NHS, NHS II, and HPFS are prospective cohort studies of US health professionals. In 1976, NHS enrolled 121 701 female registered nurses between the ages of 30 and 55 years to investigate the long term health effects of various contraceptive methods in women. 19 20 NHS II includes 116 430 female registered nurses between the ages of 25 and 44 years and began in 1989 with the goal of investigating oral contraceptives, diet, and lifestyle factors in a population younger than NHS. 20 HPFS is the male counterpart to NHS and NHS II, and included 51 529 male health professionals between the ages of 40 and 75 years at study inception in 1986. In all three cohorts, participants completed validated questionnaires every two years that captured information on disease diagnosis, disease risk factors, drug use, and lifestyle characteristics, with follow-up rates exceeding 90%. For the current analysis, baseline was defined as the year when diet was first assessed in the cohorts: 1980 in NHS, 1986 in HPFS, and 1991 in NHS II. Of the participants who completed a baseline food frequency questionnaire (NHS, n=98 047; NHS II, n=97 813; HPFS, n=51 529), we excluded those who reported a diagnosis of cardiovascular disease, cancer, or type 2 diabetes, or had coronary artery surgery before completion of the baseline; these diagnoses could result in changes in diet. 21 We also excluded participants with missing age at baseline and those who reported implausible energy intake on the food frequency questionnaire (<500 or >3500 kcal/day for women and <800 or >4200 kcal/day for men). Additionally, we excluded participants who left more than 70 items blank on the food frequency questionnaire, and those who had missing information on baseline egg intake. The final sample included 83 349 participants in NHS, 90 214 participants in NHS II, and 42 055 participants in HPFS. Supplemental figure 1 shows the flow chart of participants.

Whole egg intake was reported every two to four years, beginning in 1980, 1991, and 1986 for NHS, NHS II, and HPFS, respectively, by using a validated semiquantitative food frequency questionnaire. The questionnaire had 61 items in 1980 and 126-131 items in subsequent versions. 22 23 24 Participants were asked how often on average they consumed whole eggs with yolk in the past year. Reported intake excluded eggs in baked goods (eg, cake), liquid eggs, and egg whites. Among 173 women in NHS, food frequency questionnaires were validated against four seven day weighed diet records. The deattenuated correlation coefficient between the food frequency questionnaire and the weighed food record for whole egg intake was 0.77. 25 A similar validation study conducted among 127 men in HPFS showed a high correlation between whole egg intake reported from food frequency questionnaires and weighed food records (deattenuated correlation coefficient=0.80). 26 Consumption of liquid eggs and egg whites was not assessed in the questionnaire. However, we computed intake of eggs included in baked goods such as cakes, cookies, pancakes, muffins, sweet rolls, and donuts.

Deaths were identified by reports from next of kin, the US postal service, state vital statistics departments, and systematic searches of the National Death Index. Follow-up for deaths was more than 98%. 30 Myocardial infarction deaths were confirmed by autopsy or electrocardiographic findings and enzyme changes characteristic of myocardial infarction before death according to medical records. We did not include sudden deaths of unknown cause in our analyses. Participants with confirmed fatal coronary heart disease included those in whom the underlying cause of death was listed as coronary heart disease on the death certificate and evidence indicated a history of coronary heart disease. 30 31 When medical records were not available but confirmation was provided through interview or letter, participants were considered to have probable cardiovascular disease. For the current analysis, we included participants with confirmed and probable cardiovascular disease. Analyses that included only participants with confirmed cardiovascular disease produced virtually identical results.

The primary endpoint for this study was incident cardiovascular disease, which we defined as non-fatal myocardial infarction, fatal coronary heart disease, and fatal and non-fatal stroke that occurred after baseline. Participants (or next of kin for deceased) who reported a primary endpoint were asked for permission to have their medical records reviewed by study physicians who were blinded to the participants’ exposure and risk factor status. We used World Health Organization criteria of typical symptoms plus either elevated enzymes (including troponin) or diagnostic electrocardiographic findings to diagnose myocardial infarction. 27 Stroke was confirmed using criteria from the National Survey of Stroke, which required evidence of a neurological deficit with sudden or rapid onset that persisted for more than 24 hours or until death. 28 A physician reviewed stroke events and classified them by stroke subtype or cause using the Perth Community Stroke Study criteria (subarachnoid hemorrhage, intraparenchymal hemorrhage, ischemic stroke (thrombotic or embolic), or stroke of unknown causes). 29

We used the main biennial follow-up questionnaires to collect and update information on age, ethnicity (assessed once in 1992 in NHS, 1989 in NHS II, 1986 in HPFS), family history of myocardial infarction, body weight, cigarette smoking, physical activity, and multivitamin use. We also gathered information on menopausal status (NHS and NHS II), use of postmenopausal hormones (NHS and NHS II), oral contraceptive use (NHS II only), and history of hypercholesterolemia and hypertension. We considered participants to have hypercholesterolemia or hypertension when they reported these conditions on the biennial questionnaire or when they reported use of lipid or blood pressure lowering drugs. We determined alcohol intake through food frequency questionnaires. Detailed descriptions of the validity and reproducibility of self-reported body weight, physical activity, and alcohol consumption have been published elsewhere. 32 33 34

Statistical methods

We calculated person time from the return of the baseline questionnaire (1980 for NHS, 1991 for NHS II, and 1986 for HPFS) to the diagnosis of cardiovascular disease, death, or the end of follow-up (30 June 2012 for NHS, 30 June 2013 for NHS II, and 31 January 2012 for HPFS), whichever occurred first. We did not censor participants lost to active follow-up because fatal events were included in the outcomes. When we restricted the analyses to non-fatal events and censored participants because of loss to follow-up we had similar results (data not shown).

We computed cumulative averages of dietary variables, including egg intake, to reduce within person variation and to represent long term diet.21 For instance, for the 1999-2001 risk set in NHS II, dietary variables in 1991, 1995, and 1999 were averaged to predict subsequent cardiovascular disease risk. Within each cohort, we divided participants into predefined categories of egg intake (less than one egg per month, one to less than four eggs per month, one to less than three eggs per week, three to less than five eggs per week, five to less than seven eggs per week, and at least one egg per day). The group that consumed less than one egg per month served as the reference group. We used Cox proportional hazard models to examine the association between categories of egg intake and cardiovascular disease. In the main analyses, we used whole egg intake as the exposure. Analyses were first conducted within each cohort separately, and then by pooling data from the three cohorts.

The regression model included age in months as the time scale, stratified by calendar time in two year intervals, and allowed for possible interaction between calendar time and age in the baseline hazards to be accounted for non-parametrically (model 1). In the pooled analysis, we also stratified by cohort, which allowed concomitant stratification for sex. In model 2, we additionally adjusted for race or ethnicity (white, other), family history of myocardial infarction (yes, no), baseline history of hypertension (yes, no), and baseline history of hypercholesterolemia (yes, no). Model 2 also included the following time varying covariates updated every two years: body mass index (<21.0, 21.0-22.9, 23.0-24.9, 25.0-26.9, 27.0-29.9, 30.0-34.9, ≥35.0); smoking status (never, former, current); physical activity (<3.0, 3.0-8.9, 9.0-17.9, 18.0-26.9, ≥27.0 metabolic equivalent of task hours per week); alcohol consumption (g/day in fifths); multivitamin use (yes, no); postmenopausal status and postmenopausal hormone use (premenopausal, never, former, current, NHS and NHS II only); and use of oral contraceptives (never, former, current, NHS II only).

In our final model (model 3), we additionally adjusted for total energy intake and consumption of foods associated with egg intake in the US (eg, red meat, bacon, other processed meat, refined grains, potatoes and French fries, fruits, vegetables, full fat milk, fruit juices, sugar-sweetened beverages, and coffee). As with egg intake, we used a cumulative average update for all dietary variables. We tested for a possible linear trend in the hazard ratios across categories of egg consumption by using the median of each category of egg intake as the dose of egg consumption.35 Total cardiovascular disease was analyzed separately from coronary heart disease and stroke.

We used statistical models to estimate the effect on risk of total cardiovascular disease of replacing one whole egg per day with one serving of other foods that are common alternatives (unprocessed red meat, processed red meat, poultry, fish, legumes, nuts, refined grains, whole grains, potatoes, low fat cheese, high fat cheese, reduced fat milk, full fat milk, and yogurt; information on yogurt fat content was not collected in our food frequency questionnaires). The covariates in model 3 were used, and eggs and the alternative foods were included as continuous variables in the same multivariable model. We computed the differences between the β coefficients, variance, and the covariance of eggs and the alternative food to estimate hazard ratios and 95% confidence intervals for the replacement effect.36 In these analyses, we assumed that total consumption of different foods is constrained to a certain level for each person (the amount of food is held constant); that the association of egg intake with cardiovascular disease risk is independent of the association with the alternative food intake; and that the intake of other foods in the diet remains constant.37

We performed stratified analyses defined a priori by updated body mass index, physical activity, self-reported hypertension (including use of antihypertensive drug treatment), self-reported hypercholesterolemia (including use of lipid lowering drug treatment), family history of myocardial infarction, statin use, smoking status, age, Alternative Healthy Eating Index score,38 and self-reported prevalent type 2 diabetes. For each of these variables, we tested for potential effect modification by using likelihood ratio tests for interactions.

We performed several sensitivity analyses. We examined the association of egg intake with cardiovascular disease risk by estimating the risk of incident cardiovascular disease across seven categories of egg intake; at least two eggs per day was the highest category of intake. The risk of cardiovascular disease was also estimated for total egg intake (that is, consumption of whole eggs plus eggs in baked goods). Additionally, we modeled egg intake by using baseline diet only and by using the most recent diet. Because diagnosis of an intermediate endpoint of cardiovascular disease could result in changes in diet or in diet reporting, we evaluated associations when diet updating was stopped: after diagnosis of type 2 diabetes, hypertension, hypercholesterolemia, or angina; also after coronary artery bypass graft, or the start of statin treatment.39

We repeated the main analysis by replacing foods associated with egg intake (red meat, bacon, other processed meat, refined grains, fruits, vegetables, potatoes and French fries, fruit juices, full fat milk, sugar-sweetened beverages, and coffee) with the Alternative Healthy Eating Index score in the multivariable model. Instead of pooling the data, we used fixed effect meta-analysis to estimate the overall association of egg intake and cardiovascular disease risk among the three cohorts. Finally, we calculated E values to determine the minimum strength of association on the risk ratio scale that an unmeasured confounder would need to have with egg consumption and cardiovascular disease risk to fully shift the observed association toward a significant association.40 All P values are two sided and statistical significance was considered at P values less than 0.05. Statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC).