The aim of this project is to investigate the relationship between exposures during pregnancy and subsequent breast density, a strong determinant of breast cancer risk. The project will be undertaken within the Avon Longitudinal Study of Parents and their Children (ALSPAC) cohort. The specific objectives of the proposal are to investigate the relationship between each of two measures of breast density, described in detail below, and the following exposures:

i. Biological markers of the insulin-like growth factor (IGF) axis during pregnancy

ii. Nutritional intake during pregnancy

iii. Pregnancy weight patterns

iv. Diabetes or indications of hyperglycaemia in pregnancy

v. Blood pressure (BP), hypertensive disorder of pregnancy and pre-eclampsia

vi. Smoking during pregnancy

We also plan to investigate the relationship between IGF gene variants and IGF levels, with future plans to relate each of these to breast density.

The data collection phase of the project requires obtaining, digitising and analysing the mammograms of eligible mothers from the ALSPAC cohort. We will only include women who have a mammogram taken under the NHS Breast Screening Programme, which offers free mammography to women aged 50 to 70. As a result of the current study, we are not proposing to expose any woman to unnecessary radiation.

Eligibility criteria: The following women will be eligible to take part in the study; those who

i) have had a mammogram under the NHS Breast Screening Programme, or have one prior to the end of 2011 (eight months prior to study end);

ii) have not previously had a mammogram or MRI investigation of the breast

iii) consent to us accessing their mammograms (only requested from women aged 48 years or over, as per ethical approval);

iv) currently reside in Avon (this restriction is necessary, since we will only obtain mammograms from the Avon Breast Screening Programme)

Obtaining consent: Women who attend the Focus on Mothers clinic will be requested to consent to us obtaining and analysing their mammograms. Women who do not attend the clinic will be contacted by post and asked to give consent to us accessing their mammograms. Other than this, we will not have any further contact with the women.

Obtaining the mammograms: We will set up a flagging system, so that whenever a cohort member who has given us consent to access their mammogram attends screening, the research team will be informed. We will purchase an Array digitiser, and install it at the Avon Breast Screening Clinic (the Central Clinic). The research associate (RA) will then spend time at the Central Clinic each week, digitising the relevant films (both cranio-caudal and medio-lateral oblique images will be obtained), and transferring digital images to the coordinating centre. Dr Lis Kutt of the Central Clinic has writte a letter of collaboration agreeing to this.

Analysing breast density: Breast density will be performed in two ways: i) using a validated, computer-aided, threshold method, and ii) an automated volumetric method (SMF).

Exposure measurements: The following exposure data will be analysed.

From obstetric records: blood pressure and proteinuria in pregnancy; pregnancy weight measures; pregnancy diabetes / hyperglycaemia; offspring birthweight (from birth notification and/or obstetric data).

From questionnaire data: self-reported pre-pregnancy weight and waist/hip measures, (12 weeks' gestation); food frequency questionnaire (32 weeks' gestation); self-reported regular smoking during the 1st and 3rd trimesters; mother's weight at 8 weeks post-partum;

Biochemical measures: The earliest serum or plasma sample taken during pregnancy will be used to for analyses of: insulin, insulin-like growth factors (I and II) and relevant binding proteins (BP3).

Genetic measures: We plan to investigate 14 SNPs which have previously been demonstrated to be related to IGF-I or IGFBP-3 levels . Thirteen are listed below; one further relevant SNP (rs2854744) has already been genotyped. If other SNPs are identified from the literature duting the study period, we may request that analyses of further SNPs are performed. Funding to cover this eventuality will be requested in the initial grant proposal.