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Posted by Dr. Alison McAllister on Friday, 23 December 2016

A Year of Saliva Testing in Research


I wanted to share a few of the studies using saliva testing that were published in the literature in 2016.

Just a hint, if you want to search for articles on saliva testing, use and search saliva estradiol (or progesterone, testosterone etc.).

Be aware that saliva hormone testing is so standard in the research world that rarely is it mentioned in the titles of the papers. Over 310 studies were published in 2016 using saliva testing – 61 studies for testosterone and 189 studies on cortisol, while poor progesterone was neglected with only 14 studies this last year.

It’s always good to know that the work that ZRT has been doing for almost 20 years in the field of saliva testing is still cutting edge and useful. 

Estradiol Study

Fjeldheim FN et al. Polymorphisms in the estrogen receptor alpha gene (ESR1), daily cycling estrogen and mammographic density phenotypes. BMC Cancer. 2016;16(1):776.

Take home: Women who have genetic SNPs, even in premenopausal status, have higher estradiol levels and higher breast density. Genetic SNP for CYP19 1A1 (aromatase gene) and breast density and CYP17 was associated with increased metabolic risk factors (obesity etc). Approximately 40% of the world’s population appears to have some mutation for the CYP19A1 gene. This study also made nice mention of the fact that measuring estradiol this way measures the unbound bioavailable estradiol and that salivary immunoassays correlate nicely with LCMS levels.



Single nucleotide polymorphisms (SNPs) involved in the estrogen pathway and SNPs in the estrogen receptor alpha gene (ESR1 6q25) have been linked to breast cancer development, and mammographic density is an established breast cancer risk factor. Whether there is an association between daily estradiol levels, SNPs in ESR1 and premenopausal mammographic density phenotypes is unknown.


We assessed estradiol in daily saliva samples throughout an entire menstrual cycle in 202 healthy premenopausal women in the Norwegian Energy Balance and Breast Cancer Aspects I study. DNA was genotyped using the Illumina Golden Gate platform. Mammograms were taken between days 7 and 12 of the menstrual cycle, and digitized mammographic density was assessed using a computer-assisted method (Madena). Multivariable regression models were used to study the association between SNPs in ESR1, premenopausal mammographic density phenotypes and daily cycling estradiol.


We observed inverse linear associations between the minor alleles of eight measured SNPs (rs3020364, rs2474148, rs12154178, rs2347867, rs6927072, rs2982712, rs3020407, rs9322335) and percent mammographic density (p-values: 0.002-0.026), these associations were strongest in lean women (BMI, ≤23.6 kg/m2.). The odds of above-median percent mammographic density (>28.5 %) among women with major homozygous genotypes were 3-6 times higher than those of women with minor homozygous genotypes in seven SNPs. Women with rs3020364 major homozygous genotype had an OR of 6.46 for above-median percent mammographic density (OR: 6.46; 95 % Confidence Interval 1.61, 25.94) when compared to women with the minor homozygous genotype. These associations were not observed in relation to absolute mammographic density. No associations between SNPs and daily cycling estradiol were observed. However, we suggest, based on results of borderline significance (p values: 0.025-0.079) that the level of 17β-estradiol for women with the minor genotype for rs3020364, rs24744148 and rs2982712 were lower throughout the cycle in women with low (<28.5 %) percent mammographic density and higher in women with high (>28.5 %) percent mammographic density, when compared to women with the major genotype.


Our results support an association between eight selected SNPs in the ESR1 gene and percent mammographic density. The results need to be confirmed in larger studies.

Progesterone Study

Lovick TA et al. A specific profile of luteal phase progesterone is associated with the development of premenstrual symptoms. Psychoneuroendocrinology. 2017;75:83-90.

Take home: Women (46 of them at least) who had PMS actually had stable progesterone levels throughout the luteal phase until 3 days prior to menses, when progesterone levels sharply dropped. Women without PMS had a gradual decline in progesterone in the 8 days prior to menses. Both groups of women had approximately the same upper and lower levels of progesterone. This shows that it is likely the dramatic shift in progesterone levels that contributes to PMS symptoms rather than a drop to a certain level.


Neither maximum nor minimum concentrations of progesterone in the two groups were related to the appearance or severity of premenstrual symptoms.

There is a consensus that the development of premenstrual dysphoric states is related to cyclical change in gonadal hormone secretion during the menstrual cycle. However, results from studies seeking to link symptom severity to luteal phase progesterone concentration have been equivocal. In the present study we evaluated not only the absolute concentrations of progesterone but also the kinetics of the change in progesterone concentration in relation to development of premenstrual symptoms during the last 10days of the luteal phase in a population of 46 healthy young adult Brazilian women aged 18-39 years, mean 26.5±6.7years. In participants who developed symptoms of premenstrual distress, daily saliva progesterone concentration remained stable during most of the mid-late luteal phase, before declining sharply during the last 3days prior to onset of menstruation. In contrast, progesterone concentration in asymptomatic women underwent a gradual decline over the last 8days prior to menstruation. Neither maximum nor minimum concentrations of progesterone in the two groups were related to the appearance or severity of premenstrual symptoms. We propose that individual differences in the kinetics of progesterone secretion and/or metabolism may confer differential susceptibility to the development of premenstrual syndrome.

Testosterone Study

Afrisham R et al. Salivary Testosterone Levels Under Psychological Stress and Its Relationship with Rumination and Five Personality Traits in Medical Students. Psychiatry Investig. 2016;13(6):637-643.

Take Home: Men respond to stress by increasing testosterone; women respond to stress by decreasing testosterone. There was some correlation with emotional management, testosterone, and gender in how individuals responded to stressors that bears further study.



The purpose of this study was to evaluate the salivary testosterone levels under psychological stress and its relationship with rumination and five personality traits in medical students.


A total of 58 medical students, who wanted to participate in the final exam, were selected by simple random sampling. Two months before the exam, in the basal conditions, the NEO Inventory short form, and the Emotional Control Questionnaire (ECQ) were completed. Saliva samples were taken from students in both the basal conditions and under exam stress. Salivary testosterone was measured by ELISA. Data was analyzed using multivariate analysis of variance with repeated measures, paired samples t-test, Pearson correlation and stepwise regression analysis.


Salivary testosterone level of men showed a significant increase under exam stress (p<0.05). However, a non-significant although substantial reduction observed in women. A significant correlation was found between extroversion (r=-0.33) and openness to experience (r=0.30) with salivary testosterone (p<0.05). Extraversion, aggression control and emotional inhibition predicted 28% of variance of salivary testosterone under stress.


Salivary testosterone reactivity to stress can be determined by sexual differences, personality traits, and emotional control variables which may decrease or increase stress effects on biological responses, especially the salivary testosterone.


Ge F et al. Concurrent and prospective associations between HPA axis activity and depression symptoms in newlywed women. Psychoneuroendocrinology. 2016;73:125-132.

Take home: DHEAS is an important tool to assess HPA axis in regards to measuring hormones and depression and must be used in addition to cortisol. This study definitely doesn’t answer all questions regarding stress, and in fact suggested that individuals with higher levels of depression may have more HPA axis activity during stressors just as much as those with HPA axis dysfunction may be more prone to depression. One thing I liked about this study was that they pointed out that they measured DHEAS in saliva because it is the most biologically active molecule as compared to DHEA. They also pointed out that salivary flow rate was not a problem during collection which has been a common criticism of using DHEAS as a measurement tool.


We investigated the extent to which individual differences in activity of the hypothalamic pituitary adrenal axis (HPA) are associated with depressive symptoms among newlywed couples. Participants were 218 couples (M age 28.4 years; 94% White) who provided 5 saliva samples (later assayed for cortisol and DHEA-S) before and after participation in a discussion of a major area of disagreement in their relationship. Depressive symptoms were assessed initially, and approximately 19- and 37-months later. Results revealed an interactive effect suggesting that concordant levels of cortisol and DHEA-S (either both high or both low) were concurrently and prospectively associated with higher depression scores. Interestingly, this interactive effect was observed for wives only - not for husbands. These observations underscore contemporary theoretical assumptions that the expression of the association between HPA activity and depression is dependent on factors related to the interaction between characteristics of the person and features of the social environment, and moderated by co-occurring variation in endocrine milieu.

Cortisol Study

Poole L et al. Depression 12-months after coronary artery bypass graft is predicted by cortisol slope over the day. Psychoneuroendocrinology. 2016;71:155-8.

Take home: I purposely pulled an article regarding the diurnal rhythm of testing cortisol.  I believe that in the quest to understand cortisol and the HPA axis dysregulation that can occur, practitioners are commonly getting confused and thinking that diurnal rhythm testing isn’t important vs. diurnal circadian rhythms. There are several interesting articles on night shift work changing circadian rhythms of cortisol, but I wanted to pull an article on a different aspect of cortisol rhythms.


Alterations in the diurnal profile of cortisol have been associated with depressed mood in patients with coronary heart disease. The relationship between cortisol output and depressed mood has not been investigated prospectively in coronary artery bypass graft (CABG) patients before. We aimed to study the relationship between cortisol measured pre- and post-operatively and depression symptoms measured 12 months after CABG surgery. We analysed data from 171 patients awaiting first-time, elective CABG surgery from the pre-assessment clinic at St. George's Hospital, London. The Beck Depression Inventory (BDI) was used to assess depression symptoms and saliva samples were collected to measure diurnal cortisol. Baseline assessments of depression and cortisol were obtained an average 29days before surgery, short-term follow-up of cortisol occurred 60 days after surgery and long-term follow-up of depression was assessed 378 days after surgery. Baseline cortisol slope was not associated with depression at 12-month follow-up. However, a steeper cortisol slope measured 60days after surgery predicted reduced odds of depression (BDI≥10) 12 months after surgery (odds ratio 0.661, 95% confidence interval 0.437-0.998, p=0.049) after controlling for covariates. These findings suggest interventions aimed at improving adaptation in the early recovery period may have long-term benefits in this patient group.

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Tagged in: Saliva Testing Testosterone Progesterone Adrenal / Cortisol Estrogen Research DHEA