Sex hormonal evaluation in the clinical setting is typically performed on a serum or plasma specimen (Kaufman & Vermeulen, 2005) and can provide information about total and free circulating sex hormones. This involves an invasive procedure (venipuncture). Most steroid hormones in blood are bound to specific proteins (e.g., sex hormone–binding globulin [SHBG]). A tiny fraction (less than 5%) of nonbound or free fraction of sex hormone accounts for actual sex hormone activity at the tissue level. Free sex hormone molecules are small relative to their bound, protein–steroid complex; when free, these molecules can penetrate small membranes such as those found in the salivary gland. Salivary sex hormone measures quantify free, or bioactive, sex hormone concentration (Lu, Bentley, Gann, Hodges, & Chatterton, 1999; Worthman, Stallings, & Hofman, 1990). Salivary measures offer a relatively convenient and minimally invasive approach for obtaining sex hormone data (Granger, Shirtcliff, Booth, Kivlighan, & Schwartz, 2004; E. Kaufman & Lamster, 2002; Worthman et al., 1990)—an advantage when working with older, infirm, and/or population-based health study participants. Saliva specimens can be self-collected and safely handled by lay personnel (Granger et al., 2007).

The methods used for, correlates of cooperation with, and validity of in-home salivary specimens collected from older adults in NSHAP are described.

Estrogen metabolism and synthesis in men appear to remain relatively stable across the life course (Kaufman & Vermeulen, 2005). In contrast, major changes in sex hormone metabolism occur in women around natural menopause, most notably due to a decline in ovarian estrogen production (Manly et al., 2000). Although the human lifespan is increasing, the mean age of menopause has not changed as rapidly. Life expectancy at birth for American women increased from 56.2 years for women born in 1910 to 69.4 years for women born in 1940 (158 month increase) (Bell & Miller, 2002). At the same time, the mean age at natural menopause for U.S. women born in 1915 versus 1939 increased from 49.1 to 50.5 years (16.8 month increase) (Nichols et al., 2006). Women are therefore living increasingly longer in a menopausal state.

Relative estrogen depletion in women in later life has been associated with a variety of age-related changes in physical and cognitive function as well as disease. Among men and women, serum estradiol concentration has been associated with bone turnover and risk for osteoporotic fractures (lower estrogen is associated with skeletal vulnerability) (Kuchuk et al., 2007). Likewise, estradiol concentration has been associated with cognition (Carlson et al., 2001; Maki & Resnick, 2000; Tivis et al., 2005), mood, and memory (Tivis et al., 2005) in women and, in combination with testosterone levels and other factors, with preservation of memory and cognitive function in men (Barrett-Connor, Goodman-Gruen, & Patay, 1999; Carlson & Sherwin, 2000). A number of studies have linked higher levels of serum estradiol to increased risk for developing breast cancer (Chlebowski et al., 2003; Clemons & Goss, 2001; Tivis et al., 2005) and coronary problems (Chlebowski et al., 2003; Clemons & Goss, 2001; Manson et al., 2003; Tivis et al., 2005) in women. Much less is known about the role of estrogen in social functioning in general or in later life.

Estrogen depletion in menopause and sustained, low levels of circulating estrogen throughout the postmenopausal lifespan significantly affect the condition and function of the female genital tract. Estrogen is important for maintaining skin, subcutaneous, mucosal (vaginal, bladder, and rectal) and musculoskeletal integrity, the vaginal microenvironment (pH balance and microflora), vascular flow to the vagina and clitoris, and sensory perception. Over time, in the genital tract, low estrogen results in vaginal dryness, loss of epithelial cell glycogen, shortening of the vagina, narrowing of the introitus, thinning of the labia, and diminution of the fat pad underlying the mons pubis. Though the role of estrogen in female sexuality is not fully understood, it may influence sexual desire (Dennerstein, Gotts, et al., 1994; Meston & Frohlich, 2000). Estrogen replacement therapy may indirectly enhance female sexual performance, by restoring vaginal lubrication (Meston & Frohlich) and promoting positive body image and an overall positive sense of well-being (Bachmann & Leiblum, 2004). Additionally, sexual activity and other forms of physical contact may mitigate estrogen-related decline in sensory perception and function with age and may play a role in female attractiveness. Largely due to highly publicized findings in 2002 from the randomized, controlled hormone therapy trials of the U.S. Women's Health Initiative Study (comparing use of equine estrogen alone or in combination with medroxyprogesterone to placebo among post menopausal women ages 50–79 years at baseline) (Rossouw et al., 2002), a minority of women currently initiate estrogen therapy for treatment of menopausal symptoms or preservation of sexual function (Newton et al., 2008).

Progesterone is found in both women and men, although the physiological role in men is poorly understood (Andersen & Tufik, 2006). In reproductive age females, the primary function of progesterone involves preparation and maintenance of the endometrium for implantation of fertilized oocytes (Speroff et al., 1999). In women, progesterone is synthesized from pregnenolone in the adrenal cortex, corpus luteum of the ovary, in the brain, and during pregnancy, by the placenta. Due to decline in ovarian production, progesterone production is markedly decreased following menopause. This decline may result in diminished sexual function and desire (Dennerstein, Alexander, & Kotz, 2003). Increased levels of progesterone have been found in states of stress and anxiety in men and women; this may relate to its sedative or stress-counteracting effects (Wirth, Meier, Fredrickson, & Shcultheiss, 2007; Wirth & Schultheiss, 2006). Exogenous progesterone is used in older women primarily in combination with estrogen therapy to protect against estrogen's growth-promoting effects on the uterine endometrial lining and to treat pathological processes of the uterine endometrium. In men, exogenous progesterone has been used to reduce sexual activity, a mechanism of action that may relate to its antiandrogenic properties (Andersen & Tufik, 2006).

Testosterone is the defining male sex hormone, is more abundant in males than females, but appears to play an important physiological role in sexual functioning of both. Testosterone is synthesized in the male testes, the female ovaries, and the adrenal glands in both sexes (Davis & Tran, 2001; Guyton, 1991). During the aging process, testosterone levels gradually decline in both sexes (Davis & Tran; Ellison et al., 2002; Feldman et al., 2002; Lobo, 2001; Tenover, 1997). Because the ovaries are a primary, and relatively stable, source of female testosterone production throughout the life course (Davis & Tran, 2001), premenopausal bilateral oophorectomy (surgical removal of both ovaries prior to onset of natural menopause) results in abrupt and irreversible loss of the major source of testosterone in women (Hendrix, 2005; Laughlin, Barrett-Connor, Kritz-Silverstein, & von Muhlen, 2000). About 22 million women undergo surgical menopause each year in the United States (Keshavarz, Hillis, Kieke, & Marchbanks, 2002). Because estrogen synthesis largely derives from ovarian testosterone postmenopausally, surgical removal of the ovaries also exacerbates the estrogen deficit in these women.

In both sexes, decline in testosterone levels leads to bone density loss (Davis & Tran, 2001; Jassal, Barrett-Connor, & Edelstein, 1995; Lobo, 2001; Tenover, 1997) and decline in muscle mass (Ellison et al., 2002; Lobo, 2001). Testosterone, along with dehydroepiandrosterone (DHEA), plays an important role in female libido, arousal, genital sensation, and orgasm. Loss of testosterone can exacerbate vaginal mucosal atrophy, thinning of public hair, and may compromise an older woman's sense of general well-being. Low levels of testosterone have been correlated with lower coital frequency and loss of sexual desire in men and women (Davis & Tran, 2001), although the relationship between female sexual interest in later life and testosterone levels is controversial (Davis & Tran, 2001; Dennerstein, Smith, Morse, & Burger, 1994). Testosterone administration may improve mood and well-being in both sexes, especially among men (Ellison et al., 2001; Grinspoon et al., 2000; Jassal et al., 1995). Circulating testosterone in women is a strong correlate of ovarian function and androgen production, though approximately one third of circulating levels are derived via precursors from the adrenal gland (Lobo, 2001).

Although saliva collection via passive drool may be difficult for older adults due to aging-related changes in saliva production (Hodgson, Freedman, Granger, & Erno, 2004), medication side effects causing dry mouth, and manual manipulation of the small collection devices, overall cooperation with the home-based protocol was high in this population-based sample of older adults. Blacks were less likely to provide saliva samples during the interview. The National Health and Nutrition Examination Survey (NHANES) has also reported relatively lower participation of this racial group in biological measure protocols, specifically collection of DNA from blood. In that study, removal of language in the consent process that linked blood collection to genetic research increased participation in every demographic category (McQuillan, Pan, & Porter, 2006). Cooperation with the NSHAP saliva collection protocol was higher than that accomplished for HIV testing, blood spots, or vaginal swab collection in the NSHAP study (Lindau et al., in press; Williams & McDade, in press). Although, in the case of vaginal swab sampling, differences in cooperation were found by age and education (older age, less educated were less likely to participate) but not by race/ethnicity (Lindau et al., in press). This may be due to the relative invasiveness of the collection protocols or other factors and requires more investigation.

Internal validity of the salivary sex hormone measurements is suggested by the expected findings of higher testosterone concentrations in men versus women (Shirtcliff et al., 2002) and similar estradiol concentrations in women and men (Greenblatt, Oettinger, & Bohler, 1976; Shirtcliff et al., 2002). The proportion of individuals using exogenous hormones was small in the NSHAP sample, as is true in the population as a whole (Wysowski, Golden, & Burke, 1995). Salivary testosterone concentrations in ovariectomized women and in men using androgens were also shifted in the expected direction (Adashi, 1994; Cappola et al., 2007). Estrogen use in women was significantly correlated with higher salivary estradiol concentrations as reported by other authors (Tivis et al., 2005). Obesity is associated with lower production of sex hormone binding globulin (SHBG) in both women and men; consistent with this, and as has been observed previously (Cappola et al., 2007; Lukanova et al., 2004; Schilling et al., 2007), obese women in NSHAP exhibit higher levels of free salivary testosterone. The expected relationship between obesity and free testosterone in men is less clear, and in NSHAP, obese men do not demonstrate significantly different levels of free testosterone than non-obese men. This may be because both SHBG and total testosterone are lower in obese as compared with other men (Allan, Strauss, Burger, Forbes, & McLachlan, 2006; Derby, Zilber, Brambilla, Morales, & McKinlay, 2006; Giagulli, Kaufman, & Vermeulen, 1994).

Normative data on salivary sex hormone concentrations in later life are scarce. As a result, external validation of NSHAP data is particularly difficult for progesterone and DHEA in men and estradiol, DHEA, and testosterone in women. Most existing data derive from small, clinical populations of young volunteers. One population-based study collected salivary specimens from 48 “randomly selected,” healthy, heterosexual, cohabiting men ages 50–80 years, from Mannheim, Germany, and 50 similar men from State College, Pennsylvania (Krause et al., 2002). In that study, salivary measures of DHEA-S (but not DHEA), testosterone, and estradiol were obtained (see Table 7). The concentrations of testosterone and estradiol are similar to those obtained in the NSHAP study. Some studies report lower levels of estradiol (Schultheiss, Dargel, & Rohde, 2003; Tivis et al., 2005) than those obtained in the NSHAP study, although other authors report similar levels of salivary estradiol (Emaus et al., 2008; Nunez-De La Mora, Bentley, Choudhury, Napolitano, & Chatterton, 2008; Rantala, Enksson, Vainikka, & Kortet, 2006). Estradiol measurements in the NSHAP study contain a small number of cases with very high values. For this reason, median (rather than mean) values of salivary estradiol in the NSHAP study show better agreement with the reported data from other studies (see Tables 3, ​,6,6, and ​and77).

Mean progesterone values for women in NSHAP are similar to those reported by Salimetrics for postmenopausal women using the same assay procedures (Nallanathan et al., 2007). As in the case of estradiol, progesterone measurements contain outliers, so median levels of salivary progesterone in the NSHAP study (29.8 pg/ml for men and 24.6 pg/ml for women) demonstrate better agreement with the reported data from other studies (Celec et al., 2006; Jasienska & Jasienski, 2008; Wirth et al., 2007) compared with mean values (see Tables 6 and ​and77).

Salivary DHEA measurements reported for younger adults by other studies (Granger, Schwartz, Booth, Curran, & Zakaria, 1999; Harris et al., 2000; van Niekerk, Huppert, & Herbert, 2001) are higher than found in the NSHAP study, as expected due to age-related decline of DHEA (Ahn, Lee, Choi, Kwon, & Chun, 2007; Granger et al., 1999). DHEA concentrations measured in serum have generally been reported to be similar in men and women, although DHEA levels in men are nearly always higher than in women (Rehman & Carr, 2004). In the NSHAP sample, men exhibit consistently higher concentrations of DHEA compared with women. This may be related to more rapid decline of DHEA levels with age in women than in men, although more studies are needed to clarify this gender difference.

Compared with younger adults (Brown et al., 2008; Flegr, Lindova, & Kodym, 2008; Shirtcliff et al., 2002), testosterone levels obtained in the NSHAP study are lower. For older men, the levels of salivary testosterone obtained in the NSHAP study and in another population-based study (Krause et al., 2002) are very similar (see Tables 6 and ​and7).7). Overall, due to varying assay techniques, study populations and study conditions, salivary hormone measurements obtained across studies are difficult to compare; those reported here are of similar magnitude to findings in the most comparable studies.