This study is based on the Norwegian Mother and Child Cohort Study conducted by the Norwegian Institute of Public Health, with enrollment from 1999-2008.19
The majority of pregnant women in Norway were invited to participate in conjunction with a routine ultrasound exam around 17 weeks of gestation. A total of 39% of invited women participated in the study. Further details can be found at www.fhi.no/morogbarn
. The Norwegian Mother and Child Cohort Study was approved by The Regional Committee for Medical Research Ethics and the Norwegian Data Inspectorate, and informed consent was obtained from each participant.
At the time of enrollment, women completed a questionnaire regarding demographic and lifestyle factors, and medical and reproductive history, including breastfeeding history for previous children. In particular, women were asked whether they had planned the index pregnancy, and were asked “how many months did you have regular intercourse without contraception before you became pregnant?” Women chose “<1 month”, “1-2 months” or “3 months or more”. Women who answered “3 months or more” were further prompted to provide the number of months. Women’s responses to these questions defined their self-reported time to pregnancy. For the present study, we defined subfecundity as self-reported time to pregnancy of greater than 12 months. Additionally, information regarding the interval between women’s previous delivery and the beginning of the index pregnancy (interpregnancy interval) was obtained from the Medical Birth Registry of Norway.
Eligibility was restricted to women who delivered a live-born child and provided a plasma sample around 17 weeks of gestation. We further restricted eligibility to women enrolled from 2003 to 2004. Compared with the earliest years of the Norwegian Mother and Child Cohort Study, the volume of plasma stored for participants was greater during these years. Furthermore, levels of PFCs appear to be declining in Norway,12
and average PFC levels were likely higher during these years compared with later years.
Among eligible women, we randomly selected 400 who planned their pregnancy and were subfecund (i.e. who reported time to pregnancy greater than 12 months). Additionally, we randomly selected 550 cohort members who reported a time to pregnancy of any duration. Nine women originally selected as cases reported a time to pregnancy longer than their interpregnancy interval in the Medical Birth Registry of Norway. Two of these women had an interpregnancy interval less than or equal to 12 months, making it clear that their time to pregnancy could not be more than 12 months; these two women were reclassified as controls and their time to pregnancy recoded as equal to their interpregnancy interval. For the other seven women, the interpregnancy interval was greater than 12 months; these women were excluded from the analysis. Eleven of the randomly-selected cohort subjects were also excluded; nine did not report planning their pregnancy and two inconsistently reported their time-to-pregnancy. The case definition was then applied to the remaining 541 subjects in the random cohort sample, resulting in 30 additional cases. The remaining 502 women in the random cohort sample plus the two women originally selected as cases met the control definition (women with a planned pregnancy and time to pregnancy no longer than 12 months) for a total of 421 cases and 511 controls. Lastly, 163 cases (39%) and 7 controls (1%) reported receiving fertility treatment for the index pregnancy; the seven control women were excluded, leaving 421 cases and 504 controls.
Maternal plasma samples were shipped from the collection site to Oslo by mail at ambient temperature. Because PFCs are chemically stable,20
changes in PFC plasma concentrations while in transit are believed to be negligible. PFOS and PFOA concentrations were measured from 150 μl of plasma using high-performance liquid chromatography/tandem mass spectrometry at the Norwegian Institute of Public Health. For quantification of PFOS, the total area of the linear and branched isomers was integrated. Further details about the analytic method have been previously published.21
All values of PFOS and PFOA were above the limit of quantification (0.05 ng/ml). A total of 50 quality assurance/quality control (QA/QC) samples from a single pool were analyzed in 17 sample batches alongside the case and control specimens. Each batch contained an approximately equal proportion of case and control specimens. The lab technicians were blinded to their identity, and the QA/QC samples were indistinguishable from the plasma samples from Norwegian Cohort subjects. The within- and between-batch coefficients of variation (CV) were calculated for PFOS and PFOA based on the 50 QA/QC samples. For PFOS, the within-batch CV was 4.5% and the between-batch CV was 11.3%. The within- and between-batch CVs for PFOA were 3.5% and 6.7%, respectively. PFOS and PFOA concentrations were categorized into quartiles, with the lowest quartile as the reference category.
We used logistic regression to estimate ORs and 95% CIs for each quartile of PFOS and PFOA, separately. We adjusted a priori for maternal age and prepregnancy body mass index (BMI). To determine the magnitude of other potential confounding, we examined the following variables using a backwards deletion strategy22
: maternal plasma albumin concentration, calendar year of blood draw, maternal smoking, maternal alcohol intake, maternal fish consumption, maternal education, paternal age, paternal education, maternal diseases (endometriosis, sexually transmitted diseases, and fallopian tube infection), menstrual cycle irregularity, and frequency of sexual intercourse. The deletion of any of these variables did not change the ORs for the association between subfecundity and PFOS (conditional on maternal age and prepregnancy BMI) by more than 10%. However, the deletion of maternal alcohol intake changed the OR for the association between subfecundity and PFOA by 10%. Therefore, all PFOS analyses are adjusted only for maternal age and prepregnancy BMI, while PFOA analyses are additionally adjusted for maternal alcohol intake. Due to missing values for covariates, the final sample size for the PFOS analyses included 416 cases and 494 controls, while the PFOA analyses included 412 cases and 488 controls. Results were also stratified by parity (nulliparous vs. parous). We chose not to assess parity as a potential confounder because parity is influenced by a woman’s underlying fecundability (which will influence her time to pregnancy), and it may also affect exposure through declining PFC body burden during previous pregnancies and lactation. To compare our results with those presented in the Danish cohort,16
we performed a sensitivity analysis using the same exposure categories as in the Danish study.
Among parous women, the interval between the two most recent pregnancies (interpregnancy interval), the number of previous pregnancies, and duration of breastfeeding may have influenced measured levels of PFCs. The effect of these variables on PFC levels may either bias the association between PFCs and subfecundity (measured by self-reported time to pregnancy) or, if women with longer time to pregnancy have higher levels due only to a longer time since their previous pregnancy, result in reverse causation. To explore whether these factors have biased the association, we examined models for parous women adjusted for interpregnancy interval (after subtracting out the time to pregnancy) and breastfeeding.
Also, to further understand how the interpregnancy interval (minus time to pregnancy) and breastfeeding may have affected PFC plasma concentrations in our study, we conducted a third sensitivity analysis. Among parous women, a simple linear regression model was fit to estimate PFC plasma concentrations based on previous pregnancy variables. The dependent variable in this model was the natural log of either PFOS or PFOA. The independent variables included interpregnancy interval and duration (in months) of breastfeeding the previous child. Other factors related to the index pregnancy (maternal age at the pregnancy attempt, prepregnancy BMI, maternal education, and maternal drinking) and parity (0, 1, or 2+ previous births) accounted for little of the variance and were not included in the final model.
The beta coefficient corresponding to the interpregnancy interval represents the change in ln(PFC) levels per month, while the beta coefficient corresponding to the breastfeeding variable represents change in ln(PFC) levels per month spent breastfeeding. We used these beta coefficients to explore how much of the differences in average PFC levels between cases and controls might be explained by differences in time to pregnancy and breastfeeding. By multiplying the difference in average time to pregnancy between cases and controls by the beta coefficient for the interpregnancy interval, we estimated the percent difference in average PFC levels between cases and controls due to differences in time to pregnancy. Similarly, by multiplying the difference in average months spent breastfeeding between cases and controls by the beta coefficient for breastfeeding, we estimated the percent difference in average PFC levels due to differences in breastfeeding duration. We compared these estimated values with the observed difference in PFC levels between cases and controls.