Thyroid status and mortality in nonagenarians from long-lived families and the general population

Characteristics of study populations

Characteristics of the nonagenarian participants of the Leiden Longevity Study and the Leiden 85-plus Study are presented separately for the 384 men and 680 women in Table 1. For the Leiden Longevity Study, male participants were younger (91.4 years) than the female participants (93.6 years, P<0.001). Circulating levels of fT3 were significantly higher in men (4.1 pmol/L) than in women (4.0 pmol/L, P=0.001). The women had lower fT3/fT4 ratios (0.25) compared to the men (0.26, P<0.001). The other parameters were not significantly different. In the Leiden 85-plus Study, measurements performed at age 90 years were taken as baseline for both men and women. Levels of high-sensitivity C-reactive protein (hsCRP) were significantly different between men and women (men 3.7 mg/L, women 2.8 mg/L, P=0.02). All other measured parameters were similar for men and women.

Thyroid status and mortality in long-lived families

In Table 2 the associations between thyroid status parameters and mortality rate in the participants of the Leiden Longevity Study are presented. For levels of TSH, no association with mortality rate was found. For levels of fT4, a more than 20 percent increased mortality rate was observed in the highest tertile compared to the lowest, however statistical significance was lost after correction for hsCRP (model 1 hazard ratio (HR) 1.22 (95% CI 1.01-1.48) P=0.04, model 2 HR 1.21 (95% CI 0.99-1.45) P=0.06). Higher levels of fT3 were associated with lower mortality in both models (model 1 HR 0.70 (95% CI 0.58-0.85) P=2.33x10^-4, model 2 HR 0.73 (95% CI 0.60-0.88) P=1.33x10^-3). The fT4xTSH product and the fT4/TSH ratio were not associated with mortality in either model. The highest tertile of the fT3/fT4 ratio had more than 30 percent reduction in mortality rate compared to the lowest tertile (model 1 HR 0.65 (95% CI 0.54-0.79) P=1.65x10^-5, model 2 HR 0.68 (95% CI 0.56-0.83) P=1.13x10^-4). The results were similar for men and women in the stratified analyses (data not shown).

Thyroid status and mortality in the general population

The association between thyroid status parameters and mortality rate in participants of the Leiden 85-plus Study are presented in Table 3. Similar to the results in the participants of the Leiden Longevity Study, levels of TSH were not associated with mortality. Higher levels of fT4 were associated with an increased mortality rate in both models, though statistical significance was not reached (model 1 HR 1.36 (95% CI 1.00-1.84) P=0.05, model 2 HR 1.27 (95% CI 0.93-1.73) P=0.14). The highest tertile of fT3 had a significantly lower mortality rate than the lowest tertile (model 1 HR 0.66 (95% CI 0.49-0.90) P=0.01, model 2 HR 0.72 (95% CI 0.53-0.98) P=0.04) The fT4xTSH product and the fT4/TSH ratio were not associated with mortality rate. The fT3/fT4 ratio was associated with a more than 40 percent decrease in mortality rate in both models in the highest compared to the lowest tertile (model 1 HR 0.55 (95% CI 0.40-0.75) P=1.44x10^-4, model 2 HR 0.59 (95% CI 0.43-0.81) P=9.66x10^-4). The results were similar for men and women in the stratified analyses (data not shown).

Thyroid status and mortality independent of study population

The results of the stratified analyses on thyroid status and mortality were similar for both nonagenarians of long-lived families and of the general population. When interaction was tested between study population and parameters of thyroid status on mortality, no significant interaction was found (all P-values>0.7, Supplementary Table 1).

Generalized results for nonagenarians

Since the associations between thyroid status and mortality were similar in both nonagenarian populations, the results could be combined to provide a generalizable association for nonagenarians. For this purpose, the results for the Leiden Longevity Study and the Leiden 85-plus Study populations were combined, through a fixed-effects inverse-variance weighted analysis on the associations of thyroid parameters and mortality rate with adjustments as described in model 2 (Supplementary Table 2). The hazard ratios (HRs) of the highest compared to the lowest tertile in both studies and the weighted averages of these HRs are displayed in Figure 1. Higher levels of TSH were not associated with mortality rate in either study or in the pooled analysis (HR 0.91, 95% CI 0.78-1.07, P=0.25). Higher levels of fT4 were associated with higher mortality rate in both studies, leading to an increase of 22 percent in mortality rate in the highest compared to the lowest fT4 tertile in the pooled analysis (HR 1.22, 1.04-1.43, P=0.02). Inversely, higher levels of fT3 were associated with lower mortality rates in both studies, resulting in a decrease in mortality rate of more than 25 percent in the highest compared to the lowest tertile (HR 0.73, 95% CI 0.62-0.86, P=1.31x10^-4). The fT4xTSH product and the fT4/TSH ratio were not associated with mortality rate. Lastly, the fT3/fT4 ratio was associated with a lower mortality rate in the highest tertiles compared to the lowest in both studies, resulting in a 35 percent lower mortality rate in the pooled analysis (HR 0.65, 95% CI 0.55-0.77, P=5.64x10^-7).

Figure 1. Mortality rate in the Leiden Longevity Study and the Leiden 85-plus Study. Hazard ratios and 95% confidence intervals for mortality of the highest sex-specific tertiles compared to the lowest sex-specific tertiles are shown for the Leiden Longevity Study participants, the Leiden 85-plus Study participants and the pooled Hazard ratios. LLS = participants of the Leiden Longevity Study, 85-plus = participants of the Leiden 85-plus Study, LLS and 85-plus = combined estimates of the Leiden Longevity Study and the Leiden 85-plus Study.

Various sensitivity analyses

Sensitivity analyses performed on participants with normal levels of hsCRP (hsCRP<10 mg/L), participants with mortality within the first two years or past the first year of follow up did not materially change the results (data not shown). The results were similar in participants with TSH levels within the normal range as in the main analyses (data not shown).

Leiden Longevity Study

As described previously in more detail, the aim of the Leiden Longevity Study is to identify genetic and phenotypical factors contributing to familial longevity [8,9]. Families were included if at least two proband siblings had lived to exceptionally old age, being 89 years or older for men and 91 years or older for women. No exclusion criteria based on health or demographics were applied. The Medical Ethical Committee of the Leiden University Medical Centre approved the study and written informed consent was obtained from all study participants. Between July 2002 and May 2006, 421 Dutch Caucasian families were recruited, comprising 944 nonagenarian participants.

Of these 944 nonagenarians data on TSH, fT4 and fT3 were available for 859 participants. Three participants were excluded from the analyses because serum TSH levels were outside the detection range (two participants with TSH <0.005 mU/L and one participant with TSH >100mU/L). Four participants were excluded because their fT3 level was below the reference range (<2.5 pmol/L), as a possible sign of euthyroid sick syndrome. Lastly, 16 participants were excluded because their fT4 levels were outside the reference range (six participants with <10.0 pmol/L and ten participants with >24.0 pmol/L), because of presumed thyroid disease or medication use affecting the thyroid function. Subsequently, participants with (log transformed) TSH or fT4 or one of the composite measurements ((log transformed) fT4xTSH product, (log transformed) fT4/TSH ratio, or fT3/fT4 ratio) that deviated more than three standard deviations from the population mean were excluded. Of the included 805 participants, 749 had passed away on the 1^st of February 2014. The median follow up time was 3.49 years, with interquartile range of 1.53 to 4.97 years.

Leiden 85-plus study

The Leiden 85-plus Study is a prospective, population-based study aimed at investigating determinants of successful aging [6]. Inclusion criteria were being 85 years old and living in Leiden, the Netherlands, between September 1997 and September 1999. No exclusion criteria based on health were applied. Out of 705 eligible individuals 599 participants were enrolled, since 92 people had refused and 14 were deceased before the assessment visit. Over a follow-up period of five years, participants were assessed annually until death or refusal. The study was approved by the Medical Ethical Committee of the Leiden University Medical Centre and all participants provided oral informed consent.

To investigate a similar age group as the Leiden Longevity Study, the current study used data of the participants of the Leiden 85-plus Study at the age of ninety years. Out of the 599 participants enrolled at 85 years, 52 had refused to continue participation and 270 were deceased before the assessment visit following their 90^th birthday. From the total of 277 nonagenarian participants who were eligible, three participants did not provide a blood sample and three had missing data on TSH level. One participant was excluded because the serum fT3 level was below the reference range (<2.5 pmol/L) and two participants were excluded for having serum levels of fT4 below the reference range (10.0 pmol/L). Of the remaining 268 participants, those with thyroid status parameters (where appropriate log transformed) that deviated more than three standard deviations from the population mean were excluded. In total, 259 participants were included, of which 254 had passed away on the 1^st of February 2014. The median follow up time was 3.84 years, with interquartile range of 1.90 to 5.89 years.

Chemical analyses

For both the Leiden Longevity Study and the Leiden 85-plus Study, non-fasted blood samples were collected from all participants between 09:30 hours and 17:00 hours at the assessment visit. Measurements of TSH, fT4 and fT3 were performed using the Modular E170 from Roche, Almere, the Netherlands. The reference ranges at our laboratory are 0.3-4.8 mU/L for TSH (detection range 0.005-100 mU/L), 10.0-24.0 pmol/L for fT4 (detection range 1.3-100 pmol/L) and 2.5-5.5 pmol/L for fT3 (detection range 0.400-50.0 pmol/L). High-sensitivity C-reactive protein (hsCRP) (detection range 0.3-350 mg/L) was measured using Cobas Integra 800 from Roche, Almere, the Netherlands. All coefficients of variation were below 5%. The measurements were performed in a single batch at the Department of Clinical Chemistry of the Leiden University Medical Centre.

Composite measurements of thyroid status

To assess the thyroid status in more detail, three composite measurements were computed. The product of fT4 and TSH (fT4xTSH) was calculated, to allow for assessment of the sensitivity of the pituitary thyrotrophs to feedback regulation by thyroid hormones as described previously [11,28]. Furthermore, the ratio of fT4 and TSH (fT4/TSH) was calculated, as a measure of responsiveness of the thyroid gland to TSH as proposed by Jansen et al. [11]. The ratio of fT3 and fT4 (fT3/fT4) was calculated as a measure of thyroid hormone conversion.

Statistical analyses

Because of the study design of the Leiden Longevity Study in which the men are younger than the women, all analyses were performed on men and women separately. Means and standard deviations, and where appropriate medians and interquartile ranges, were computed for parameters of thyroid status and age and hsCRP for male and female participants of both the Leiden Longevity Study and the Leiden 85-plus Study. Differences between men and women were tested using the T-test, Chi-Square test, or Mann-Whitney U test. Secondly, the participants of both studies were divided over sex-specific tertiles based on TSH, fT4, fT3, the fT4xTSH product, the fT4/TSH ratio, or the fT3/fT4 ratio. Survival analyses were performed using Cox regression on combined tertiles of men and women in both studies and in men and women separately using two statistical models. In model 1 for the Leiden Longevity Study, the analyses were adjusted for differences in age at entry by left truncated analysis and adding age at entry as an independent variable. Furthermore, robust standard errors were used to correct for siblingship by clustering on family number of the Leiden Longevity Study participants. For the Leiden 85-plus Study, model 1 was a crude analysis. In model 2, hsCRP was added as an independent variable, to adjust for inflammation, for both the Leiden Longevity Study and the Leiden 85-plus Study. The outcomes were presented as hazard ratios (HRs) for tertiles with the lowest tertile as a reference. To formally test whether the associations between parameters of thyroid status and mortality were similar for nonagenarians of long-lived families and the general population, interaction between study population (long-lived families or the general population) and parameters of thyroid status on mortality was tested by adding an interaction term and population of origin as covariates to the model 2 analysis excluding familial clustering. To assess a generalizable result for nonagenarian populations, the results were analyzed using a fixed-effects inverse-variance weighted analysis based on the coefficient for the highest tertile compared to the lowest tertile and its Standard Error in Cox regression. The output was transformed to HR. As sensitivity analyses we performed the same analyses in participants with hsCRP <10 mg/L, in participants who had passed away in the first two years of follow up, in participants who had survived the first year of follow up, and in participants with TSH levels within the normal range. In the analyses values of P<0.05 were considered significant. For data analyses Statistical Package for the Social Sciences (SPSS) version 23, the Stata Data analysis and Statistical Software for Windows version 12.0 SE and R version 3.2.4 for Windows were used. The graph was created using GraphPad Prism version 6.05 for Windows.