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  • The study sample was drawn from two neighbouring indigenous rural Kaqchikel Mayan communities located in the southwest highlands of Guatemala. All of the participants were Kaqchikel Maya with at least five generations of traceable ancestors and a high degree of endogamy, which reduces genetic variability among individuals in this population compared to that of industrialized settings. Lifestyle was relatively homogenous amongst the participants. Specifically, women had similar diets, levels of physical activity, education, and socio-economic status. This homogeneity in lifestyle reduces the potential effects of confounding factors, thereby increasing our statistical power to detect the relationships of interest. Participants were originally recruited in the year 2000 for the Society, Environment and Reproduction (SER) study, a prospective, naturalistic, longitudinal study that focused on the relationship between “real life” daily stress and women’s reproductive function [27, 28]. Recruitment in 2000 was conducted using the following inclusion criteria: women had to be parous, cohabitating with a male partner and not using any form of chemical contraceptive method [27, 28]. Of the 107 original SER participants, 94 volunteered to partake in the current study. In 2013 (13 years after the onset of SER), those 94 women ranged in age from 29 to 53. None of the participants smoked. Data collection and analysis in 2000 were approved by the University of Michigan’s Institutional Review Board. Secondary analysis of data collected in 2000 was approved for this study by Simon Fraser University’s Ethics Review Board. Data collection and analysis in 2013 were approved by Simon Fraser University’s Ethics Review Board and the University of British Columbia Clinical Ethics Review Board. Informed consent was obtained orally from illiterate individuals and in written form from literate ones. In all cases the consent document was read in Kakchiquel Mayan by a female research assistant to each prospective participant and signed by the participants with a cross, finger print or name initials, according to their individual preferences. The University of Michigan’s Institutional Review Board approved the 2000 consent procedure, and Simon Fraser University’s Ethics Review Board approved the one used in 2013. In 2013 participants were asked to complete a short verbal demographic interview. The interview was administered in Kaqchikel (the native local language) by trained, local, bilingual female research assistants, and all answers were recorded by hand. The demographic interview included questions about each participant’s age, age at her first birth, maternal parity, inter-birth intervals, offspring survival, family income, diet, alcohol consumption and smoking habits. This questionnaire was used to determine each participant’s reproductive life history. “Change in number of surviving offspring” was defined as the number of children alive in 2013 that were born between 2000 and 2013. “Total number of surviving offspring” was defined as the number of children born to a woman since the onset of her reproductive career who were alive at the end of our observation period in the year 2013. Of the 94 women who volunteered for the current study, 75 completed the 2013 demographic interview and provided biospecimens in both 2000 and 2013. TL was assessed for each participant at two time points 13 years apart (2000 and 2013). This is a sample of convenience. In the year 2000 only salivary specimens were collected, while in 2013 all participants provided a buccal epithelial cell sample. Thus, TLs in 2000 were measured in salivary specimens, while TLs in 2013 were measured in buccal epithelial cells. Importantly, saliva and buccal samples both contain a large number of epithelial cells [29], and TL in different tissues within an individual are known to be highly correlated [30–34]. Salivary specimens were collected by passive drool, transported on ice to the field laboratory, aliquoted into 2ml cryo-vials and stored at -10°C within 4 hours of being collected. The specimens were then transported from the field site to our laboratory on dry ice where they were stored at -80°C until analysis. Buccal specimens were collected using a SK-1 Isohelix Buccal Swab (Cat. No: SK-1S) with a Isohelix Dri-Capsule (Cat. No: SGC-50) to ensure the long-term stability of buccal DNA on the swab head. Buccal specimens were collected according to the manufacturer’s instructions. Briefly, the sterile swab was removed from the tube, inserted into the mouth of the participant and rubbed firmly for 1 minute against the inside of the right cheek. The swab was then placed back into the tube along with a Dri-Capsule and the tube was closed and secured. Buccal samples were transported to the field laboratory and stored at room temperature in a dry, dark cabinet for approximately 2 months before being transported at room temperature from the field site to our laboratory where they were stored following the same storage protocol until analysis. Genomic DNA was extracted from saliva and buccal samples. Saliva cells were washed with sterile PBS prior to a standard ethanol precipitation extraction using the Gentra PureGene Cell Kit (Qiagen, Hilden, Germany). Buccal samples were extracted with the DDK DNA Isolation Kit (Isohelix Ltd, Kent, UK), following the manufacturer’s instructions. All DNA samples were diluted to 10ng/uL in 10mM Tris, 0.1mM EDTA buffer. Average relative TL was measured using quantitative PCR (qPCR) as previously described [35]. Although measurement of TL by Southern blot of the terminal restriction fragment (TRF) is considered the ‘gold standard’ in the field, qPCR is also a widely used and accepted technique for assessing TL. qPCR is particularly useful in population-based studies, such as this one, because it requires much smaller amounts of DNA [36]. An advantage of the qPCR-based method is its high reproducibility [37, 38], which has resulted in a recent increase in the use of qPCR for TL measurement [36]. Briefly, SYBR green was used to quantify the amplification of telomeric repeats relative to the single-copy gene, 36B4. The design of the telomere primers allows for amplification of the smallest possible amplicon (76bp); as a result, the qPCR amplification (CT value) is proportional to the amount of primer binding sites in the genomic DNA template [39]. qPCR does require attentive preparation and stringent criteria to be reproducible due to higher inter-assay variation in comparison to other methods [37, 38]. For that reason, a stringent criteria was applied to our methodology: samples were run in triplicate in each 96-well plate, with at least two independent replicates. Furthermore, we used a rigorous threshold for the variation between replicates as CT values with a standard deviation greater than 0.20 between triplicates were excluded. If the coefficient of variation between the two independent replicates was less than 0.15, the replicates were averaged to obtain a relative TL for each sample. For those samples that did not meet these criteria, two additional independent replicates were performed using the same quality control thresholds. Average relative TL for each sample was expressed as the ratio of the telomere repeat copy number to the single-copy gene copy number (T/S ratio). The T/S ratio has previously been shown to be proportional to the average TL of a given sample [39]. We have previously validated the reproducibility of our methods by calibrating and optimizing our qPCR methodology by blind comparison with sample measurements obtained by the flow FISH technique (r = 0.96; [35]). Two separate multiple regression models were used to evaluate the relationships between TL in 2013 and 1) “change in number of surviving offspring (2000–2013)”, maternal age at first birth and average inter-birth interval, as well as 2) “total number of surviving offspring”, maternal age at first birth and average inter-birth interval using JMP (version 12; SAS Institute). We tested for multicollinearity amongst the variables in each multiple regression model by examining each variable’s variance inflation factor (VIF) [40]. All VIF values were less than 10, indicating that these variables were not correlated with each other [40]. As TL in 2000 and 2013 were measured in different tissues, we decided to take a conservative approach and include salivary TL in 2000 as a covariate in our model to control for each participant’s initial TL in 2000 rather than to use it to calculate change in TL between 2000 and 2013. Saliva and buccal samples contain a large number of epithelial cells [29], and TL in different tissues within an individual are known to be highly correlated [30–34]. Women’s ages in 2013 were also included as a covariate in both models. Significance levels were set at α = 0.05.
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