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Step 1 - Do not eat or brush your teeth 60 minutes prior to saliva sample collection.Step 2 - Rinse and swill mouth out thoroughly three times with tap or bottle water. Female Salivary daily Testosterone rhythm throughout the menstrual cycle for A) pre-menopausal women (age group 19-29, 30-39 years) and B) postmenopausal women (age groups 44-49, 50-59 and 60-69). Female salivary testosterone levels in women at 40-49 years showing episodic fluctuations of individual data points exceeding the 09.00 hours levels on some days (Data are mean of 12 females). Summary of Salivary Testosterone results in females: Average testosterone concentration per day in pmole. Step 3 - Chew a quarter of a stick of sugar free gum provided, as this will aid the production of saliva. Sharp2[1] Dietetics, Nutrition and Biological Sciences, Queen Margaret University, Edinburgh, UK[2] School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, UK1. Step 4 - Retain the sugar free gum in your mouth, and spit away the first mouthful of saliva into the waste container provided. IntroductionTestosterone (T) is one of the most important naturally circulating steroid hormones. This will remove unwanted cellular elements in the mouth and from the chewing gum.Step 5 - Continue chewing the gum and spit into the collection container provided, until you have deposited 3-5 millilitres (ml) of saliva.
Clinical contribution of monitoring salivary T in females for pathological and physiological conditions3. Exerting both androgenic and anabolic activities it is secreted into the blood and in men is produced primarily by the testes. In women, by contrast, production occurs in the ovaries and particularly from peripheral conversion of the T precursors androstenedione, DHEA and DHEA-S (Burger, 2002). T is no longer regarded as a male only hormone, and similarly estradiol is no longer a female only hormone (Fausto-Sterling, 2000).
Storage of saliva samplesIt is now known that bacterial growth can occur if saliva samples are stored above 4?C (i.e. While it is true that men generally have higher levels of T and lower concentrations of oestrogen and progesterone than women, all these sex steroid hormones play essential roles in both sexes (Ullis et al., 1999). Testosterone has now been well established as having an essential function in wide-ranging areas of female health.
In a recent report, it was found that bacterial growth in saliva increased by incubation at room temperature over 10 days, and this caused a significant decrease in salivary levels of both cortisol and T (Whembolua et al., 2006). By the end of 4 weeks, there was an increase of 330% in T concentration using a Salimetric assay.
The majority of these studies on T were conducted in men and the data published were conflicting. Clearly, in order for researchers to have confidence in their assay results a good deal of attention has to be paid to issues of collection, storage and compliance.5.
These equivocal findings resulted, at least in part, from different study designs and protocols (fibre content and type, subject compliance, lack of control affecting other dietary components) and the complex mechanisms involved in steroid metabolism. Subsequently, we have performed a pilot study to investigate the effect of increasing dietary fibre and found a moderate increase in urinary T excretion of healthy women taking a mixed high fibre diet for two weeks (unpublished observations), suggesting a potential correlation between dietary fibre intake and androgen status.
In-direct techniqueSolid phase and coated-technology assays can adopt either a direct or an in-direct approach to determining levels of T. Whilst several assays, including commercially available kits, employ a direct method (that is, salivary samples are not treated in any way prior to assay) there are a number of limitations with this approach, especially when examining female androgens. Whilst the benefits of utilising saliva over serum have previously been described, saliva is a far from inert substance; it contains a variety of contaminants, such as bacteria, leukocytes, mucins, and very importantly for enzyme assays, endogenous enzymes. As a consequence, salivary samples are rendered extremely susceptible to interfering agents such as pH imbalance which yields results that are, unpredictably, either too high or low (alkaline samples, for example, tend to yield low results). However, early attempts at determining concentrations of circulating T, primarily for clinical purposes, traditionally utilised plasma or serum. As blood samples require time-consuming and often stressful venipuncture, obtaining invasive multiple samples over a period of hours can be painful and this procedure can be unattractive to participants (Dabbs, 1990). At a round table discussion of sex hormones and corticosteroid assays, Adlercreutz (1990) citing a range of studies, noted that T assays do not work well in non-extracted plasma. Moreover, measurement tends to be of the total rather than free, biologically active, fraction of T. Faced with these challenges there has been a growing awareness of the potential value of utilising saliva for measuring hormone concentrations (Mandel, 1993; Collins, 2000). Consequently, the use of saliva as a diagnostic tool in clinical and bio-behavioural research has grown significantly during the last two decades (Quissell, 1993). This has prompted some authors to doubt the validity of female T assays and suggest perhaps guessing levels in women to be cheaper and more accurate!
Moreover, little is known about detailed daily patterns in female T throughout the menstrual cycle (Davis, 1999). One of the aims of this review article is therefore to discuss the importance of estimating salivary T in women and the challenges posed by its measurement in female saliva, including a summary of our in-house ELISA technique and the optimisation needed for the estimation of salivary T. Finally, we would like to highlight the importance of sampling protocol; multiple female salivary T sampling versus single saliva sampling. The importance of the cross-reactivity findings depends not only on the % cross-reactivity, but on the relative concentration of the compound compared against T. Using the extraction procedure removes the ability of DHEA-S to interfere with the ‘in-house’ assay optimised in our laboratory.6. Epidemiology and clinical importance of abnormal testosterone levels in femalesFemale sexual dysfunction is thought to affect over 40% of women in the United States, according to a study by Laumann and colleagues (1999).
Development of ELISA based assays for the measurement of testosterone in salivaA simple, reliable, easy to perform, sensitive and highly specific ELISA type assay for the measurement of female salivary T has been developed in our lab and utilized in several wide-ranging research projects investigating T levels in the menstrual cycle, circadian rhythm and bio-behavioural studies. As experts evaluate women with potential sexual interest disorders, there is a growing body of literature to guide them in how to understand, diagnose and treat these problems. This has enabled us to screen large number of samples within a short time at relatively low cost and with high sensitivity and assured specificity.
Androgens are known to be involved in women’s arousability, response, intensity and ease of orgasm, as well as in initial spontaneous desire, the active neurovascular smooth muscle response of swelling, increased lubrication and genital sexual sensitivity. T is thought to be the most important hormone for maintaining sex drive or libido in women and a deficiency can cause impaired sexual function (Snyder, 2001). Essentially, the T present in salivary samples competes with a fixed and limited amount of T coated on the micro-titre plate, for binding sites on an antibody.
In this regard, T may decrease vaginal atrophy as well as inflammation, itching and pain of the vulva (Leiblum et al., 1983).
Because the concentration of the T coated to the wells is held constant, while the concentration of T in the salivary samples vary, the amount of enzyme labelled second antibody bound to the first antibody is inversely proportional to the concentration of the unlabelled analyte present in the sample.Extensive experimentation was conducted in our laboratory to optimise this assay.
Because of the often extremely low levels of T in female saliva, one of the particular requirements for quantitative determination is that the assay be especially sensitive. However, the combined therapy is associated with a higher incidence of hair growth, acne and a reduction in high-density lipoprotein (HDL) cholesterol.

However, the ELISA process sits within a complex web of inter-locked parameters, and achieving this sensitivity requires a constant balancing act between reagents and conditions to arrive at and maintain the final protocol.
These adverse events may vary with differing doses, routes of T administration and individual differences. For example, one of the ways to minimise the impact of interfering factors is to extract the samples prior to assay. Reflecting on the theoretical and conceptual challenges of relating specific hormone levels to behaviour, there is no doubt that a relationship has not been established between levels of T and symptoms of sexual dysfunction in women. Indeed, high levels of T exert a significant negative effect on mood, personal sense of well being, interpersonal relationships, self-confidence and self-worth, and depression is a major symptom associated with low levels in women (Sands & Studd, 1995).
Optimized ELISA Procedure100µL of standard and previously extracted and reconstituted sample into wells in duplicate.
It has been suggested that postmenopausal women who are not receiving some T therapy may have greater risk of developing coronary heart disease (Rako, 1998).
The effects associated with administering exogenous T are not especially straightforward however. For example, when T is administered alone, it can increase the risk of atherosclerosis and decrease HDL levels (Crook & Seed, 1990). Body composition measures were found to be significantly and positively associated with total T concentrations in a dose-response manner (Sowers et al, 2001). Inter-assay imprecision of 8.7% was determined from the mean of averaged duplicates for 50 separate runs for male and female aliquots.
Both inter and intra assay coefficients of variation are at levels comparable with the best commercially available assay kits.
In light of this, T replacement may markedly decrease osteoporosis in postmenopausal women, and together with oestrogens the steroids can preserve and rebuild the cartilages between bones (Tremollieres et al., 1992). In the skin, T can improve the overall skin appearance by preserving collagen and protecting against thinning of the skin as well as sebaceous glands activity that lubricates the skin (Brincat et al., 1987).
As we begin to more fully understand the above actions, the therapeutic use of exogenous T in women is becoming increasingly widespread, although not always without often unwanted side effects. Thousands of women have been treated with T, the majority experience symptom improvement, improved sexual well-being (Davis and Davison, 2012). The applications of our salivary T ELISA can be seen in a wide-range of clinical and bio-behavioural studies (e.g. Other possible beneficial effects of T therapy (reduced fracture risk, improved cognitive and cardiovascular function), necessitate further investigation. This marked fall in peripheral androgens is associated with a number of conditions including metabolic syndrome and osteoporosis (Davy & Melby, 2003). In addition, the Women’s Health Initiative (2002) published their results from a prospective randomised prevention trial of more than 16000 healthy postmenopausal women which was intended to study the long term effects of HRT given as a combination of conjugated equine oestrogens and medroxyprogesterone acetate. Although decreased risks of colorectal cancers and hip fractures were reported, many women came off their hormone supplements and explored alternatives because of the reported increased risk of stroke and invasive breast cancers.
Likewise, a large-scale UK study (Beral, 2003) found that current use of HRT, but not past use, was associated with an increased risk of breast cancer. Clinical contribution of monitoring salivary T in females for pathological and physiological conditionsTo date, a definite relationship has not been established between a specific level of T and symptoms of T excess or deficiency such as sexual dysfunction in women and premenstrual symptoms.
There is no established level of free T below which a woman can be said to be deficient, nor any level to which a woman should be restored that determines she is replete. Thus the diagnosis of these disorders due to low testosterone remains a clinical diagnosis of exclusion. There is a paucity of research which investigates salivary female T in pathological and physiological conditions.
For this reason we would like to highlight the following examples of clinical monitoring of female salivary T. Salivary T has been used to monitor treatment of children with congenital adrenal hyperplasia (CAH), Salivary T was found to be a useful additional biochemical marker with 17OHP to indicate the levels of free T (Perry et al., 2005). In a study comparing salivary and total plasma testosterone levels in healthy controls and patients with Klinefelter’s syndrome (Wellen et al.,1983) provided indirect evidence that in Klinefelter patients levels of salivary T and androstenedione correlated well with the reported free plasma levels. This suggests that measurement of salivary steroids may be useful in evaluating endocrine function in both healthy and disease states. These findings corroborate earlier studies, which found higher post-exercise plasma sex steroid levels. Several studies have published data on the applicability and clinical value of salivary T measurements for the diagnosis and follow-up of therapy of idiopathic hirsutism, late-onset hypogonadism and androgen deficiency in end-stage renal disease (e.g. Manni and colleagues (1985) indicated that albumin-bound T was available to tissues such as the brain in conditions where the free T level was negligible and in the absence of albumin. This hypothesis was contested by Ekins (1990) and Mendel (1989) based on the assumption that steroids can act only through their genomic mechanism.
As an adjunct to this latter point it should be noted that around 50% of pre-menopausal (and almost 100% of post-menopausal) T is synthesised by peripheral conversion, distinct from any endocrine function (Labrie et al., 2000). In addition, the fact that many steroids are now shown to exert some effects through the non-genomic pathway (see below) leads us to suppose that some actions, particularly in the female, are mediated by the albumin-bound fraction of T. Moreover, in a recent review Lepage (2006) demonstrated clearly the importance of measuring the bio-available T as compared to total and free T. Thus, free and non-SHBG-bound (sometimes called bio-available) T measures are likely to be the most reliable indicators of tissue exposure to T (Collins, 2000). It was therefore concluded that the measurement of androgen glucuronides instead of T perhaps better reflects the androgenic activity in women (Labrie et al., 2006).
Total concentrations of T in peripheral tissue and body fluids are mainly dependent upon the levels of binding proteins such as sex hormone binding globulin (SHBG) and albumin.
These binding proteins can act as a reservoir for the steroid and protect it against extensive metabolism of active (free) steroids during passage of the blood through the liver (Mendel, 1989). It is now widely accepted that the free steroid form represents the biologically active fraction. T, like other steroids, may exert its action in living cells by either the well-known genomic pathway, involving hormones binding to a cytosolic receptor and subsequent modulation of gene expression followed by protein synthesis (genomic actions).
Or through pathways that do not act on the genome (non-genomic actions) (Losel et al., 2003).
T has recently been reported to exert effects through interactions with receptors in the cell membrane (Heinlein & Chang, 2002).
Once a medium in which to analyse T has been selected (saliva in this case) the question then becomes what type of assay should be employed? In both saliva and blood, RIA was until comparatively recently the method of choice for determining concentrations of circulating T.
Despite a number of limitations, traditional analogue RIA is still widely used (Rosner, 2001). This situation arises because most T RIA's were designed for the measurement of serum levels in men and thus lack the sensitivity required for the precise measurement of the low levels prevalent in women.Even now, simple and routine methods for determining free steroid concentrations in plasma have not been fully developed and widely validated. All are mathematical formulae of varying complexity for working out the free component of T based on the measurement of total T and, as a minimum, SHBG.

Faced with these challenges, there has been a growing awareness of the potential value of utilising saliva for measuring hormone concentrations (Mandel, 1993). In a clinical paper on screening for androgen insufficiency, Guay (2002) argued that a major problem in assessing female T is the inaccuracy of the measurements by current assays. This issue of measurement therefore acts as one potentially serious limiting factor in the confidence that we can place in the results of hormone-behaviour studies, clinical studies and for female’s screening before T therapy. For example, reagents are cheap in comparison with RIA, the laboratory in which our research is undertaken employs staff with substantial expertise in ELISA development, validation and trouble-shooting. Moreover, there is no hazard of radiation, and because of the low concentrations of antigens in saliva HIV and hepatitis infections are much less of a danger from saliva than from blood (Major et al., 1991). Indeed, unless visibly contaminated with blood, human saliva is not considered a class 2 biohazard, affording researchers and institutions administrative and safety benefits. As a consequence of these factors the ELISA method stands out as the most appropriate routine method of choice for use in the majority of research institutes conducting bio-behavioural research and clinical work on T.There are a number of features that are required of an effective assay.
As Kemeny and Chantler (1988) note, the type of assay should be closely tailored to the particular task for which it is required; and the requirements of diagnostic laboratories are often very different than those of bio-behavioural research laboratories.
For example, in bio-behavioural studies that require assessing chronobiological changes in the very low levels of free salivary T in females, issues such as ease of use and speed are less important characteristics than sensitivity and accuracy. Subsequently, one of the additional aims of this chapter is to describe the development, optimisation, and validation of an extremely sensitive in-house ELISA, designed specifically for determining salivary T in women.
In particular, the ELISA is evaluated for its accuracy, specificity, and precision.Salivary sampling regimens have several obvious and distinct advantages over blood sampling.
They accommodate frequent and easy collection by non-invasive, relatively stress free-techniques, thereby facilitating short term dynamic tests, pharmacokinetic analyses, and studies of chronobiological changes (Riad-Fahmy et al., 1982).
In addition, it has been reported that the majority of subjects find little difficulty in salivating directly into collection containers, providing adequate volumes (between 3mL and 5mL) for determining a steroid hormone profile in less than 5 minutes (Dabbs, 1991). Nonetheless, a number of challenges and problems associated with saliva methodology do exist (Granger et al., 2004). For example, compliance with salivary sampling protocols has been identified as a potential challenge. Compliance to collection protocols is essential for accurate determination of T levels in saliva and concerns amount collected, condition of sample and precautions taken prior to collection (i.e. Our own experience is that engagement with participants and the provision of detailed oral and written instructions alongside the opportunity to practice with an investigator present to answer questions and provide guidance ameliorates significantly issues of compliance.Although diurnal and monthly patterns of salivary T generally parallel serum values, absolute ranges show variability across several studies. Few studies of normal individuals, controlling for known variables, such as pH, time of day, month and medications, have been performed using the recently developed high sensitivity enzyme immunoassays, such as those sold by Diagnostic Systems Laboratories (Webster, TX), Salimetrics (State College, PA) and American Laboratory Products Co. Blood-saliva correlationAs Albumin is a small protein, with a molecular weight of approximately 69,000, it can pass through the salivary membrane.
Conversely, SHBG (to which the majority of circulating T is bound) is a large carrier protein with a molecular weight of 150-200,000. Shirtcliff and co-workers (2002) published a study which appears to cast doubt on the veracity of this correlational relationship in females, stating that regardless of assay method, salivary T levels are modestly correlated with serum levels for males but not necessarily females. The absolute concentration, whilst reflecting accurately the unbound fraction in the plasma (and also the fraction which is not bound to SHBG) is approximately twice the concentration of free-T in plasma, in contrast to the findings in male subjects (Baxendale, Jacobs & James, 1982).
One major implication of these findings is that substitution of saliva for serum T levels in at least bio-behavioural studies may estimate the T-behaviour relationship differently for females than males because substitution of saliva assay results for serum values markedly underestimates known T-behaviour associations.
In addition, there might be some ethnic variation in total and free T concentration in that Heald et al.
Contamination of salivary samplesClinical researchers have established that collection techniques can affect the integrity of salivary samples, which subsequently interferes with ability to accurately determine hormone levels. For example, given the difference in T concentration between blood and salivary samples, leaking of blood or serum into the mouth (i.e.
In response to a question about the incidence of haemoglobin contamination in salivary samples, at a round table discussion on assay development and collection procedures (Tenovus workshop, 1982), Schurmeyer suggested that researchers might reasonably expect a contamination incidence of around 5-10%.
The majority of bio-behavioural studies investigating the role of T in women propose that single time-point or very limited sampling is sufficient. Whilst this issue is explored in more detail later in this chapter, it is worth noting here that potential contamination of salivary samples by blood further complicates the use of single samples. In addition, it is noted that where samples have been found with aberrant levels, researchers have on occasion been treated by diluting the samples (i.e. Whilst this step may be appropriate for male salivary samples, dilutions of female salivary samples which may already contain levels of T at the utmost sensitivity of the assay are rendered un-determinable.Whilst the contamination of salivary samples by blood is widely recognized as a potential problem in the scientific fields driving assay development there appears to be something of a malaise regarding the issue in a wide range of bio-behavioural studies.
Indeed, with only a handful of notable exceptions a large number of authors have neglected to report how they have dealt with this serious confound. In utilising measurement strips called Hemastix® they attempted to ascertain which, if any, of their salivary samples may have become contaminated with blood. Other methods have recently been developed to assess blood contamination in salivary samples.
For example, Salimetrics now offer an assay to determine blood contamination in salivary samples (Schwartz & Granger, 2004), although this approach adds substantially to the cost of assaying samples. Perhaps the best approach to reducing blood contamination is avoiding its occurrence in the first place.
In our own lab, and following suggestions by Adlercreutz (1990), our subjects were asked to adhere to a number of steps including refraining from brushing their teeth prior to sample collection, rinsing their mouths thoroughly several times prior to collection, not consuming large meals, and not smoking or drinking caffeine. Collection methodsWith the increased use of salivary measures in clinical practice, bio-behavioural and clinical research, several research teams have sought to advance our understanding of the circumstances and conditions that may influence the validity of salivary assessments (e.g.
Granger and co-workers (2004) found that materials commonly used in the literature to absorb saliva (cotton and polyester swabs) or stimulate saliva (powdered drink-mix crystals, citric acid and chewing gum) have the potential to change salivary T results.
Typically, saliva is collected by having a participant deposit between 3-5mL into a collection container (less may be required in some circumstances); this step is usually reported as taking between 3-8 minutes. While clinical subjects may be willing and able to provide un-stimulated samples that can be immediately frozen, this protocol is often unpractical for field collection (Lipson & Ellison, 1989). As such, collection of saliva samples under these conditions may necessitate certain changes from common clinical practices. Although collection of saliva is often referred to as un-stimulated, this is somewhat misleading. If the salivary gland is un-stimulated it does not produce saliva; what is meant essentially is saliva produced by minimal stimulation (Read, 1989). Small amounts of saliva can be collected without the need to externally stimulate production, but the amount usually collected often requires subjects to stimulate saliva production in some way.
This issue is particularly pertinent when investigating changes in concentration of salivary hormones over a certain period of time or prior to competition, where time constraints exist and feelings of stress or anxiety may make saliva production more troublesome.
We have optimised our own salivary samples collection method (see Table 1) that ensures the quality of saliva, reproducibility, minimises blood contamination and stress during collection.

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