120 TOWNSEND LETTER for DOCTORS & PATIENTS – JANUARY 2004
As I frequently write on the topic of laboratory testing and
analysis of steroid hormones in saliva, I have chosen to collect
and augment my writings for purposes of this article, to
encompass the advantages and disadvantages of the
methodology as it compares with serum. I will also discuss one
of the most perplexing issues surrounding salivary hormone
testing: why transdermal delivery of hormones through the skin
results in a remarkable and measurable increase in salivary
hormone levels, but little change in blood serum levels. Finally,
I will talk about the “ideal” hormone laboratory report and its
clinical value in helping doctors and patients evaluate and
individualize appropriate therapies to restore hormone balance.
What is saliva and how do hormones enter saliva?
Saliva originates from the salivary glands located under the
tongue and along the sides of the mouth. Saliva is composed of
a complex mixture of mucins, enzymes, antibodies, electrolytes,
and hormones, all of which serve to begin the process of digestion
and protect the oral mucosa.1
The formation of saliva in the salivary ducts begins with
electrolytes (particularly sodium) being actively pumped into
the ducts by an energy dependent process. Water then diffuses
by osmosis into the duct to reestablish a physiological osmolality.
Blood components enter the watery fluid of the salivary duct by
one of three processes: active transport, ultrafiltration or passive
diffusion. Antibodies such as IgA and IgG are actively secreted
into saliva by an energy dependent process. Small charged
molecules like glucose enter saliva by ultrafiltration, the rate
of entry of which is inversely related to molecular size. Steroids
and other small neutral molecules not bound by blood proteins
freely pass through the membranes of the salivary gland into
saliva by passive diffusion.
Passage of neutral steroids from blood into the salivary ducts
is about 10 times faster than the flow rate of saliva.1,2 Because
of the rapid passive diffusion of steroids into the saliva ducts,
saliva hormone levels are not altered significantly when the
flow of saliva is increased with stimulants such as chewing gum.
However, when a steroid is rendered more polar by metabolic
conjugation (eg, sulfation, glycosylation) its rate of flow into
saliva is significantly slower resulting in lower concentrations
of the conjugated steroid as the saliva flow rate increases with
use of stimulants.
Only a small fraction of steroids in blood are bioavailable
In blood, 95-99% of the steroids are bound up by binding
proteins such as sex hormone binding globulin (SHBG), cortisol
binding globulin (CBG), and albumin.3-5 The small fraction of
steroids not bound is considered the free fraction, or that which
is bioavailable as blood percolates through the capillary beds of
tissues. The free or bioavailable fraction of steroids in blood
enters the salivary gland and the salivary duct by passive
diffusion just as they enter other tissues of the body. Hence,
saliva provides a convenient diagnostic fluid from which to
monitor, noninvasively, the bioavailable fraction of steroid
hormones circulating in the bloodstream and entering tissues.
“Rule-of-thumb” – saliva hormones represent 1-5% of serum.
When steroid hormones are produced endogenously or are
taken orally the salivary level of any particular steroid generally
ranges from about 1-5% of the levels found in serum. Shown
below in Figure 1 are examples of serum and expected saliva
levels of the same steroid, assuming 2% of the blood steroid is
bioavailable. The exception to this “rule-of-thumb” is when
steroid hormones are delivered topically through the skin, where
salivary hormones often exceed levels measured in serum. I will
discuss this anomaly in a later section.
Saliva Laboratory Testing:
The Advantages and Disadvantages
There are numerous advantages to using saliva to test for
steroid hormones compared to blood serum or plasma. But there
are also disadvantages. These are discussed below and outlined
in Figure 2.
The ease and simplicity of collecting saliva any place or time
of day under stress-free conditions in a private setting certainly
has advantages over collecting blood at restricted times in a
doctors office or clinic under stressful conditions. Steroid
hormones are exceptionally stable in saliva, allowing for
convenient collection and shipment at room temperature by
inexpensive couriers. Serum, on the other hand, requires
Saliva Hormone Testing
by David Zava, PhD
Figure 1 Serum Level Expected Saliva Level
Estradiol 100 pg/ml 2 pg/mi
Progesterone 10 nglml 0.2 ng/ml = 200 pg/mi
Testosterone 6’ 500 ng/dL =5 ng/ml 0.1 ng/ml = 100 pg/mi
Advantages of saliva hormone testing
•Noninvasive (no needles)
•Less expensive/more convenient for health care provider and
•Optimized for collection any time of day/month, any place
•No special processing (eg, centrifugation, ice-packs) prior to
•Hormones stable in saliva for prolonged period of time
•Convenient shipment by regular US mail
•More representative than serum of total bioavailable steroid
Disadvantages of saliva hormone testing
•Technically more challenging: need 10-20 x sensitivity
•Interfering substances-food, beverages
•Saliva easily contaminated with topical hormones on lips or
•Spurious results with peridontal disease (more problem with
•Lack of familiarity (serum is gold standard)
•Sublingual use of hormones often leads to inaccurate (high)
•No proficiency testing (CAP, AAB) that reflects saliva hormone
•Topical application of hormones results in saliva levels higher
TOWNSEND LETTER for DOCTORS & PATIENTS – JANUARY 2004 121
qualified personnel (phlebotomist), special procedures (needles
and serum collection tubes) and equipment (centrifuge and lab
space to house it) as well as specialized shipping vessels (ice
pack) and express delivery (overnight). Moreover, hormone
concentrations can vary depending on the time of day and month
making it especially difficult to schedule optimal collection times
with serum. Overall, the conveniences in testing salivary
hormones translate into lower overall costs both for the patient,
health care provider, and insurance carrier.
More technically challenging than serum testing of steroids.
Although salivary testing has many advantages over serum
testing, there are still some disadvantages to saliva testing that
should be acknowledged. Technically, saliva testing is more
challenging than blood testing, limiting the number of
laboratories that are capable of performing the tests.6 Blood
levels of steroid hormones are, on average, about 10 to 100 times
higher than saliva levels and commercial test kits and ranges
are based on these higher serum hormone levels.7,8 For example,
most commercial test kits for estradiol provide standards and a
sensitivity range within the expected serum levels of about 10
to 200 pg/mi. Salivary estradiol levels range from about 0.5 to 5
pg/mi, much lower than the standards and assay sensitivity
provided by commercial kits. Therefore, laboratories performing
saliva testing must have the technical expertise to either create
their own tests or modify commercial test kits. Significant
technical hurdles beyond the technical expertise of most
commercial testing laboratory personnel must be overcome to
make this transition.
Interfering Components. Foods, beverages such as coffee, and
drugs taken just before collecting saliva can interfere with test
results or cause a transient shift in hormone levels.9 Mucins in
saliva can interfere with the test, causing spuriously high levels.
Some chewing gums and cottons used for saliva collection
contain substances that interfere with some saliva tests,10
resulting in erroneously high hormone levels. Sugar in regular
chewing gum can interfere with some saliva tests. Chewing gum
or other physical agents (eg, parafilm) used to increase the flow
of saliva can also cause more bleeding of the gums, especially if
the individual has advanced periodontal disease. Since 95-99%
of hormones are blood-bound, small amounts of blood in saliva
could cause significant changes in hormones that are not truly
representative of the bioavailable fraction of the hormone.
Contamination of the saliva collection tube with topical
hormones. The saliva can be contaminated inadvertently during
collection with hormones present on the hands or lips from use
of topical hormones. This results in an inordinately “false-high”
hormone level. Care must be taken to avoid the use of topical
hormones on the face and neck the day before collection. If topical
hormones are used, hands should be washed thoroughly to avoid
potential contamination of the tube.
False-elevated results with use of sublingual hormones. Use
of sublingual hormone troches can cause spuriously high
hormone test results if the hormone has not been given ample
time to clear from the oral cavity. Individuals who use hormones
sublingually at night and collect saliva in the morning will
almost certainly get false-high saliva test results. We recommend
at least 36 hours between use of hormone troches and saliva
No proficiency testing. Saliva and serum testing laboratories
are governed by the CLIA (Clinical Laboratory Improvement
Act) regulatory agency. However, both saliva and serum testing
laboratories use serum, not saliva, for proficiency testing. Saliva
testing laboratories are, therefore, not being tested for their
competence to reproducibly and accurately measure hormones
in saliva, which is a much greater challenge than measuring
the hormones in serum.
Perceived disadvantage. Another “perceived” disadvantage
to saliva testing is that when a hormone is administered topically
via creams or gels, salivary hormone levels often are very high,
even higher than serum levels. Out of frustration, many health
care providers are left to conclude that this anomaly in high
salivary hormone levels is the result of an unexplained artifact
of saliva testing unique to topical hormone delivery. For lack of
explanation in the medical literature concern has arisen over
the validity of saliva hormone testing when hormones are
delivered topically. This odd phenomenon, and the failure of some
saliva testing laboratories to reset the expected ranges, has
driven many health care providers away from saliva testing. As
I will discuss below, when steroids are delivered topically, serum
testing grossly underestimates bioavailable hormone levels and
is not reflective of either tissue uptake or response. Saliva
hormone levels, on the other hand, closely parallel tissue uptake
of the hormone.
The Paradox: Salivary Hormone Levels Often are
Higher than Serum Levels when Hormones are
One of the most perplexing issues surrounding saliva testing
is the odd phenomenon that topically delivered steroids cause a
dramatic increase in salivary hormones without a concomitant
increase in serum levels.11,12 For example, 30 mg topical
progesterone supplementation results in an average rise in
salivary levels from about 50 pg/ml (0.05 ng/ml) to 500 to 3000
pg/ml (0.5 to 3 ng/ml), a 10 to 60-fold increase. This increase is
proportionally even greater when progesterone is supplemented
at 100 to 200 mg, a common topical dose used by many doctors,
resulting in salivary progesterone levels rising to as high as
10,000 to 100,000 pg/ml (10-100 ng/ml). Under the same
conditions, serum progesterone levels only increase about 4-fold,
from about 0.5 to 2-3 ng/ml.12,13 The same disproportionate
increase in salivary hormone levels is seen with topical delivery
of all the other steroid hormones (eg. estradiol. testosterone.
The remarkable increase in salivary hormone levels observed
with topical hormone delivery raises a number of questions.
First, if salivary hormones are derived from blood, how could
saliva hormone levels be higher than serum or plasma levels?
The numbers just don’t add up. Second, if this is some
unexplained artifact, how does the hormone get into saliva and
by what mechanism?
To answer these questions it is worthwhile to take a step
back and question the assumptions made when testing hormones
in saliva and serum. It is assumed that hormones measured in
serum or plasma represent all the hormones in whole blood.
Serum (or plasma) comprises only about half of the blood volume,
the remainder is made up of blood cells, mostly erythrocytes,
which are removed by sedimentation. It is assumed that the
blood cells are inert and play no role in hormone transport in
the bloodstream. This, in fact, is not so.
Blood cells as hormone transporters
Blood cells, particularly erythrocytes, play an important role
in steroid hormone transport. In fact, early studies with red
blood cells demonstrated that when progesterone was added
Saliva Hormone Testing
122 TOWNSEND LETTER for DOCTORS & PATIENTS – JANUARY 2004
directly to whole blood, about 80% associated with erythrocytes
and was removed from serum by sedimentation.14 In contrast,
only about 5% of aldosterone associated with the erythrocytes
under the same conditions. Progesterone is a non-polar steroid
that seeks out a non-polar, lipophilic environment such as the
plasma membrane of the red blood cells. In contrast, aldosterone
is a more oxidized and polar version of progesterone and would
more likely find its way to a hydrophilic portion of blood, such
Koeford and Braun15 studied the permeability of red blood
cells to steroids and concluded that steroids bind avidly to red
blood cells and serve as a transport mechanism for them. They
determined that when steroid-laden red blood cells enter
capillaries of tissues much of the steroid payload is delivered to
adjacent tissues within milliseconds. From this, it is not difficult
to envision a nonpolar steroid entering the bloodstream directly
through the skin, hitching a ride on red blood cells, and being
delivered almost instantaneously to tissues throughout the body.
One of these tissues would be the salivary gland, duct, and
Studies show topical delivery of hormones results in increased
tissue hormone levels without parallel increase in serum levels.
Several studies support the evidence that topical
progesterone supplementation significantly increases tissue
levels of progesterone without a parallel increase in serum levels.
Three of these studies are discussed briefly below. These studies
are important because they emphasize that serum hormone
testing does not reflect tissue response when hormones are
The first study by Chang and de Lignieres16 demonstrated
that topical application of estradiol, progesterone, or
combinations of these hormones to the breasts of women resulted
in a dramatic increase in breast tissue levels of these hormones
without a concomitant increase in serum levels. In this study,
women applied either a gel containing no hormones, or gels
containing 25 mg progesterone, 1.5 mg estradiol, or a
combination of estradiol and progesterone. After 10-13 days
tissue biopsies of the breast were obtained and analyzed for
hormone content and cell growth patterns. The tissue content
of progesterone and estradiol increased about 100- and 200-fold,
clearly demonstrating that tissue uptake of these hormones
occurred. Pathologists then assessed the replication rate of
breast ductal cells and found that estradiol increased and
progesterone decreased the rate of breast cell replication. This
demonstrated that steroids delivered topically to the breasts
not only were taken up by breast tissue but had a clear biological
impact on the growth rate of breast ductal cells.
Oddly, these remarkable changes in the tissue content of
progesterone and estradiol and cell growth rate were not
paralleled by a statistically significant increase in serum
estradiol or progesterone. Although salivary progesterone was
not monitored in this study, we know from thousands of saliva
tests in my laboratory,11 and from similar clinical studies,12 that
the 25 mg of topical progesterone gel used in this study results
in a dramatic rise in salivary progesterone levels, as much as
A second study by Rachel Miles, MD, and associates17
demonstrated that serum testing of progesterone is not reflective
of tissue uptake when progesterone is used as a vagina
suppository. In this study, progesterone levels in serum and
tissue were compared after intramuscular and vaginal delivery
of progesterone. After treatment with either intramuscular
injection of progesterone or vaginal progesterone suppositories,
serum and uterine biopsies were taken to measure blood and
tissue uptake of progesterone. Serum levels of progesterone
(measured by RIA) were three times higher with intramuscular
delivery of progesterone than with vaginal delivery. In striking
contrast, tissue uptake in the uterine biopsies was 10 times
higher with vaginal delivery of progesterone than with
intramuscular injections. For lack of a better understanding,
these authors attributed the tissue differences to a “first pass
effect,” a term used to describe local diffusion of progesterone
from the vagina to the uterus without significant systemic
delivery to other tissues.18 However, these authors could not
prove or disprove what they described as a “first pass effect”
because they did not biopsy other tissues.
Vaginal delivery of progesterone into the body is, in essence,
through the epithelial layer of skin and does not differ in this
regard from other forms of topical progesterone delivery.
Therefore, a more likely explanation for the discrepant serum/
tissue results is that when progesterone is delivered vaginally
it is rapidly delivered to all tissues throughout the body. The
manufacturers of Crinone, a vaginal suppository progesterone
gel, make the same claims of a “first pass effect,” yet also claim
that women have significant improvement in well being,
indicating that progesterone also finds its way from the vagina
to the brain as well as the uterus.19
A third study performed by a group of Italian investigators
showed that progesterone delivered to the nasal cavity caused
remarkable changes in uterine morphology but only marginally
affected serum progesterone levels.20 Following a period of
estrogen priming, progesterone was administered as a nasal
spray at about 8 mg/spray 4 times per day (total dose of about
30 mg). After using progesterone nasally for a week, uterine
biopsies were obtained and analyzed for changes in
morphological features. Nasal progesterone induced changes
that were consistent with a secretory endometrium, meaning
that progesterone found its way from the nasal cavity to the
uterine lining. These clinicians would be hard pressed to explain
their results by a “first pass effect.”
These three studies clearly reveal that when progesterone is
delivered topically through the skin, serum testing of steroid
hormones does not reflect tissue uptake. Although saliva was,
unfortunately, not measured in any of these studies, our
unpublished results of thousands of saliva tests reveal that this
same concentration (about 15-30 mg) of progesterone (or any
other steroid hormone) results in a dramatic increase in salivary
progesterone. Therefore, when steroid hormones are delivered
topically, saliva provides a more realistic and accurate reflection
of tissue hormone uptake and biological response.
It is unfortunate that many physicians refuse to allow their
patients to use topical hormones based on the failure of serum
to demonstrate significant increases in hormone levels. I am
confronted daily with letters and phone calls from women who
have benefited enormously from use of about 15-30 mg of OTC
topical progesterone but whose physicians refuse to write
Saliva Hormone Testing
The ideal test report should contain the following information
on the patient
•menopausal status: pre vs peri vs post, menstrual cycle:
follicular vs luteal, hysterectomy/oophorectomy, status use of
•delivery (oral vs topical)
•time since last used (hours vs days) symptoms
TOWNSEND LETTER for DOCTORS & PATIENTS – JANUARY 2004 123
prescriptions for it based on their experience with serum testing.
Perhaps it is time to question the assumptions we have made
about serum testing of steroid hormones, especially when they
are delivered through the skin.
The Ideal Saliva Hormone Test Report
Ideally, a hormone test report will contain information that
serves as a guide to help an individual and his or her health
care provider better understand how hormonal imbalances could
be affecting health and well being (Figure 3). The hormone test
report should also help health care providers and their patients
come to an educated decision about the most effective treatment
strategy (hormonal, nutrition, exercise, stress reduction, etc.).
Gender, age, menopausal status, menstrual cycle information,
and whether or not a woman has had her ovaries removed all
affect hormone levels and should be included on a test report.
The use of hormones (types, delivery systems such as oral vs.
topical, dosage) and when they were last used all impact test
results and ranges and should be included in the test report.
Progesterone levels differ during the phases of the menstrual
cycle (follicular vs. luteal) and with different delivery systems
when taken as replacement therapy (oral vs. topical). Test results
should reflect these differences; otherwise the results can lead
to confusion. For example, a postmenopausal woman
supplementing with topical progesterone will have much higher
salivary progesterone levels than a postmenopausal woman not
supplementing. Therefore, if no information is provided about
route of administration (oral, sublingual, topical, vaginal), or
Saliva Hormone Testing
Figure 4: Typical Symptoms of Hormonal Imbalance – EX=Excess / DEF = Deficiency
Estrogen Progesterone Androgen Cortisol
EX DEF EX DEF EX DEF EX DEF
Hot flashes x x x
Night sweats x x x
Vaginal dryness x x x
Incontinence x x
Foggy thinking x x x x
Memory lapses x x x x
Tearful x x x
Depressed x x x x x
Heart palpitations x x x x x
Bone loss x x x x
Sleep disturbances x x x x
Aches/pains x x
Morning fatigue xxx
Evening fatigue xxx
Sensitivity to chemicals xxx
Stress x x x x x
Cold body temperature x x x x
Sugar craving x x x x
Elevated triglycerides x x x x
Weight gain (waist) x x x
Decreased libido x x x x x
Loss of scalp hair x x x
Increased facial/body hair x x x
Mood swings (PMS) x x
Tender breasts x x x x
Bleeding changes x x x
Nervous x x x
Irritable x x x x
Anxious x x x
Water retention x x x x
Fibrocystic breasts x x
Uterine fibroids x x
Weight gain (hips) x x
Endometriosis x x
Candida x x x
124 TOWNSEND LETTER for DOCTORS & PATIENTS – JANUARY 2004
the ranges are not shifted to a supplemented level, test results
can lead to confusion and the impression that the patient has
some hormonal imbalance when, in fact, she may be enjoying
enormous benefit from the hormonal therapy.
The importance of matching symptoms with
The majority of individuals who test their hormones in saliva
or blood have a reason; they are suffering from symptoms that
impact the quality of their life. They recognize that these
symptoms often are based on hormonal imbalances. For
example, a woman suffering from PMS and fibrocystic breasts,
which have been getting progressively worse as she approaches
menopause, understands that this is due to hormonal changes.
She often wants to verify this hormonal imbalance by hormone
testing to justify some form of intervention. Or a man who suffers
from low energy, muscle loss, apathy, sagging sex drive, and
has lost that “get up and go” as he approaches his 50’s recognizes
something is wrong and seeks hormone testing to determine if
his problems are related to dwindling testosterone.
There is little question that an individual’s symptoms are
the primary driving force that compels them to seek professional
help, and spend money on hormone testing. Documenting these
symptoms and understanding their relationship to hormonal
levels has numerous advantages over just simply knowing an
individual’s hormone profile. Matching hormone profiles with
symptoms helps confirm that the hormonal imbalance is causing
the symptom. For example, if a woman has normal levels of
estradiol, but low progesterone during the luteal phase of her
menstrual cycle and she suffers with symptoms of estrogen
dominance (fibrocystic breasts, water retention, irritability,
PMS) these symptoms help confirm the hormonal imbalance
and point to treatment strategies that can be used to correct
Another reason it is useful for the testing laboratory to
document symptoms is because the health care provider ordering
the hormone test often does not fully understand how to
interpret the test results. This frequently results in a phone
call to the testing laboratory to help shed light on the hormone
test results. For a productive and meaningful discussion about
the test results, the health care provider and the professional
at the testing laboratory must both know the patient’s age,
menopausal status, what hormones they are taking, and their
symptoms. If these parameters are not documented on the test
report, time-consuming conversation often ensues in order to
provide a mutual understanding of the hormone test results in
relationship to the patient’s primary complaints. A patient’s most
pressing symptoms in relationship to hormonal imbalances are
often overlooked in a conversation where both parties do not
have access to this information.
Numerous studies and books21 have documented symptoms
in relation to hormonal imbalances. Some of the most common
symptoms in relationship to hormonal excesses and deficiencies
in women are listed in Figure 4.
In summary, saliva hormone testing is more convenient, less
stressful, more cost-effective, and more representative of the
bioavailable fraction of hormones in blood than serum hormone
testing. When hormones are delivered topically, serum hormone
testing grossly underestimates the bioavailable fraction of
hormones in blood and tissue hormone uptake and response.
This often leads to continued escalation of dosing despite tissue
saturation and symptoms of hormone excess. The ideal saliva
hormone test report should contain pertinent information not
only about an individual’s hormone level but how this relates to
symptoms associated with hormonal imbalance. Such
information helps both health care providers and their patients
come to a more educated decision about the most effective
treatment strategy (hormonal, nutrition, exercise, stress
David Zava, PhD
1815 NW 169th Place, Suite 5050
Beaverton, Oregon 97006 USA
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11. Zava, DT. Unpublished results, ZRT Laboratory.
12. O’Leary P, Foddema P. Chan K, Taranto M, Smith M, Evans, S. Salivary, but
not serum or urinary levels of progesterone are elevated after topical
application of progesterone cream to pre- and postmenopausal women.
13. Burry KA, Patton PE, Hermsmeyer K. Percutaneous absorption of
progesterone in postmenopausal woman treated with transdermal
estrogen.’Am J Obstet Gynecol 180: 1504-11, 1999.
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15. Koefoed P. Brahm I. The permeability of the human red cell membrane to
steroid sex hormones. Biochim Biophys Acta 1195: 55-62, 1994.
16. Chang K-I, Lee LTY, Linares-Cru.z G, Fournier 5, de Lignieres B. Influences
of percutaneous administration of estradiol and progesterone on human
breast epithelial cell cycle in vivo. Fertility Sterility 63: 785~791, 1995.
17. Miles RA, Paulson RI, Lobo RA, Press MA, Dahmoush L, Sauer MV.
Pharmacokinetics and endometrial tissue levels of progesterone after
administration by intramuscular and vaginal routes: a comparative study.
Fertility and Sterility 62: 485490, 1994.
18. Bulletti C, de Ziegler D, Flamigni C, Giacomucci E, Polli V. Bolelli G,
Franceschetti F. Targeted drug delivery in gynaecology: the first uterine pass
effect. Hum Reprod 12(5): 1073-1079, 1997.
19. Fanchin R, de Ziegler D, Bergeron C, Righini C, Torrisi C, Fydman R.
Transvaginal administration of progesterone. Obs Gyn 1997;90:396-401.
20. Cicinelli E, Petruzzi D, Scorcia P. Resta L. Effects of progesterone
administered by nasal spray on the human postmenopausal endometrium.
Maturitas 18: 65-72, 1993.
21. Lee, JR. Natural Progesterone; The Multiple Roles of a Remarkable Hormone.
BLL Publishing, Sebastapol, CA, 1993.
Saliva Hormone Testing
Reprinted with permission
Townsend Letter for Doctors & Patients
911 Tyler Street, Pt. Townsend WA 98368
360-385-6021 • www.townsendletter.com