Specific Aim - To conduct analyses of existing data to assess the effects of in-utero lead exposure on timing of puberty in children enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC).

Background and Significance - Recent literature suggests that lead appears to have endocrine disrupting potential in humans. Cross-sectional analyses of the United States Third National Health Examination and Nutrition Survey showed associations of blood lead levels with delayed breast and pubic hair development and menarche in girls 1 2 A cross-sectional study from Russia reported a delayed onset of genital development among boys with blood lead levels >= 5 micro-g/dL 3. Overall, the literature of the impact of lead on pubertal timing is sparse and the mechanisms leading to these associations are not known.

The Centers for Disease Control and Prevention (CDC) is currently conducting a study in ALSPAC participants to assess effects of exposure to substances with endocrine-disrupting potential in pubertal onset and progression. Analytes being tested in maternal serum and urine and children urine samples originate mainly from pesticides or plasticizers. The analytes are: organochlorine pesticides, polychlorinated biphenyls (PCBs), brominated flame retardants (PBB and PBDE), polyfluoroalkyl chemicals (PFCs), triazine herbicides (atrazine), environmental phenols, phthalates, and phytoestrogens.Data on pubertal development have been collected via self-administered questionnaires beginning at age 8 up to age 14.

In our study we are not testing samples for lead levels. However, ALSPAC have tested maternal blood samples for lead levels to study the effect of this exposure on other health outcomes. Because of the evidence that lead may impact the pace of pubertal development we want to obtain from ALSPAC the data on maternal blood lead levels to assess the effects of in-utero lead exposure on timing of puberty onset.

Due to the sparse, yet suggestive, reports on the association of lead with delayed pubertal development in boys and girls the proposed analysis of existing data would be an important contribution to the literature on the effects of environmental contaminants on endocrine function.

Research Design and Methods - As indicated previously outcome and exposure data has already been collected for other purposes. Among 8-14 year old singletons who responded to the puberty questionnaire, there were 3945 girls with valid data on breast and pubic hair Tanner stage and menarche; 3938 boys with valid data on pubic hair Tanner stage; and 2877 boys with valid data on genital Tanner stage.

We will construct survival models to assess the effect of lead on puberty. For girls, we will estimate the difference in timing of menarche attainment and of transition into stage 2 of breast and pubic hair development in exposure groups after adjustment for confounders. For boys, we will estimate the same for transition into stage 2 of genital and pubic hair development. We found inconsistencies in genital stage reporting in a large proportion of boys. Because of the reported association between delayed genital development and lead exposure we believe that it is important to assess this association in our study group. We have included the analyses of this association in the proposal as a possibility whose validity would be explored further.

Lead levels were tested on samples taken at time of enrollment in approximately 4000 mothers whohad whole blood stored in an acid washed vaccutainers. Data collection on parents and children enrolled in the ALSPAC cohort was conducted by the University of Bristol. The University of Bristol assigned a unique identification number to the puberty data they sent to CDC. We will provide the identification numbers of children with valid puberty data to ALSPAC staff to obtain their mother's lead levels and the gestational age when the blood sample was obtained. They will not be sending blood samples to CDC for lead testing. They will use the CDC assigned IDs to know for which children we want to obtain results of the blood lead testing they conducted. What we expect from them is an updated de-identified dataset of children with valid puberty data that will include results of their blood lead testing and the age the child was tested. Only the University of Bristol has access to information on personal identifiers. Data transfer will be conducted according to the confidentiality assurances and procedures that the University of Bristol have implemented as part of their study to prevent any breach in confidentiality.

The final number of children with both exposure and outcome values is not yet known. The number of maternal samples tested is large and may yield sufficient children to detect statistically significant differences among exposure groups. As a comparison, of the 1127 children from the Children in Focus Cohort seen at 25 months, 368 girls responded to at least one puberty questionnaire. If we apply this ratio (368/1127) to the number of mothers with lead data (~4000) we may have about 1300 girls with both lead and puberty data for analyses.

We conducted power calculations for different sample size scenarios of the study outcomes. For menarche attainment we obtained power estimates for sample sizes ranging from 250 to 1500 in increments of 250 at a level of significance of 0.05, a proportion of lost to follow-up of 13%, and a prevalence of delayed menarche of 3.2 2 for unequal sample sizes in comparison groups. Estimates were obtained using the PASS software.

Power calculations

Sample

size

Difference in proportion of girls with

menarche in comparisons groups

3.0

5.0

7.0

9.0

250

0.29

0.52

0.71

0.84

500

0.50

0.81

0.95

0.99

750

0.67

0.94

0.99

1.00

1000

0.80

0.98

1.00

1.00

1250

0.88

0.99

1.00

1.00

1500

0.93

0.99

1.00

1.00

With a sample size of 500 we will be able to attain a power of 80% to find a difference in the proportion of menarche attainment >= 5.0. It is likely that effects of lead in puberty are of a smaller magnitude; however, it seems reasonable to expect that the number of girls with lead and puberty data will be larger. We will address the potential for selection bias by exploring differences between those children with and without lead data. The proposed analyses of existing data seem feasible and will make an important contribution to current knowledge on effects of lead on endocrine function.

References

1. Selevan SG, Rice DC, Hogan KA, Euling SY, Pfahles-Hutchens A, Bethel J. Blood lead concentration and delayed puberty in girls. N Engl J Med 2003;348(16):1527-36.

2. Wu T, Buck GM, Mendola P. Blood lead levels and sexual maturation in U.S. girls: the Third National Health and Nutrition Examination Survey, 1988-1994. Environ Health Perspect 2003;111(5):737-41.

3. Hauser R, Sergeyev O, Korrick S, Lee MM, Revich B, Gitin E, et al. Association of blood lead levels with onset of puberty in Russian boys. Environ Health Perspect 2008;116(7):976-80.