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Hormone Testing

Hormone Testing

Measuring hormone levels is essential for the proper diagnoses of perimenopause, menopauseandropause or other hormone related disease states such as hypothyroidism and adrenal exhaustion (chronic fatigue syndrome), which all exhibit similar and overlapping symptoms making an accurate diagnosis based on symptoms alone very difficult. Hormone level testing also enables you to closely monitor your hormones ensuring they all remain adequately balanced and within the optimal physiological range.

The hormone test results used in conjunction with any symptoms you have are invaluable tools when designing an Individual Bioidentical Hormone Replacement Therapy (IBHRT) regime. It is very surprising, not to mention dangerous, how many women and men on HRT have never had their hormone levels tested. Hormonal imbalances that are not accurately identified and appropriately treated may lead to inappropriate treatments with very serious side effects so the importance of monitoring their levels cannot be overstated.

There is currently some criticism against hormone testing by some whom claim they are worthless and that it is best to diagnose and dose hormones based on symptoms alone. We do not agree with this argument as many conditions share similar symptoms making it difficult to differentiate between them making an accurate diagnosis difficult. In addition some people can tolerate very high hormone levels or hormone imbalances and display no symptoms yet these imbalances in the long term could potentially create a problem such as cysts, fibroids or cancer. The critics of hormone testing usually pay little to no attention to the appropriate balance of hormones which can only be accurately determined by hormone testing.

A couple of common examples we see regularly are given to illustrate the necessity for testing. Firstly if a women presents with hot flushes many practitioners will assume she is menopausal and requires estrogen when in actual fact the hot flushes may by high cortisol levels. By giving estrogen to her is an inappropriate and possibly dangerous treatment which may cause further hormone imbalances and side effects. Secondly, again a women presents with hot flushes which were caused by excessively high levels of estrogen which resulted in a down regulation of her estrogen receptors causing her hot flushes. By assuming it is caused by an estrogen deficiency and giving estrogen to this women, which would be the most common diagnosis based on her symptoms, would make her condition worse. Finally in order to reduce the long term risks of hormone replacement an appropriate balance of the three estrogens is essential and also their balance with progesterone. Therefore ongoing monitoring is essential in order to ensure a healthy hormone balance is maintained throughout therapy. These can only be determined by hormone testing. Here are only a couple of examples we see in our practice which emphasize the need for hormone testing.

Finally another criticism of hormone testing is that women's sex hormone levels fluctuate throughout the month and thus testing is useless. Sex hormone levels do indeed fluctuate in pre-menopausal women yet if the menstral cycle is correctly understood it is easy to test during the luteal phase of the cycle (around day 21) where progesterone levels peak, being the most significant time of the cycle progesterone wise, and also avoiding the mid cycle estrogen surge and thus this problem is easily overcome with a little knowledge of the menstral cycle. Post menopausal women's sex hormones do not fluctuate nor do any of the adrenal or thyroid hormones with the menstral cycle.

Types of Hormone Tests - Blood Tests v 24 hour Urine Tests v Saliva Tests

(1) Blood (serum) tests - are commonly used by conventional doctors to determine total hormone levels in the serum. This type of hormone testing is considered the least accurate of all methods available. These serum tests are 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 are within normal limits but once the free and active levels are tested deficiencies are identified. In addition 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. They are also only a snap shot of your levels at a single point in time and do not reflect total daily output. And finally 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 thus unable to give any indication of an appropriate estrogen balance.

VERY IMPORTANT: When monitoring hormone levels while on transdermal (creams) hormones 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 little to no increase in progestreone levels after weeks of applying progesterone cream whereas other methods show an increase only after a couple of hours! This often results in overdosing hormones. 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!

The facts are there has never been a single study correlating serum values for topically administered hormones to actual tissue levels or to long term effectiveness. Most mainstream physicians have accepted serum testing as the "gold standard" without any science which is based on some false assumptions. You should never use serum testing for judging effectiveness or hormone levels from topical application -  Stanzyck’s article proves this for progesterone and clinical observation sees it occuring with most other hormones as well.

NOTE: If you are using troches then urine (preferable) or blood tests are recommended as hormone residues in the mouth can affect saliva test results for up to 36 hours after your last troche dose. It is not wise to wait 36 hours and then do the saliva test as these hormones have a short half life and will be mostly eliminated from your system so you will only be measuring baseline levels. Serum tests will detect progesterone when it is taken orally or by troche.

Unlike sex and adrenal hormones serum testing still remains the method of choice for thyroid hormone monitoring.

(2) 24 hour Urine Hormone Testing is one of the most accurate methods of all and ideally should be used whenever available. It measures the free and active form of most sex and adrenal hormones, gives a 24hour reading so relays more information about total daily output, is able to measure all important hormones including many of their metabolites and is easy and convenient to do. Supplementation with exogenous hormones and improvement in symptoms is reflected in values seen on follow-up tests. The main problems with urine testing is the inconvenience of 24 hour urine collection and they are significantly more expensive compared to saliva testing.

(3) Saliva testing measures the free and therefore biologically active form of most sex and adrenal hormones. Saliva tests have been proven to be an accurate reflection of hormone levels present inside cells, where the hormone action takes place, whereas blood tests measure hormones outside the cells. It is considered to be the gold standard for hormone analysis by the World Health Organisation (WHO) and even used by NASA. Despite the WHO's recommendation to use saliva hormone testing and all the research to back up its accuracy it is often the most criticized and neglected method by medical practitioners, many whom refuse to order such tests as they are not informed of the differences and usually state there is no research to validate saliva testing. Click here for references that validate saliva testing.

Saliva hormone testing is a convenient method which can be performed in the comfort of your own home. They are however also a snap shot in time of your hormone levels however multiple daily measurements can be made to overcome this draw back. Saliva measurements are greatly affected by the use of exogenous hormones. Transdermal pro­gesterone and testosterone, in particular, can result in supra-physiological levels in saliva testing due to lymphatic accumulation which is reflected by saliva testing. These results normally confuse many practitioners unfamiliar with this method of testing.

We can arrange any of these tests for you with a consultation if your doctor refuses to do so. Both urine and saliva hormone testing involves a test kit being sent to your home with printed instructions. Samples are taken and placed into provided containers at one or more specific times of the day, then sent directly to the laboratory for analysis. The results are then sent back to us where we can interpret the results and determine the appropriate therapy.

Timing of Tests in Relation to your Last Dose

The best time of the day to collect a sample for any baseline hormone analysis for diagnosis is in the early morning (except for 24 hour urine analysis) before breakfast, and the best time of the month for menstrating women is between days 19 to 23 of a 28 day menstral cycle (day one is first day of mensus). This is when progesterone levels are apt to be highest (luteal phase) during the entire cycle. Men can do the test any day that is convenient but again before breakfast.
When doing subsequent follow up tests to monitor hormone levels while on any hormone replacement it is best to take the sample 8 to 12 hour after the last dose if your using creams and capsules. This is very important in order to obtain meaningful results. If your using troches then a serum test should be done approximately 4 hours after your last troche dose. Be consistant so all follow up tests can be compared to each other. If the timing of the test is mixed on each occasion the test is performed then the results are not comparable!

For thyroid testing if a patient is on T4 (oroxine) therapy, it is best to test about 8 to 12 hours after the last dose of medication to get the best correlation of levels produced by the T4 replacement. With T3, the best time to test is 1.5 to 4 hours after the last dose. For thyroid extract (Armour thyroid), which is a combination of T3 and T4, you should test 4 hours after the last dose. Any sooner could produce a peak of T4 and any later could produce a drop off from the T3. All repeat tests should use the same timing as previous testing so results can be directly compared. If not then no direct accurate comparison can be made between tests.

Many doctors are critical of any form of hormone testing for women as they claim hormone levels fluctuate so testing is therefore useless. This is true, hormone levels do naturally fluctuate on a monthly cycle in menstarting women. However if that cycle is clearly understood the tests should be performed on a specific day of the cycle (luteal phase), as mentioned above, where we are well aware of the appropriate hormone levels for that time of the month and thus the results are meaningful. If periods are irregular it is more difficult to accurately assess hormone levels with just one sample. Therefore take samples on two different occasions before starting IBHRT to increase the chance that testing will reveal natural biological variations. Post menopausal women do not experience hormonal fluctuations so tests can be done on any day.

What to Test For

As a generalisation I recommend:

WOMEN: Test for estriol, estradiol, estrone, progesterone, testosterone, DHEA, cortisol and thyroid (T3,T4 and reverse T3) hormones. The first five hormones make up the basis of IBHRT while measuring DHEA, cortisol, aldosterone and thyroid hormones may indicate if adrenal exhaustion or hypothyroidism are present, which have been implicated in perimenopause, menopause and fatigue type conditions.

MEN: Test for estradiol, estrone, progesterone, testosterone, DHEA, cortisol, DHT,  aldosterone and thyroid (T3,T4 and reverse T3) hormones. Ideally levels should be tested first thing in the morning before breakfast as elevated sugar levels after meals are reported to reduce testosterone levels thus giving an inaccurate result.

Important note for women: If you are still cycling then it is very important that the test is done between days 19 to 23 of your cycle (luteal phase) assuming it is a 28 day cycle. If the test is done outside this range then the results obtained are of only limited use. In addition make sure that the estrogens and progesterone are ordered and do not settle for FSH and LH tests which is what the majority of doctors will order as these values are of little value when comparing hormone balance

Your current signs and symptoms will help determine exactly what tests are appropriate to preform so you may not need all these tests mentioned. The tests required is often very individual. All of these ideally should be measured before you start any IBHRT which will give you a good baseline assessment of your overall hormone status. From these initial results it can be determined which hormones need supplementation and the appropriate starting dose of each hormone.

It should be clarified that test results must be used in conjunction with signs and symptoms and not be totally relied upon 100% for a diagnosis and latter on to determine appropriate dosages. There is always a general optimal physiological level we try to achieve with however these levels can vary in some patients and this must be taken into account and can only be done so by also using symptoms to go by. Every day I am sent hormone test results from people wanting my advice on what to take and the doses required. I cannot responsibly offer any advice without an appropriate background into their signs, symptoms, history, etc in conjunction with their test results to help make a final decision on what is best.

Interpreting Test Results

A major problem with hormone testing is the interpretation of test results. Practitioners with little experience in hormonal matters often observe results that lie at the low end of the so called “normal range” and determine that no hormone imbalance or deficiency exists thus determine no action is required. A major problem is that laboratory test “normal” ranges are defined and standardised according to statistical norms instead of physiological optimal levels. That is, mathematics rather than patient symptoms define “normal” hormone levels.

Instead of using “normal” laboratory ranges we prefer to use optimal ranges which as a general rule lie within the upper one third of the normal laboratory range. This general rule is only a guide as it does not take the appropriate balance between certain hormones into account which is also very important. Therefore it is important that someone with experience and knowledge on appropriate hormone balance views your test results for an accurate diagnosis. Often there is a significant improvement in symptoms when levels at the low end of the normal range are increased to the upper end of the normal range with supplementation. For a more detailed explanation refer to Limitations of Lab Test Reference Ranges blog on this website.

Once you have started IBHRT it is essential to retest your hormone levels after four to eight weeks to ensure your hormone levels remain within the upper one third of the normal physiological range and also to ensure the supplemented hormones are absorbed and utilized by the body. If you use lozenges to deliver your hormones you must have blood tests as saliva test will be adversely affected if performed within 36 hours after your last dose. After your hormone levels have stabilized to suitable levels continue to retest at least annually for the rest of your life.

Further Monitoring

In addition to saliva/urine analysis we also recommend that both men and women have their 24 hour urine estriol, estradiol, estrone, 2-hydroxy-estrone, 4-hydroxy-estrone, 16-hydroxy-estrone levels checked. The goal is to assess and reduce any potential risk of disease by monitoring which estrogen metabolites are being produced, the relationship between metabolites, and how replacement affects metabolite levels. For more information on an unfavorable estrogen metabolite balance possibly causing breast and prostate cancer please refer to appropriate sections.

Annual breast screening and endometrial ultrasounds may also be important tests to have, in certain cases, to detect if any problems arise from hormone therapy.


Any of these tests discussed may be arranged during a consultation with Dr Michael Serafin. Click here for details about our consultations. Alternatively discuss them with your health care provider.


Frank Z. Stanczyk, PhD, Richard J. Paulson, MD, and Subir Roy, MD.  Percutaneous administration of progesterone: blood levels and endometrial protection.  Menopause: The Journal of The North American Menopause Society Vol. 12, No. 2, pp. 232-237

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