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Saliva Hormone Testing The Evidence

Saliva hormone testing has been used in research scientific testing for decades and has been proven to be highly accurate. The World Health Organization has used this method of hormone testing for worldwide comparisons of hormone levels in women with breast cancer living in industrialized vs. non-industrialized countries and they recommend it as the gold standard of hormone testing. In addition there is ample evidence which suggests standard blood tests are unable to effectively evaluate hormone levels. Despite the evidence many medical doctors refuse to accept the validity of saliva testing, and even claim there is no evidence to support its use, preferring to stick with conventional blood tests. The problem with blood tests are:

(1) Serum testing is unable to distinguish the protein-bound, and therefore inactive form of the hormone, from its free and biologically active form, thus giving only a rough estimate of your hormone levels. This may lead to inappropriate diagnosis as quite often total hormone levels may be within normal limits but once the free and active levels are tested deficiencies are identified.

(2) Serum test results only reflect hormone levels outside the cells in the serum and do not reflect levels else where in the body where they are actually active.

(3) Serum tests are not widely available for estriol and estrone in Australia. Therefore two very important estrogens will go undetected when using serum analysis and quite often we see test results where estradiol is normal however estrone is elevated which is a problem that would go undetected by blood tests.

(4) VERY IMPORTANT: When monitoring hormone levels while on transdermal (creams) replacement therapy serum testing is not accurate. This is because once absorbed through the skin into the blood stream hormones bind to red blood cell membranes in order to minimise unfavorable interactions with the aqueous water as most hormones are fat loving and prefer to bind to fats. Once your blood sample is taken it is centrifuged and the red blood cells along with the hormones are removed prior to analysis. This phenomenon was described by Frank Z. Stanczyk (see references) for transdermal progesterone but it also seems to occur to a lesser extent for all other hormones.

Serum tests show no increase in progestreone levels after weeks of applying progesterone cream whereas saliva tests show an increase only after a couple of hours! This is a significant problem monitoring levels if you are using transdermal hormone creams. To illustrate this problem further clinical trials performed by the American Academy of Anti-Aging on over 300 patients revealed that every patient whose hormone levels were deemed at optimal levels by serum blood tests had in actual fact excessive levels based on saliva tests. The doses used to achieve optimal serum levels were higher than standard physiological doses which was all that was required to achieve optimal levels by saliva tests. In every case the patients doses were reduced until saliva tests reflected optimal levels. In our own practice we see this same phenomena on a regular basis with those patients being monitored by serum blood tests that is their current doses are too high!

Listed below are some references supporting the use of saliva testing as opposed to blood tests.

Salivary References

The following references are articles found by searching the peer reviewed literature that address the benefits of saliva over serum.

    • Percutaneous administration of progesterone: blood levels and endometrial protection.Stanczyk FZ, et al. Menopause (2005), 12(2): 232-237.A review of the issues related to the effectiveness of topical administration of progesterone on the endometrium and the disparity between saliva and serum levels. The RBC carrier theory is validated


    • Salivary, but not serum or urinary levels of progesterone are elevated after topical application of progesterone one cream to pre- and postmenopausal women.
      OLeary P, et al. Clin Endo (2005) 53: 615-620.


    • Researchers applied 64mg of progesterone topically to 6 each pre- and postmenopausal women. The continuous 3hr serum and 24hr urine (including pregnanediol-3-glucuronide metabolite) samples showed no significant level changes; whereas, remarkable elevations were noted in the saliva. Authors question clinical organ response without a measurable serum level, though organ delivery was obvious. They also suggest that the lymphatic system delivers the hormones rather than RBCs.


    • A study to evaluate serum and urinary hormone levels following short and long term administration of two regimens of progesterone cream in postmenopausal women.
      Carey BJ, et al. British J Obstetrics and Gynecology (2000) 107:722-726.


    • Authors evaluated serum and urine levels in 24 pre and postmenopausal women following the topical application of 40mg of progesterone either bid divided dosage or qid. Conclusion: Transdermal progesterone (40mg) per day for 42 days causes a small increase in serum progesterone concentration, although there is wide variation. Whether such levels are of clinical benefit remain to be seen. There was no change in the metabolite


    • Topical progesterone cream has an antiproliferative effect on estrogen-stimulated endometrium. Leonetti HB, et al. Fertility and Sterility (2003) 79:221-2


    • Authors monitored endometrial biopsies proliferative activity in 32 postmenopausal women following 0.625 CEE and given either bid daily 0, 1.5% or 4% progesterone topically. Endometrial biopsy evaluation after 2 weeks of progesterone clearly showed an antiproliferative effect of topical progesterone. The antiproliferative effect was essentially the same for the 1.5% and 4% dosages. Regarding serum testing, the authors comment: The plasma concentrations of progesterone were low and varied greatly among individuals. However, elevated serum levels are irrelevant, provided one obtains the desired clinical outcome. Micronized transdermal progesterone and endometrial response.


    • Wren BG, et al. Lancet (1999) 354: 1447-8.
      Authors randomized 27 estradiol exposed (Climara 100 weekly) postmenopausal women into 16mg, 32mg or 64mg groups. Serum levels and endometrial biopsies were monitored. Summary: The use of transdermal progesterone for 14 days over three cycles, even at concentrations as high as 64 mg daily, did not increase circulation blood progesterone concentrations sufficiently to induce any evidence of secretory effect in the endometrium.
      Hormones in Saliva.Vining RF and McGinley RA. Critical Reviews in Clinical Laboratory Sciences. (1986) 23(2):95-146.


    • A review article looking at the constituents of saliva. Conclusion: Saliva flow rate does affect saliva pH and the concentration of many salivary ions. This has led many clinicians to assume that it would also affect all salivary steroid levels. This is not the casea number of clinically important steroids, such as cortisol, testosterone, estriol and progesterone, have salivary concentrations which are not appreciably affected by saliva flow rate. However, the conjugated steroids (e.g., DHEAS) and some unconjugated (e.g., cortisone) may exhibit marked flow rate dependence.Salivary cortisol: a better measure of adrenal cortical function than serum.
      Vining RF, et al. Ann Clin Biochem (1983) 20:329-35


    • Prospective study: three groups (ages 24-32) consisting of 7 healthy men and woman and 10 third trimester pregnant woman). Advantages of saliva: reflects bio-available cortisol and unaffected by CBG level, which rises with BCP and during pregnancy. Stress free and easy to collect. Lends itself to multiple samples. IV cortisol injection shows salivary rise within 5 mins. Routine serum samples at 0900 and 1700 do not accurately reflect adrenal dysfunction.


    • Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Chang KJ, et al. Fertil Steril (1995) 63(4):785-91


    • Randomized placebo controlled study of 40 Premenopausal women scheduled for excisional biopsy of benign lesions. Study groups were given either Pg 25mg or E2 1.5mg or both topically qd to the surgical breast (10-13 days before surgery. Findings: Both E2 and progesterone readily penetrated the skin, increasing the progesterone level x100. Progesterone induced a major reduction in the acinar cell proliferation rate whether used alone or in combination with E2. The serum levels did not reflect the topical hormone supplementation.


    • Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Gozansky WS, et al. Clin Endocrin (2005) 63:336-341.


    • Author compared salivary and serum cortisol levels between 12 individuals under various conditions: exercise stress, dexamethasone suppression or CRH stimulation. EIA was the salivary test method compared to serum RIA. Conclusion: Therefore, assessment of salivary cortisol should be considered over serum total cortisol because more physiologically relevant data are obtained, particularly when the cortisol response to an HPA axis stimulus exceeds
      Direct assay for progesterone in saliva: comparison with a direct serum assay.
      Webley GE, Edwards R. Ann Clin Biochem (1985) 22:579-585.


    • Study compares direct serum and saliva assays for sensitivity, precision and recovery. Twenty women in various stages of their menstrual cycle were compared using serum and saliva. Conclusion: Saliva showed a significant correlation (r=0.71, P<0.001) compared to serum with the added advantages of convenience and reduced stress (no needles).


    • Human Erythrocyte Membrane Uptake of Progesterone and Chemical Alterations.
      Devenuto F, et al. Biochem. Biophys. Acta (1969) 193:36-47.


    • Study RBC membrane uptake to progesterone, corticosterone and cortisol in fresh and 42 day stored (blood bank) blood. Findings: progesterone showed a much greater affinity for RBC constituents (6 to 8 times greater) than the glucocorticoid hormones. Furthermore, there is a likely direct relationship with the amount of bound progesterone and the viability of RBCs in storage, e.g., female blood may be more stable in storage. Also, indirectly this data supports the RBC as a carrier medium for topical applied progesterone.

Additional References:

    • Seasonality of reproductive function and weight loss in rural Nepali women. Painter-Brick C, Lotstein DS, Ellison PT. Hum Reprod May 1993; 8 (5): 684-690.


    • The ecological context of human ovarian function. Ellison PT, Painter-Brick C, Lipson SF, ORourke MT. Hum Reprod Dec 1993; 8 (12): 2248-2258.


    • Measurements of salivary progesterone. Ellison PT. Ann NY Acad Sci Sept 20 1993; 694: 161-176.


    • Menstrual variation in salivary testosterone among regularly cycling women. Campbell BC, Ellison PT. Horm Res 1992; 37 (4-5): 132-136.


    • Reference values for luteal progesterone measured by salivary radioimmunoassay. Lipson SF, Ellison PT. Fertility and Sterility May 1994; 61 (3): 448-454.


    • Metabolism of progesterone and testosterone in human parotid and submandibular salivary glands in vitro. Bloom T, Ojanotko-Harri A, Laine M, Huhtaniemi . J Steroid Biochem Mol Biol Jan 1993; 44 (1): 69-76.


    • For Good Evidence Concerning the Absorption of Steroids Through Human Skin
      Permeation of steroids through human skin. Johnson ME, et al. J Pharmaceutical Sci 1995; 84: 1144-1146.

The Evidence of Red Blood Cell Transport of Progesterone

    • Human erythrocyte membrane: Uptake of progesterone and chemical alterations Devenuto F, et al.. Biochim Biophys Acta, 1969;193:36-47.


    • Permeability of human red cell membrane to steroid sex hormones. Koefoed P, Brahm J. Biochim Biophys Acta 1994; 1195: 55-62.


    • Direct Comparison of Plasma and Saliva Levels After Topical Progesterone Application
      Absorption of progesterone after topical application: plasma and saliva levels. Dollbaum CM, Duwe GF Presented at the 7th Annual Meeting of the American Menopause Society, 1997



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