We propose a program project that will permit the processing and analysis of all consented ALSPAC placentas that have follow-up data on at least one of the three outcomes proposed for the program project, as described below.

The program project will include an Administrative Core (details to be provided by ALSPAC).

A "Placental Core" will be headed by Dr Craig Platt, Senior... at BRI, with Dr Carolyn Salafia as Co-PI at Placental Analytics LLC. Dr Platt will be responsible for supervision and quality control of placental examination, photography, tissue sampling, and sample processing to wax blocks, Placental Analytics LLC will be responsible for image analysis of placental photographs, and preparation of all histologic slides deemed essential to study goals including routine (hematoxylin and eosin) and special (e.g., immunohistochemistry) stains, and will digitize all such slides in a standard and accessible format. At the completion of the program project, all data extracted from photographs of placentas and digitized slide files, plus the digitized slide files, slides and remainders of wax tissue blocks will be returned to ALSPAC/BRI (?) as a permanent archive.

Program subprojects:

Neurocognitive: "Placental structure and pathology and neurocognitive outcome"

PI Prof Jean Golding, Co-Investigators D Misra, J Miles, C Platt, C Steer, C Salafia

We propose to test the hypothesis that detailed markers within the placenta are related to specific cognitive, behavioural and psychological outcomes from early childhood to school leaving (see Annex, below). A range of statistical approaches including structural equation modelling, a method well known to the psychometric literature, will be used to determine the predictive value of the placental measures in regard to the outcomes. Particular attention will be paid to ways in which the placenta varies in regard to factors that have been shown to be related to adverse neurocognitive outcomes such as influenza in mid-pregnancy, alcohol in early pregnancy, maternal anxiety, maternal prenatal levels of lead and mercury, and paternal omega-3 intake. We will also assess the ways in which placental variation is observed in placentas of children with minor congenital defects (minor congenital malformations are found in excess among children with many different neurocognitive outcomes).

All the neurocognitive outcomes will be available, but the minor congenital malformations will need to be identified from the neonatal clinical records - for which this grant would budget to be extracted from existing medical records.

The placenta provides two routes of analysis that we propose will account for variation in neurocognitive development/developmental trajectory:

1. Placental branching growth is determined by the branching growth of its vascular tree. The "neurovascular hypothesis of disease" summarizes recent recognition that the gene families, growth factors and molecular signals that promote and maintain neuronal health are the same factors that promote and maintain vascular health. We propose that measurement of the placental vascular tree, in terms of 2D chorionic surface, 3D placental volume, chorionic surface vascular networks and finer architecture of the placental fetal and terminal villi correlate with fetal neuronal growth and health and will therefore account for variability in neurocognitive outcome/developmental trajectory.
2. Placental pathologies of inflammation and injury patterns that may indicate oxidative stress can be identified in clinically abnormal and in clinically "normal" pregnancies. These pathologies are measured by histologic features identified in tissue sections of the placenta. We propose that measurement of these features will provide quantitative estimates of fetal exposures to inflammation and oxidative stress, via fetal effects on the developing brain, will account for variation in neurocognitive outcome/ developmental trajectory.

Pulmonary: "Placental structure and pathology and pulmonary outcome"

PI Dr John Henderson, Co-Investigators D Misra, J Miles, C Platt, C Salafia

There is evidence from cohort studies that have measured lung function sequentially from childhood that decrements of lung volumes (FEV1) and flows (FEF25-75 and maximal flows at 75%, 50% and 25% remaining FVC) may become established by early childhood and then 'track' into adult life. The effect of this would be to diminish peak measures of lung function prior to the natural decline in lung function with age, thus hitting a threshold at which disease becomes manifest at an earlier age. This paradigm is compatible with additional insults, such as smoking, leading to more rapid decline of lung function & exacerbating early life deficits. Thus, there may be additive effects of early life factors and later insults leading to diseases such as COPD.

Birth cohort studies that have measured lung function shortly after birth have demonstrated differences in measures of airflow associated with different patterns of symptom presentation in early childhood (e.g. transient early wheezing is associated with decrements in V'maxFRC shortly after birth (before symptoms are manifest) but regresses to the mean during the first years after birth whereas children who develop wheeze in later childhood have normal V'maxFRC in infancy but lower values of FEV1 in later childhood than never-wheezers) and with in-utero exposure to maternal tobacco smoke - children exposed in utero have lower values of lung function after birth and are more likely to develop wheeze and asthma during childhood.

Following the work of Barker and others, who demonstrated that adult respiratory diseases and lung function (FEV1) is associated with birth weight, there is considerable interest in the notion that fetal growth restraint is associated with abnormalities of airway development during a critical window of exposure leading to canalisation of effects that persist through the lifecourse (but may manifest only in the context of a second insult, e.g. smoking or following the decline in lung function measures with ageing).

Again, the placenta provides two routes of analysis that we propose will account for variation in pulmonary function:

1. Placental trophoblast epithelial branching growth, determined by the branching growth of its vascular tree, parallels the branching of the pulmonary epithelium that is coordinated with the development of its parallel vascular tree. Major airways are laid down in the embryo/fetus at similar times as the major chorionic surface vessels, and more distal villous branching parallels later lung growth in utero. The close linkage of these two organ's development is highlighted by the difficulty in studying mutations of genes such as FGF and Notch that are key to lung development, as many of such muiutations are embryonic lethals because of failure of the development of the placenta. We propose that measurement of the placental vascular tree, in terms of 2D chorionic surface, 3D placental volume, chorionic surface vascular networks and finer architecture of the placental fetal and terminal villi correlate with fetal pulmonary growth and health and will therefore account for variability in pulmonary function.

2. Placental pathologies of inflammation and injury patterns that may indicate oxidative stress can be identified in clinically abnormal and in clinically "normal" pregnancies. These pathologies are measured by histologic features identified in tissue sections of the placenta. Both inflammation, especially when cytokines are inhaled in the course of intraamniotic infections, and oxidative stress may alter pulmonary branching morphogenesis. We propose that measurement of these features will provide quantitative estimates of fetal exposures to inflammation and oxidative stress that will account for variability in pulmonary function.

More specifically, ALSPAC has detailed respiratory history at approximately 12 monthly intervals from birth and has measured lung function by spirometry at 8 and 15 years, allowing associations of both attained lung function pre- and (mostly) post-puberty and trajectory of lung function growth during adolescence (with a wealth of background information on exposures during this period, such as passive and active tobacco smoke exposure, validated with measurements of tobacco metabolites (cotinine).

Subproject 3 "Maternal overnutrition and offspring fat mass, metabolic and vascular function"

Principal Investigator: Dawn Misra, PhD, Co-Investigators: D Lawlor, J Miles, C Platt, C Salafia.

Dr. Lawlor's current grant aims refer to "offspring" generally; we have explicitly separated out the neonatal (birth outcomes) within our aims given the focus on the placenta.

SPECIFIC AIMS

Specific Aim 1: To investigate the influence of maternal BMI, weight gain, and diet during pregnancy on placental anthropometric measures. Specifically, we will assess and study the following placental parameters: placental weight, thickness, diameters; umbilical cord length; shape; symmetry measures; chorionic vascular branching pattern). The following null hypotheses will be tested:

1A. Maternal BMI does not explain variation in placental size and shape.

1B. Maternal weight gain does not explain variation in placental size and shape.

1C. Maternal diet does not explain variation in placental size and shape.

Specific Aim 2: To determine whether and how placental size and shape influences birth size. The following null hypotheses will be tested:

2A. Placental size and shape are not associated with birth size (e.g. weight, birth weight ratio, length, ponderal index).

2B. Associations between maternal factors and birth size are mediated by variation in placental size and shape.

Specific Aim 3: To determine whether and how placental size and shape influence offspring growth. Offspring growth was measured at multiple time points beginning at age 1 year up to 15 years of age and includes measures of adiposity (DXA assessed fat mass and fat distribution), vascular function (blood pressure, pulse pressure and endothelial function), and metabolic function (fasting glucose, insulin and lipids). The following null hypotheses will be tested:

3A. Placental size and shape are not associated with offspring childhood adiposity.

3B. Placental size and shape are not associated with offspring childhood vascular function.

3C. Placental size and shape are not associated with offspring childhood metabolic function.

3D. Associations between placental size and shape with offspring outcomes are mediated by effects of the placenta on birth size.

Specific Aim 4: To investigate the role of genetic variation on placental size and shape. DNA was extracted from mothers and offspring in the parent study. The following null hypotheses will be tested:

4A. Genetic variation does not explain variation in placental size and shape.

4B. Interactions between genes and the selected maternal factors (BMI, weight gain, diet) do not explain additional variations in placental size or shape.

Specific Aim 5: To develop and validate a placental index to identify offspring at risk for adverse outcomes with regard to adiposity, vascular function, and metabolic function.

Pilot analyses for the program subprojects will be generated using the placental data already collected from the Children in Focus subset (CIF, N=1050), and intermediate neurocognitive outcomes (TBD by Prof Golding), intermediate pulmonary function outcomes (TBD by Dr Henderson), and BMI, waist & fat & lean mass (DXA) at age 9 as previously determined by Dr Lawlor. These pilot analyses will provide strong support for our (generally well received) October submission, and we are optimistic about receiving funding based on our July resubmission. Dawn Misra and Jeremy Miles will work together on data analysis for the 3 subprojects, with Colin Steer additionally on the neurocognitive subproject.

Annex

1. Temperament and behaviour of child: Infant temperament using the Carey

at ages 6 and 24 months; EAS temperament scale at 42 months;

Behaviour measures of hyperactivity, antisocial/conduct problems, peer

problems, emotional behaviour and prosocial behaviour as well as overall

behaviour difficulties, measured at ages 4 - 13 years

2. Child development: mental and motor development measured annually

between 6 months and 4 years

3. Cognitive measures: IQ using the WISC at age 8; short term memory;

executive function;

4. Speech and language: Vocabulary measured from 15 months to 3 years;

Grammatical understanding; Phonological problems; Use of speech

tharapist

5. Motor coordination: Balance tests; fine motor coordination; Gross motor

coordination; Functional motor impairment, including DCD

6. Childhood growth: Height; Weight; Head circumference; Hip

circumference; Waist circumference; Body mass index - all at various time

points from 4 months to 15 years

7. Educational attainments: Results of SATS tests at 7, 11 and 14; GCSE

results; Results of ALSPAC's own tests ofReading, Spelling, Mathematics,

and Science understanding.

8. Attention and hyperactivity: Hyperactive and inattentive behaviour using

questions to parents and teachers from 4 to 11 years and to the children

at 13.

9. Hearing ability: Tests of hearing under standardised conditions at ages 7,

9 and 11 - using audiometry and otoacoustic emissions; Hearing of

speech discrimination at 3.5 and 5 years

10. Visual ability: Tests of visual acuity, stereoacuity, and other features of

visual ability at various ages from 8 months to 11 years

11. Other sensory outcomes (taste and smell): Taste test (PROP) results at

age 10; Smell test results at age 11

12. Childhood psychiatric disorders: Autism; ADHD; ADD; Anorexia nervosa;

Bulimia; Depression; Anxiety disorders; Conduct disorder;