 weeks of pregnancy, 95% range 28.3–33.0).

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To assess gestational age, crown-rump length (until a gestational age of 12 weeks and 5 days) or

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biparietal diameter (from 12 weeks and 5 days onwards) were measured by fetal ultrasound

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examination. The methods for the assessments of gestational age and the standard ultrasound

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planes for fetal measurements have been described previously (14). Inter- and intra-observer

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intra-class correlation coefficients, defined as the ratio of the variance between subjects to total

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variance, were all >0.98 (14).

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Fetal head circumference, abdominal circumference and femur length were measured to the

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nearest millimeter using standardised ultrasound procedures (15). Estimated fetal weight was

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calculated using the formula of Hadlock with parameters head circumference (HC), abdominal

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circumference (AC) and femur length (FL) in cm: estimated fetal weight =

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10 × (1.326 − 0.00326 × AC × FL + 0.0107 × HC + 0.0438 × AC + 0.158 × FL). Gestational-age-

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adjusted standard deviation scores (SD-score), representing the equivalent of z-scores, were

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constructed for all fetal growth measurements based on reference growth curves from the whole

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study population (15).

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Pregnancy outcome

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ACCEPTED MANUSCRIPT 9 Information about infant gender, duration of pregnancy, and weight, length and head

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circumference at birth and placental weight was obtained from community midwifery and

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hospital registries. Birth weight (n=6,974) and placental weight (n=5,100) were measured

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immediately after delivery and expressed in kilograms (kg).

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We expressed birth weight by gestational age and gender adjusted SD-scores that were

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constructed based on the distributions in the Generation R cohort (15). Fewer measurements of

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length and head circumference than of birth weight were available because these were not

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routinely measured at birth (n=4,409 and n=3,771 for length and head circumference at birth,

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and=6,974 for birth weight, respectively in the current study population). Preterm birth was

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defined as a duration of pregnancy of <37 weeks at birth (n=369, 5.3%). Information on

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spontaneous and induced delivery was available from medical records. SGA was defined as a

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gestational-age-adjusted birth weight below the 10th percentile (-1.4 SD-score) in the study

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cohort.

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Covariates

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Possible confounders of the association between maternal PUFAs and pregnancy outcomes that

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were derived from the literature (16) are the following: At enrollment (median gestational age

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14.7 weeks (SD 3.6)) we obtained information on maternal age, national origin, educational level,

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parity, prenatal smoking, alcohol use and folic acid supplement use by self-report questionnaires.

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National origin of the mother was based on the country of birth of her parents. This classification

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has been composed by Statistics Netherlands as method of choice to classify common ethnic

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groups in the Netherlands. Educational level of the pregnant woman was assessed by the highest

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completed education and categorised as primary school only, secondary school or higher

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education. Maternal prenatal smoking and alcohol use were classified as ‘no use’, ‘use until

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ACCEPTED MANUSCRIPT 10 pregnancy was confirmed’ and ‘continued use during pregnancy’. Folic acid supplement use was

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classified as ‘no use,’ ‘use started during the first 10 weeks of pregnancy’ or ‘use started

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preconceptional’. Information on pregnancy complications, e.g. gestational diabetes, pregnancy

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induced hypertension or pre-eclampsia, was obtained from community midwifery and hospital

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registries. To assess psychological symptoms, mothers completed the Brief Symptom Inventory

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at 20 weeks of pregnancy, which is a validated self-report questionnaire consisting of 53 items

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(17). All covariates passed the 5% threshold of change in effect estimated for the association

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between n3-:n-6 PUFA ratio with birth weight (18). The same selection of covariates was entered

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a priori in all analyses.

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153 Statistics

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First, to test our main hypothesis, we performed analyses of the association between n-3:n-6

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PUFA ratio with growth velocity using repeatedly measured fetal weight, length and head

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circumference as assessed during pregnancy and at birth. These outcome variables are referred to

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as growth velocity of the fetal weight, length and head throughout the manuscript.

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For the repeated measurement analyses Linear Mixed Models were used. Mixed models have the

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advantage to allow correlated random effects in individuals and to accommodate missing data

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points of the repeated measurements (19).

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Because body length cannot be measured reliable by fetal ultrasound, we used SD scores of

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femur length as well as total length at birth in our growth models of fetal length. As these

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measurements reflect different body parts we also analyzed the association of the n-3:n-6 PUFA

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ratio with fetal length growth velocity as indicated by femur length in mid and late pregnancy

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only.

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ACCEPTED MANUSCRIPT 11 Second, we examined the association between n-3:n-6 PUFA ratio with birth weight and duration

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of pregnancy as continuous outcomes using regular multivariable linear regression analyses. To

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examine the independent effects of maternal PUFAs on duration of pregnancy we additionally

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adjusted the analyses for birth weight. The population for the analyses of the association between

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PUFAs and duration of pregnancy was restricted to women who gave birth after spontaneous

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onset of labor. This restriction might introduce bias as the study population may include a

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selection of healthier women. To test whether this has influenced our results we also examined

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the association between the maternal n-3:n-6 PUFA ratio and duration of pregnancy in the total

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study population (n=6,974).

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We chose a hierarchical approach to reduce the risk of type I error and performed an initial

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overall analysis of the calculated maternal n-3:n-6 PUFA ratio. If any association between n-3:n-

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6 PUFA ratio with birth weight or duration of pregnancy in the overall analyses was observed

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(first level), the possible specific effects of total n-3, total n-6 PUFAs and individual n-3 and n-6

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PUFAs and on these pregnancy outcomes (second level) were tested. To disentangle the effect of

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duration of gestation from that of intrauterine growth, birth weight is analysed using SD-scores

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adjusted for sex and gestational age. However, there is some evidence that vice versa, intrauterine

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growth is associated with the duration of pregnancy (20).

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Subsequently, if any association between maternal n-3:n-6 PUFA ratio with the continuous

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outcomes birth weight and duration of pregnancy was established, we ran logistic regression

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analyses using dichotomous outcomes for clinical interpretation. To this aim we then explored

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whether the maternal n-3:n-6 PUFA ratio was associated with adverse pregnancy outcomes using

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the dichotomous outcomes SGA (SD-score birth weight < p10) and preterm birth (<37 weeks

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duration of pregnancy).

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ACCEPTED MANUSCRIPT 12 To give more insight in the shape of the association between n3:n6 PUFA ratio and the

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investigated outcomes, we also provided results of analyses with n3:n6 PUFA ratio divided in

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quintiles. The highest n3:n6 PUFA ratio was used as the reference category.

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In supplemental analyses, we present the association between n-3:n-6 PUFA ratio and placental

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weight as the placenta may play a role in initiating the mobilization of fatty acids from the

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maternal adipose tissue (21).

In a sensitivity analyses, we included only women of Dutch national origin (n=3.439,

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50.1%), as intake of PUFAs may differ between ethnic subgroups. This group is the largest ethnic

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subgroup in our study and consists of women with both of her parents born in the Netherlands.

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All analyses were adjusted for all potential confounders. Missing data on covariates was handled

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in two ways: for categorical variables, the missing value was recoded as a dummy variable; for

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continuous variables, the missing item was replaced by the mean value. Data on exposure (n-3

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and n-6 PUFA’s) or outcome (birth weight) was not imputed, as availability of these data was a

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criterion for selection of the population for analyses. If one or more of the data points of the

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growth measurements was missing, this was accommodated by the linear mixed model as

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described above. Analyses were performed using SPSS software, version 21.0 (IBM-SPSS,

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Chicago, IL, USA).

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ACCEPTED MANUSCRIPT 13 RESULTS

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Descriptive statistics

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Characteristics of mothers and infants in the study are presented in Table 1. Mothers had a mean

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age of 29.8 (SD 5.2) years, 50.8% was higher educated and 18.7% continued smoking during

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pregnancy. Infants were born after a median of 40.1 (interquartile range 1.9) weeks of pregnancy

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and the average birth weight was 3.4 (SD 0.6) kg.

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214 Maternal plasma n-3 and n-6 PUFAs and fetal growth velocity

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Table 2 shows the association between maternal n-3:n-6 PUFA ratio and growth velocity of fetal

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weight, length and head based on measurements in mid and late pregnancy and at birth. A higher

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n-3:n-6 PUFA ratio was associated with a higher fetal growth velocity from mid pregnancy

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onwards, indicated by the slope of fetal weight, length and head circumference. The association

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with growth velocity of the fetal weight delineates a dose-depend association of the maternal n-

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3:n-6 PUFA ratio with this growth velocity.

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Growth velocity of fetal length was built up using both femur length in mid and late pregnancy

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and total body length at birth. To rule out the possibility that the association between n-3:n-6

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PUFA ratio and fetal length was driven by fetal body length, we repeated the analyses removing

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total body length at birth. We found consistent results of the association of the n-3:n-6 PUFA

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ratio and fetal length excluding the measure of body length at birth: Slope (SD-score/week) β

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0.086, 95%CI 0.052; 0.119, P<0.001).

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In an additional analysis, where analyses were rerun among Dutch women only, results did not

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change materially although the associations were less clear for length and head circumference

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(Table S2).

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ACCEPTED MANUSCRIPT Table 1. Maternal and infant characteristicsa

14 All women

Dutch women only

(n=6,974)

(n=3,325)

Age, years

29.8 (5.2)

31.3 (4.4)

Gestational age at blood sampling, weeks

20.7 (1.2)

20.6 (1.1)

Maternal characteristics

NA

Dutch

50.9

other Western

11.2

non Western

37.9

Parity (% primiparous)

59.9

24.8 (4.5)

Educational level (%) 6.0

Secondary

43.2

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Primary

High

24.3 (4.1)

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Body Mass Index, kg/m

55.6 2

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National origin (%)

0.5

33.6

50.8

65.9

72.4

73.2

8.8

9.3

18.7

17.5

43.0

56.2

Started during the first 10 weeks of pregnancy

31.9

33.0

Never

25.1

10.8

73.6

70.7

14.0

16.3

12.4

13.0

1.9

1.6

3.7

5.1

Gender (% boys)

50.5

50.6

Birth weight, kg

3.4 (0.6)

3.5 (0.6)

Gestational age at birth, weeks (median, interquartile range)

40.1 (1.9)

40.3 (1.8)

Smoking during pregnancy (%) Never Until pregnancy was recognized Continued Folic acid supplement use (%)

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Started preconceptional

Mode of delivery (%) Spontaneous

Caesarean section Pre-eclampsia (% yes)

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Instrumental

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Pregnancy-induced hypertension (%) Infant characteristics

a

Values represent means (SD) unless otherwise indicated. *Differences in characteristics between mother-child pairs of Dutch (n=3.325) and non-

Dutch national origin (n=3.649).

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ACCEPTED MANUSCRIPT 15 Table 2. Associations of maternal mid-pregnancy plasma n-3:n-6 PUFA ratio with features of fetal growth velocity (all women) N=6,974

Growth velocity of the fetal weight Change in (estimated) fetal weight (SD-score) a P-valueb

Intercept

Slope (SD-score/week

P-value b

n-3:n-6 PUFA ratio, SD

-0.119 (-0.163; -0.074)

<0.001

n-3:n-6 PUFA ratio, quintiles

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(95% CI)) 0.004 (0.003; 0.005)

<0.001

0.290 (0.150; 0.430)

<0.001

-0.010 (-0.014; -0.006)

<0.001

2 (1:7-1:6)

0.225 (0.087; 0.363)

0.001

-0.009 (-0.013; -0.005)

<0.001

3 (1:6-1:5)

0.229 (0.093; 0.365)

0.001

-0.007 (-0.011; -0.003)

<0.001

4 (1:5-1:4)

0.109 (-0.028; 0.245)

0.12

-0.005 (-0.009; -0.001)

0.02

5 (1:4-1:2)

reference

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1 (1:14-1:7)

reference

N=6,961

Growth velocity of the fetal length

Intercept

n-3:n-6 PUFA ratio, SD

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Change in fetal length (SD-score) a P-valueb

Slope (SD-score/week

P-value b

(95% CI))

-0.124 (-0.176; -0.071)

<0.001

-0.004 (0.002; 0.006)

<0.001

1 (1:14-1:7)

0.332 (0.168; 0.495)

<0.001

-0.011 (-0.016; -0.006)

<0.001

2 (1:7-1:6)

0.290 (0.129; 0.451)

<0.001

-0.010 (-0.015; -0.005)

<0.001

3 (1:6-1:5)

0.225 (0.066; 0.384)

0.006

-0.007 (-0.012; -0.002)

0.005

0.77

-0.002 (-0.007; 0.003)

0.49

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n-3:n-6 PUFA ratio, quintiles

4 (1:5-1:4)

0.024 (-0.134;0.183)

5 (1:4-1:2)

reference

N=6,954

reference

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Growth velocity of the fetal head

Change in fetal head circumference (SD-score) a

Intercept

Slope (SD-score/week

P-value b

(95% CI))

-0.083 (-0.137; -0.028)

0.003

0.175 (0.004; 0.345)

0.04

-0.005 (-0.011; 0.000)

0.06

0.160 (0.004; 0.345)

0.07

-0.006 (-0.011; -0.001)

0.046

0.114 (-0.053; 0.281)

0.18

-0.005 (-0.010; 0.001)

0.08

0.009 (-0.157; 0.178)

0.92

-0.008 (-0.006; 0.005)

0.79

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n-3:n-6 PUFA ratio, SD

P-valueb

0.003 (0.001; 0.004)

0.003

n-3:n-6 PUFA ratio, quintiles 1 (1:14-1:7) 2 (1:7-1:6) 3 (1:6-1:5) 4 (1:5-1:4) a

Values are based on Linear Mixed Models and reflect the change in fetal growth velocity in age-adjusted SD-score/ week based on measurements in mid and b late pregnancy and at birth, per maternal n-3:n-6 fatty acid quintile compared to the reference group of n-3n-6 fatty acid ratio in the highest quintile. P–value reflects the significance level of the estimate. Analyses are adjusted for gestational age at blood sampling, infant gender, maternal age, national origin, parity, educational level, body mass index, alcohol use, smoking, folic acid supplement use, psychological symptoms and pregnancy complications.

Using the maternal n-3:n-6 PUFA ratio as the independent variable, the model can be written as follows: SD-score of head size= β0 + β1 × gawks +β2 × n-3:n-6 ratio + β3 × gawks × n-3:n-6 PUFA ratio + ε (β0 = intercept; gawks = gestational age in weeks; ε includes additional covariates for the adjusted model and error; similarly, formula for n-3 and n-6 PUFAs as the independent variable, or estimated fetal weight or length as dependent variables, can be defined). The term ‘β2 × n-3:n-6 PUFA ratio ’ tests the difference in intercept for each 1-SD higher n3:n6 PUFA ratio or comparing the n-3:n-6 PUFA ratio quintile group to the reference group (quintile 5).

ACCEPTED MANUSCRIPT 16 233 Maternal plasma n-3 and n-6 PUFAs and pregnancy outcome

235

The associations between maternal n-3:n-6 PUFA ratio in mid-pregnancy and birth weight are

236

shown in Table 3. A higher n-3:n-6 PUFA ratio of the mothers during pregnancy was associated

237

with a higher infant birth weight. When analysing the quintiles of the n-3:n-6 PUFA ratios, the

238

association with birth weight was most profound in the highest quintiles (1 and 2). The observed

239

difference in birth weight between the first and reference quintile equals 170 g at term.

240

When n-3 and n-6 PUFAs were analyzed separately, only n-3 PUFAs were positively associated

241

with birth weight. In particular the n-3 PUFA Docosahexaenoic acid (DHA, C22:6n-3), and n-6

242

PUFA Arachidonic acid (AA, C20:4n6) and Dihomogamma-linolenic acid (DGLA, C20:3n6)

243

seem to explain the association between n-3:n-6 PUFA ratio and birth weight (Table 3).

244

When comparing with unadjusted estimates, the effect of adjustment for socio-economic

245

covariates on the effect estimates in the association with birth weight varies quite substantially

246

between the specific PUFAs (Table S3).

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The association of n-3:n-6 ratio and the specific n-3 and n-6 PUFAs with placental weight showed a consistent pattern as the results for birth weight (Table S4). A higher n-3:n-6 PUFA

249

ratio was associated with a lower risk of SGA (SD-score p<10) (Table S5).

250

A higher maternal n-3:n-6 PUFA ratio was related to a longer duration of pregnancy, in women

251

who gave birth after spontaneous onset of delivery (Table 4). The results were independent of the

252

effects of the maternal PUFAs on birth weight. This observation was partly explained by higher

253

percentages of n-3 PUFA in glycerolphospholipids, in particular higher DHA percentages.

254

In the total study population, not restricted to spontaneous onset of delivery the association

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between the maternal n-3:n-6 PUFA ratio and duration of pregnancy was comparable to results in

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ACCEPTED MANUSCRIPT 17 the original, restricted study population (β 1.51, 95%CI 0.57; 2.46, P=0.04, Dutch women: β

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1.42, 95%CI 0.12; 2.72, P=0.03, other results not shown).

258

A higher percentage of n-3 PUFA in glycerolphospholipids was also related to a lower risk of

259

preterm birth in the total study population (<37 weeks gestational age; All pregnancies with

260

exclusion of those complicated by pre-clampsia and diabetes gravidarum: adjusted odds ratio

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0.57, 95%CI 0.35; 0.91, P=0.02, other associations not tested). After adjustment for potential

262

confounders, effect estimates were reduced by 15-35%.

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ACCEPTED MANUSCRIPT 18 263 264 Table 3. Associations of maternal mid-pregnancy plasma PUFAs with infant birth weight a Birth weight SD-score

Beta (95%CI)

P-value

n-3:n-6 PUFA ratio n-3:n-6 PUFA ratio, SD

0.04 (0.01; 0.06)

0.006

1 (1:14-1:7)

-0.12 (-0.20; -0.04)

0.005

2 (1:7-1:6)

-0.11 (-0.19; -0.04)

0.003

3 (1:6-1:5)

-0.06 (-0.13; 0.02)

0.14

4 (1:5-1:4)

-0.10 (-0.17; -0.03)

5 (1:4-1:2)

reference

n-3 PUFAs

Beta (95%CI)

0.06 (0.02; 0.09)

0.008

P-value

0.003

-0.18 (-0.31; -0.06)

0.005

-0.13 (-0.24; -0.03)

0.02

-0.04 (-0.14; 0.05)

0.38

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n-3:n-6 PUFA ratio, quintiles

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Maternal fatty acids, wt%

Dutch women only (n=3,325)

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All women (n=6,974)

b

-0.09 (-0.18; 0.00)

0.06

reference

0.09 (0.01; 0.18)

0.04

0.18 (0.05; 0.30)

0.007

C18:3n-3

0.17 (-0.06; 0.41)

0.15

0.12 (-0.23; 0.47)

0.48

C20:3n-3

0.77 (-0.27; 1.80)

0.15

0.68 (-0.84; 2.20)

0.38

0.06 (-0.02; 0.14)

0.12

0.08 (-0.02; 0.19)

0.13

0.10 (0.00; 0.20)

0.04

0.20 (0.05; 0.34)

0.009

0.02 (-0.01; 0.04)

0.12

0.04 (0.01; 0.07)

0.02

-0.02 (-0.08; 0.03)

0.44

-0.08 (-0.16; 0.01)

0.09

-0.00 (-0.01; 0.01)

0.57

-0.01 (-0.02; 0.00)

0.16

0.11 (-0.52; 0.74)

0.73

0.45 (-0.47; 1.37)

0.34

0.19 (-0.06; 0.43)

0.13

-0.12 (-0.51; 0.27)

0.56

0.10 (0.07; 0.13)

<0.001

0.08 (0.03; 0.13)

0.001

-0.06 (-0.07; -0.04)

<0.001

-0.04 (-0.07; -0.02)

0.001

0.10 (-0.10; 0.31)

0.34

0.28 (-0.05; 0.60)

0.09

0.04 (-0.09; 0.18)

0.51

0.12 (-0.09; 0.34)

0.26

C20:5n-3 C22:5n-3 C22:6n-3

n-6 PUFAs

C18:2n-6 C18:3n-6 C20:2n-6 C20:3n-6 C20:4n-6 C22:4n-6 C22:5n-6 a

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Total n-6 PUFAs

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Total n-3 PUFAs

Results from multivariable linear regression models. Analyses are adjusted for gestational age at maternal blood sampling, child gender, maternal age, parity, educational level, body mass index, alcohol use, smoking, folic acid supplement use, psychological symptoms and b pregnancy complications. Also adjusted for maternal national origin.

ACCEPTED MANUSCRIPT 19 265 266 Table 4. Associations of maternal mid-pregnancy plasma n-3 and n-6 PUFAs with the duration of pregnancy

Maternal PUFA, wt%

Beta (95%CI)

P-value

n-3:n-6 PUFA ratio n-3:n-6 PUFA ratio, SD

0.06 (0.01; 0.12)

0.02

1 (1:14-1:7)

-0.11 (-0.28; 0.05)

0.18

2 (1:7-1:6)

-0.15 (-0.30; 0.00)

0.06

3 (1:6-1:5)

0.04 (-0.11; 0.19)

0.56

4 (1:5-1:4)

0.01 (-0.14; 0.16)

5 (1:4-1:2)

reference

n-3 PUFAs

Beta (95%CI)

0.07 (-0.01; 0.14)

0.93

P-value

0.07

-0.08 (-0.34; 0.19)

0.58

-0.17 (-0.38; 0.05)

0.13

0.01 (-0.20; 0.21)

0.96

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n-3:n-6 PUFA ratio, quintiles

Dutch women only (n=2,171) a

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All women (n=4,678)

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Duration of pregnancy, weeks a,b

-0.02 (-0.21; 0.18)

0.87

reference

0.23 (0.05; 0.41)

0.01

0.32 (0.06; 0.58)

0.01

C18:3n3

0.15 (-0.33; 0.63)

0.53

0.14 (-0.58; 0.86)

0.70

C20:3n3

1.99 (-0.11; 4.10)

0.06

2.03 (-1.14; 5.19)

0.21

0.07 (-0.08; 0.22)

0.37

0.18 (-0.02; 0.38)

0.08

0.12 (-0.08; 0.31)

0.26

0.21 (-0.08; 0.51)

0.16

0.06 (0.02; 0.11)

0.008

0.08 (0.01; 0.15)

0.02

-0.08 (-0.19; 0.03)

0.14

0.03 (-0.15; 0.20)

0.77

-0.00 (-0.02; 0.02)

0.73

0.02 (-0.01; 0.05)

0.19

-0.86 (-2.12; 0.40)

0.18

-1.45 (-3.29; 0.38)

0.12

0.37 (-0.13; 0.86)

0.15

1.12 (0.32; 1.93)

0.006

-0.02 (-0.08; 0.05)

0.59

-0.06 (-0.16; 0.05)

0.27

-0.01 (-0.04; 0.02)

0.55

-0.03 (-0.08; 0.02)

0.30

-0.20 (-0.61; 0.21)

0.35

-0.19 (-0.85; 0.46)

0.57

0.04 (-0.23; 0.30)

0.78

-0.06 (-0.50; 0.38)

0.79

C20:5n3 C22:5n3 C22:6n3

n-6 PUFAs

C18:2n6 C18:3n6 C20:2n6 C20:3n6 C20:4n6 C22:4n6 C22:5n6 a

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Total n-6 PUFAs

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Total n-3 PUFAs

Population for analyses is restricted to women who gave birth after spontaneous onset of labor. Values are based on linear regression models. Analyses are adjusted for gestational age at blood sampling, infant gender, birth weight SD-score, maternal age, parity, educational level, body b mass index, alcohol use, smoking, folic acid supplement use, psychological symptoms and pregnancy complications. Also adjusted for maternal national origin.

ACCEPTED MANUSCRIPT 20 DISCUSSION

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In this large population-based prospective study, we found that a higher maternal n-3:n-6 PUFA

269

ratio was associated with a better fetal health, determined by a higher fetal growth velocity

270

already from mid-pregnancy onward, a higher birth weight and a longer duration of pregnancy.

271

These findings seem to be partly accounted for by higher percentage of maternal mid-pregnancy

272

n-3 PUFA in glycerolphospholipids.

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Our main finding is that maternal PUFAs were related to growth velocity of the fetus.

274

From the second half of pregnancy, an exponentially increase in the percentage of PUFA has

275

been observed in the fetal circulation, which mostly derived from maternal blood due to a

276

transplacental gradient in favor of the fetus (21). This increase in fetal PUFAs coincides with the

277

strongest increase of fetal growth and is likely to fulfill the requirements of accelerated cell

278

division. Although adipose tissue constitutes 14% of birth weight, almost half of the variation of

279

birth weight is determined by total fat mass (22). Low birth weight children were observed to

280

have lower n-3 and n-6 PUFA status as compared to normal weight neonates (23). The fetus uses

281

its adipose tissue to store DHA, and after birth this fatty acid is quickly mobilised to the

282

circulation suggesting that fatty acids are stored to ensure optimal development during the critical

283

neonatal phase (21).

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The mechanisms by which maternal fatty acids can affect fetal growth are not clear. Fatty

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284 285

acids of the n-3 and n-6 PUFA families play major roles implicated in fetal growth including

286

energy storage, oxygen transport and cell membrane functioning. Furthermore, these PUFAs

287

have roles in inflammation and are suggested to act as important mediators of gene expression

288

(24).

289 290

The observation of the apparent opposite effects of the n-6 PUFAs DGLA and AA on birth weight have been reported previously (16, 25). Both PUFAs are suggested to have competing

ACCEPTED MANUSCRIPT 21 291

effects on inflammation as the anti-inflammatory effect of DGLA is influenced by a competitive

292

inhibition by AA (26). Evidence pointing to the association between diet-induced inflammation

293

and fetal myogenesis and adipogenesis is emerging (27). The close correlation between placental and birth weight illustrates the main importance of

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294

placental function for the growth and development of the fetus (28). As the placenta is an organ

296

of extra-embryonic origin, the observed associations with placental weight might also reflect

297

effects early in pregnancy. We describe the patterns of the relationship of n-3 and n-6 PUFAs

298

with placental weight similar to our findings with birth weight. Apart from maternal dietary

299

intake, the fetal supply of fatty acids also depends on the fatty acid release from the maternal

300

adipose tissue and the placental transport (21).

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We furthermore observed that a higher percentage of n-3 PUFAs in glycerolphospholipids, in particular DHA, was associated with a longer duration of pregnancy.

303

This finding is in line with several epidemiological and randomized controlled trials reporting

304

that higher n-3 PUFA concentrations and more specific DHA concentrations may prolong

305

pregnancy (8). Unlike the association with birth weight, the percentage of n-6 PUFAs in

306

glycerolphospholipids was not negatively related to the duration of pregnancy. As different

307

processes underlie the duration of pregnancy and birth weight (29), the PUFAs that augment fetal

308

growth, not necessarily prolong the duration of pregnancy. Prostaglandins, inducing cervical

309

ripening and uterine contractions are synthesised from AA. A high intake of n-3 PUFAs is

310

considered to depress the synthesis of AA-derived prostaglandins (30).

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Other researchers assumed that the reported increase in birth weight in response to fatty

312

acid intake is merely a consequence of a longer duration of pregnancy and not of a specific

313

growth enhancing effect (31). In contrast, our results indicate a growth promoting effect from

ACCEPTED MANUSCRIPT 22 314

mid-pregnancy onwards. In addition, the observed effects of fatty acids on the duration of

315

pregnancy were independent of birth weight.

316

Our study investigated not only the n-3:n-6 PUFA ratio, but also investigated the sums of n-3 and n-6 and separate PUFA percentages. In many epidemiological studies often only the n-

318

3:n-6 PUFA ratio was investigated. With the use of a ratio, however, it is often unclear whether it

319

is the numerator or denominator or both, that are driving the association with the outcome of

320

interest (32). The results in the current study suggest that an increase in percentage of n-3 PUFAs

321

in glycerolphospholipids may have different effects on fetal growth velocity than a decrease in

322

percentage of n-6 PUFAs in glycerolphospholipids. The approach not to consider the fatty acids

323

in each class as physiological equivalent to one another concurs with different biological

324

properties of the short- and long-chain fatty acids (32).

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The strengths of our study were the availability of percentages of plasma n-3:n-6 PUFAs in glycerolphospholipids, the large sample size, and the ability to adjust for several potential

327

confounders. Biomarkers of dietary intake can provide a more accurate measure of PUFA status,

328

than intake of fish (a major source of n-3 PUFAs) or fish oil supplementation during pregnancy

329

because they are not subject to reporting errors and less prone to overestimation of negative

330

health effects (33). Moreover, despite potential differences in dietary intake, associations

331

between n-3 and n-6 fatty acids and fetal outcomes were robust across subgroups of Dutch and

332

non-Dutch national origin.

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Several limitations should be considered in the interpretation of our data. First, as

334

maternal percentage of PUFAs in glycerolphospholipids were measured in mid-pregnancy we

335

missed information on these biomarkers during the first half of pregnancy. Longitudinal data

336

have indicated that the pattern of PUFAs changes considerably from week 10 of pregnancy

337

onwards (34). Early placentation events may be initiator of fetal growth restriction (35).

ACCEPTED MANUSCRIPT 23 338 339 340

The risk of measurement errors in growth velocity declines with accumulation of the fetal size, producing more reliable measures from the second half of pregnancy onwards (15). Along gestation there are major changes in plasma lipoprotein fractions, fatty acid profile, fatty acid composition of circulating lipids and fatty acid composition in lipoprotein

342

fractions what may complicate the representation of PUFA measurements in pregnancy (36, 37).

343

Previous literature, however, indicated that the glycerophospholipids PUFA composition is

344

representative for the FA status in a variety of physiological and pathological conditions (13) .

345

Plasma fatty acid levels reflect only very recent dietary intake within the past couple of weeks

346

(38). The single PUFA measurement in this study gives an indicative but not very precise

347

reflection the long-term nutrient status of the participating women. However, repeated

348

measurements were not feasible in this large study.

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Given that fish, a major source of n-3 PUFAs, is consumed sporadically by a meaningful proportion of the Dutch population (39) this could lead to a less precise indication of the n-3

351

PUFA status. However, our measurements permit adequate ranking of women by n-3 PUFA

352

status, as participants with a higher fish intake will also have higher n-3 PUFA concentrations

353

compared to women with low fish intake. Furthermore, others have found that the overall

354

composition of diet in general and intake of fatty acid in particular, change very little from before

355

to during pregnancy (40, 41) suggesting that this limitation may not have influenced our results

356

greatly.

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Although we were able to adjust for potential confounding by other factors, we cannot

358

rule out that the observed associations are the result of residual confounding from socio-

359

demographic and lifestyle related determinants. Furthermore, other nutrients that are associated

360

with fatty acid metabolism and growth, e.g. iron, were not included in this study (42).

ACCEPTED MANUSCRIPT 24 Finally, we cannot rule out that selective participation could have influenced our results. Data

362

were more complete in women of higher socio-economic status. If selective non response has

363

influenced our results, it might be more likely to have created under- rather than overestimation

364

of the observed effects. Nevertheless, our results should be carefully interpreted and generalized

365

to other populations.

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In conclusion, we found indications of positive associations between a higher maternal

367

mid-pregnancy n-3:n-6 PUFA ratio and several features of fetal health. Future research should

368

address underlying pathophysiological mechanisms and the long-term effects on infant health.

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ACCEPTED MANUSCRIPT 25 Supplementary Material Additional Supporting Information may be found in the online version of this article:

derivatives used for calculation of the n-3:n-6 PUFA ratio

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Table S1. List of available PUFAs measured in maternal plasma and their desaturated/elongated

Table S2. Associations of maternal n-3:n-6 PUFA ratio with features of fetal growth (Dutch women)

obtained by unadjusted and adjusted analyses

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Table S3. Associations of maternal mid-pregnancy plasma PUFAs with infant birth weight as

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Table S4. Associations of maternal mid-pregnancy plasma n-3 and n-6 PUFAs with placental weight

Table S5. Associations of maternal n-3 and n-6 PUFAs and the risk of an infant born small-for

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gestational age

ACCEPTED MANUSCRIPT 26

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consumption, fetal growth and the risks of neonatal complications: the Generation R Study. Br J Nutr. 2011;105(6):938-49. Otto SJ, van Houwelingen AC, Badart-Smook A, Hornstra G. Changes in the maternal essential fatty acid

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ACCEPTED MANUSCRIPT 30 Acknowledgements The Generation R Study is being conducted by the Erasmus Medical Centre in close collaboration with the School of Law and the Faculty of Social Sciences of

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the Erasmus University, Rotterdam; the Municipal Health Service, Rotterdam area; the

Rotterdam Homecare Foundation; and the Stichting Trombosedienst and Artsenlaboratorium Rijnmond, Rotterdam, the Netherlands. We gratefully acknowledge the contributions of the

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general practitioners, hospitals, midwives and the pharmacies in Rotterdam.

We gratefully acknowledge the contributions of the general practitioners, hospitals, midwives,

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and pharmacies in Rotterdam, and the dedicated fatty acid analysis work of Stefan Stromer,

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Munich.

ACCEPTED MANUSCRIPT 31 Source of Funding The Generation R Study was made possible by financial support from Erasmus MC, University Medical Center, Erasmus University Rotterdam, the Netherlands Organization for Health

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Research and Development (ZonMw) ‘Geestkracht’ program (10.000.1003), the Netherlands Organisation for Scientific Research, the Ministry of Health, Welfare and Sport, and the Ministry of Youth and Families. This research was additionally supported by the European Community’s

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7th Framework Program (FP7/2008e 2013, grant agreement 212652, NUTRIMENTHE project) and the European Research Council Advanced Grant ERC-2012-AdG – no.322605 META-

Research (SSWO grant number 268).

Disclosures of Interest

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GROWTH. NGM was supported by an additional grant from the Sophia Foundation for Medical

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All authors declare that they have no conflicts of interest.

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HIGHLIGHTS A higher maternal n-3: n-6 PUFA ratio in mid pregnancy is associated with a higher fetal growth velocity and a longer duration of pregnancy.

maternal percentage of n-3 PUFAs in glycerolphospholipids.

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These findings seem to be partly accounted for by, in particular, a higher mid-pregnancy

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These findings suggest that an optimal balance between n-3 and n-6 PUFA status during

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pregnancy is important for fetal health.