Biologically Identical Hormone Replacement Therapy

Transcription

1 Program Overview BHRT Prescribing Guide Hormone Treatment Centers of America Biologically Identical Hormone Replacement Therapy Comprehensive Health Care Provider Reference Guide

2 jgfjfdjkgbvm,v,khg,fbggxgfccdbdzsgd

3 Dear Health Care Provider, BHRT is a very popular and highly requested therapy, especially by women in their 30 s to 60 s. As many as an estimated 50 million women are in menopause. Additionally, increasing numbers of men want hormone therapy and evaluation. We believe that by providing BHRT you can increase your patient base and patient satisfaction, ultimately increasing your bottom line. The Hormone Treatment Centers of America, is pleased to offer a comprehensive and simplified program to assist you in providing Bio-Identical Hormone Replacement Therapy, BHRT, to your patients. Since implementing BHRT in the average medical practice can be a tedious and time consuming task, we have created an easy program that does the work for you! This comprehensive reference guide discusses BHRT, describes how to use lab and clinical work-ups to initiate HRT dosing, explains the various modes of administration available to optimize and individualize therapy, and provides illustrative case studies. As you implement BHRT into your practice, you will soon be able to develop long-term monitoring programs for your patients and thus enhance their quality of life significantly. The Hormone Treatment Centers of America partners with you every step of the way in implementing BHRT into your practice: 1. We assist in generating patient referrals to your practice. 2. We provide BHRT evaluation forms and saliva testing directly to your patients. 3. Our medical team reviews patient history, signs and symptoms, salivary assays and generates a BHRT protocol for your approval. 4. Upon your approval we ship the bio-identical hormone prescriptions to patients. 5. We provide detailed patient education to patients and handle patient questions. 6. We provide all necessary marketing materials. If you have any questions about this guide or any aspect of introducing BHRT into your practice, please contact us. We are here to be your BHRT resource and look forward to the opportunity of partnering with you and your practice. Sincerely, The Hormone Treatment Centers of America Entire Team of BHRT Specialists

4 jgfjfdjkgbvm,v,khg,fbggxgfccdbdzsgd

5 Hormone Makeover & Hormone Overhaul Program Overview The Hormone Treatment Centers of America offers two programs: The Hormone Makeover for women and The Hormone Overhaul for men. To get the process started we enroll your practice as our official provider in your geographical area. In most cases we get you jump started by offering community seminars. We then send those referrals to you. Once you have an interested patient from your current practice or from our referrals, your staff faxes us the contact sheet and we take it from there. How the Hormone Treatment Centers of America Program Works: 1. HTCA staff calls patient per physical request or potential patients call in to center. 2. HTCA staff does the mini-evaluation, tells the patient the total number of symptoms and enrolls them into the program, sends out the test kit and detailed evaluation form. 3. If patient has not had an initial physician consultation they are referred to one of our affiliates. 4. When the test results are in, the physician receives a summary of symptoms and a copy of saliva test results. A written treatment plan is prepared by a BHRT Advisor consisting of BHRT, supplements, and dietary support. This includes BHRT prescription recommendations. 5. The physician approves the recommendations and prescriptions or makes adjustments as he/ she deems necessary. 6. Once the treatment plan is approved the patient is notified by HTCA to make a follow up appointment with the provider to receive the treatment plan. The physician s staff then faxes the prescriptions to the HTCA. The patient is advised to contact an HTCA advisor to address and questions regarding the treatment plan and follow up instructions. The Hormone Treatment Centers of America Provider Marketing Support Includes: 1. Hormone Makeover and Hormone Overhaul brochures for waiting rooms, exam rooms, and any other networking location. 2. Extensive patient education including a variety of articles and online video seminars via the Hormone Treatment Centers of America website. 3. Patient education handouts and articles. 4. Patient newsletters. 5. Monthly live webinars or tele-seminars. 6. Community seminars in some locations.

6 jgfjfdjkgbvm,v,khg,fbggxgfccdbdzsgd

7 Hormone Treatment Centers of America BHRT Provider Resource Guide SECTION ONE: BHRT PRESCRIBING GUIDE Table of Contents What is BHRT?... 1 Symptoms of Hormone Deficiency Progesterone Deficiency... 2 Estrogen Deficiency... 2 Testosterone Deficiency... 3 Thyroid Deficiency... 3 Cortisol Deficiency... 3 Symptoms of Hormone Excess Excess Estrogen... 4 Excess Testosterone... 4 Excess Progesterone... 4 Excess Cortisol... 4 Symptoms of Hormone Imbalance In Men... 5 Actions & Effects of Hormones Progesterone... 6 Estrogen... 8 Estriol... 9 Testosterone Hormone Cascade Actions & Effects of Hormones In Men BHRT Dosing Forms Dosing Forms Pros and Cons A Comparative Analysis: Transdermal versus Oral Estrogen Transdermal Dosing PerQ Gel TM Cream Hormone Testing & Sample Plan Testing and Monitoring Hormone Levels HTCA Sample Treatment Plan... 16

8 Hormone Treatment Centers of America BHRT Provider Resource Guide Table of Contents, continued. SECTION TWO: BHRT TECHNICAL RESEARCH Progesterone Progesterone and Menstrual Health/PMS, Infertility, Pregnancy Progesterone and Breast Cancer Progesterone and Safety Progesterone and Progesterone and Cardiovascular Health Progesterone and Bone Health Progesterone and Relief of Menopausal Symptoms Progesterone and the Nervous System/Brain Progesterone and the Uterus Progesterone and Dosing Forms Progesterone and the Difference between Progestins Estrogen Estradiol and Estriol Andropause Androgens Testing Saliva Testing Research... 66

9 jgfjfdjkgbvm,v,khg,fbggxgfccdbdzsgd

10 What is BHRT? Simply stated, Bio-identical Hormone Replacement Therapy, BHRT, is a prescription alternative to current traditional hormone replacement therapy (HRT), but unlike HRT, BHRT utilizes hormones that are identical in molecular structure to endogenously produced hormones. Since bio-identical hormones have the exact molecular configuration, they have the same exact biological function as endogenous hormones. Bio-identical hormones fit into the same metabolic pathways as endogenous hormones. They are also converted into other hormones as well as the same important metabolites as endogenous hormones. A well known example is human recombinant insulin. Bio-identical hormones are originally derived from a molecule called diosgenin in either soy or yam. Through a series of chemical processes the plant molecule, which is structurally similar to a steroid hormone, is converted to the specific human hormone molecule in a laboratory. The final product has no trace of the plant-just the pure biologically identical hormone. These bio-identical hormones are then prepared by compounding pharmacies in the desired dosing form of transdermal creams and gels, capsules, sublingual delivery, or implants placed under the skin. BHRT is a highly effective therapy, especially for women and men experiencing symptoms of hormone imbalance and hormone deficiency. Patients may be as young as teen girls with PMS to elderly postmenopausal women or elderly men with andropause. These patients may present with PMS, peri-menopausal or menopausal symptoms, or other conditions that result from or are associated with hormone imbalance. These include but are not limited to: insomnia, mood disorders, metabolic syndrome, migraines, obesity, fatigue, hypothyroidism, bone loss, uterine fibroids, fibrocystic breasts, skin disorders, PCOS, CFS, hirsutism, and connective tissue disorders. BHRT addresses hormonal deficiencies and imbalances by accurately assessing each patient s individual hormonal status (based on medical history, symptoms, lab work, etc.) and subsequently implementing a protocol individually tailored and prescribed for each patient. Therapy is then modified or titrated in accordance with clinical and laboratory results. BHRT is generally initiated after a hormone consultation with the patient. Patient progress is typically reviewed and repeat testing submitted in 8-12 weeks. Yearly or semi-annual evaluation is expected following resolution of initial symptoms. Often therapy requires adjustments after periods of stress, significant weight loss or gain, or as endogenous hormone levels shift, etc. 1

11 Symptoms of Hormone Imbalances Symptoms of Deficient Hormone Levels Progesterone Deficiency (Female) Physical symptoms or related conditions: Weight Gain Low Body Temp Headaches Hypothyroidism Hair Loss Fluid Retention Allergies/Sinusitis Bone Loss Gynecological symptoms or related conditions: Breast Pain/Cysts PMS Fibroids Heavy Cycles Irregular Cycles Endometriosis Cramps Emotional symptoms or related conditions: Anxiety Depression Irritability Tendency to be stressed easily Estrogen Deficiency (Female) Physical symptoms or related conditions: Vasomotor Symptoms Poor Memory Insulin Resistance Bone Loss Heart Palpations Hair Loss Dry Skin/Eyes Insomnia Headaches Gynecological symptoms or related conditions: Lighter/No Cycles Vaginal Dryness Urinary Tract Infections Incontinence Emotional symptoms or related conditions: Depression Anxiety Carb Cravings Sleep Disturbances Low Libido 2

12 Symptoms of Hormone Imbalances Symptoms of Deficient Hormone Levels Testosterone Deficiency (Female) Physical symptoms or related conditions: Aches and Pains Thinning Skin Loss of Muscle Tone Fatigue Heart Palpations Insomnia Bone Loss Vasomotor Symptoms Gynecological symptoms or related conditions: Vaginal Dryness Incontinence Loss of Pubic Hair Lichen Sclerosis Impaired Sexual Function Emotional symptoms or related conditions: Loss of Libido Depression Lack of Motivation Cortisol Deficiency Symptoms (Female or Male) Aches and Pains Irritability Fatigue Heart Palpations Chemical Sensitivity Allergies Arthritis Cold/Low Body Temp Sugar Cravings Thyroid Deficiency Symptoms (Female or Male) Fatigue/Exhaustion Cold Hands and Feet Weight Gain Memory Lapse High Cholesterol Low Blood Pressure Decreased Sweating Hair Loss Aches/pain Heart Palpations Bone Loss Infertility Constipation Depression Low Body Temp Inability to Lose Weight Poor Concentration Puffy Eyes/Face Slow Pulse Dry Hair/Skin Brittle Nails Low Libido Sleep Disturbances Thinning Skin Slowed Reflexes Hoarseness 3

13 Symptoms of Hormone Imbalances Symptoms of Excessive Hormone Levels Excess Estrogen (Female) The collection of symptoms related to excess estrogen is called Estrogen Dominance. This condition occurs when there is a lack of progesterone. It is diagnosed by symptoms and lab values that show a low Progesterone-to-Estradiol ratio. Basically, in Estrogen Dominance there a lack of the differentiation action by Progesterone to sufficiently manage the proliferatory action of Estradiol. Fluid Retention Hormonal and Premenstrual Headaches Clots Irritability Breast Pain, Fibrocystic Breast Disease, Endometriosis Heavy Bleeding Irregular Bleeding Uterine Fibroids, Breast Cancer, Breast Adenomas Excess Testosterone (Female) Acne/Oily Skin Facial Hair Thinning Scalp Hair Excess Body Hair Mid-Cycle Pain Hypoglycemia or Insulin Resistance Ovarian Cysts Nipple Pain Aggression & Irritability Excess Progesterone (Female) (Note: symptoms only occur with over dosing.) Sleepiness Candida Bloating Estrogen Deficiency Symptoms Excess Cortisol (Female or Male) Hormone resistance to thyroid, insulin, estrogen, testosterone, progesterone. Sleep Disturbances Low Libido Tired and Wired Feeling Cravings Stressed Feeling Irritability Bone Loss Thinning Skin Loss of Muscle Mass Breast Cancer Headaches Heart Palpations Glycation Insomnia Cardiovascular Disease Low Serotonin 4

14 Symptoms of Hormone Imbalances Symptoms of Deficient Hormone Levels Symptoms of Androgen Imbalance: Male Low Libido Decreased Erections Acne/Oily Skin Fatigue/Burned Out Feeling Aches/Pains Foggy Thinking Decreased Flexibility Aggression Heart Palpitations Prostate Problems Irritability Symptoms of Estrogen Imbalance: Male Hot Flashes Prostate Problems Headaches Night Sweats Decreased Urine flow Low Libido Foggy Thinking Increased Urinary Urge Irritability/Anxiety Bone Loss Nervous Elevated Triglycerides Depressed Mood Weight Gain Decreased Mental Ability Arthritis Anxious Depressed Mood Sleep Disturbances Bone Loss Decreased Muscle Mass Thinning Skin Decreased Stamina Decreased Urine Flow Increased Urinary Urge 5

15 Understanding The Role of Hormones Actions & Effects of Hormones: Female Progesterone Gestation Enables Egg Implantation Protects Against Miscarriage Facilitates Fetal Brain Development Effects on Estrogen & Reproductive Functions Decreases Menstrual Bleeding Normalizes Endometrial Shedding Permits Action of Estradiol Increases Circulating Levels of Estradiol Inhibits Fibrocystic Breast Disease May Inhibit Growth of Fibroids Helps with Estrogen Metabolism Anti-Proliferatory (Promotes Differentiation) Increases Sensitivity of Estrogen Receptors and Down Regulates Estrogen Receptors. Counters Symptoms of Estrogen Dominance: Breast Tenderness, Fluid Retention, Headaches, Irritability, etc. Skin Inhibits Aging of the Skin Cardiovascular Protection Protects Against Atherosclerosis Blocks Expression of VCAM-1 Blocks Cell Surface Markers Has No Negative Affect on Lipid Profile Does Not Raise C-Reactive Protein Reduces Vascular Smooth Muscle Proliferation Blocks Cardiac Fibroblast Proliferation Enhances/Preserves Estrogenic Benefits Dilates Coronary Arteries (By Increasing Nitric Oxide) Has No Negative Affect on Blood Pressure Central Nervous System Improves Brain Structure/Function/Memory Reduces Irritability/Anxiety Promotes Normal Sleep Patterns Modulates Gaba Receptor Function Protects the Brain Promotes Myelin Repair 6

16 Understanding The Role of Hormones Actions & Effects of Hormones: Female Progesterone, continued. Anti-Mineral Corticoid Acts as a Potassium Sparing Diuretic Skeletal Stimulates osteoblast production Breast Cancer Protection Stops Breast Cell Proliferation Increases IL-12 & NK Cells Helps with Estrogen Metabolism Reduces Estradiol Induced Breast Cell Proliferation Blocks Conversion of Estrone Sulfate to Estrone Suppresses Metalloproteinases (MMPs) Miscellaneous Increases Endurance During Exercise Prevents and Reduces PMS Decreases Hot Flashes Has Anti-Inflammatory Properties Increases Metabolic Rate May Increase Libido Enables Growth/Development/Repair of Tissue Effects on other Hormones/Glands Facilitates Thyroid Hormone Utilization Blocks Aromatase Opposes Estradiol Assists Thymus Gland Function Opposes Cortisol Can be Converted to Androgens in the Presence of Insulin Has a Positive Feedback on Endogenous Production Competes with Testosterone for 5-Alpha-Reductase to Reduce Conversion of Testosterone to DHT 7

17 Understanding The Role of Hormones Actions & Effects of Hormones: Female Estrogen Effects on the Reproductive System Creates Endometrial Lining Regulates Cycles Promotes Proliferation Causes Development at Puberty Cardiovascular Protection Relaxes Vascular Smooth Muscle Acts as a Calcium Channel Blocker Lowers Fibrinogen Has Anti-Oxidant Properties Effects on Lipids Lowers LDL Cholesterol Decreases Homocysteine Decreases Lipoprotein (a) Decreases Proliferation of Smooth Muscle Cells Decreases Activity of Angiotensin Converting Enzyme Inhibits Endothelin-1 (Vasoconstrictor) Promotes Endothelium-Derived Factors (Nitric Oxide & Prostacyclin) Increases HDL Cholesterol Increases/decreases triglycerides depending on dosing form Effects on the Central Nervous System Prevents Memory Disorders Reduces Amyloid Potentiates Neuronal Responses Regulates Inflammatory Cytokines Acts as an Anti-Depressant Aids in Healing after Injury Increases Acetyltransferace Production Promotes New Growth of Nerve Connections Increases Reaction Time, Alertness, Verbal Ability Increases Glucose Transport across Blood Brain Barrier Increases Seratonin, Dopamine, Epinephrine, Norepinephrine Effects on Glucose/Insulin Estradiol (physiologic levels) improves carbohydrate metabolism. Estradiol augments pancreatic response to insulin. Increases sensitivity of muscle and adipose tissue to insulin. Excess estrogen causes impaired glucose tolerance and increases the release of insulin. 8

18 Understanding The Role of Hormones Actions & Effects of Hormones: Female Estrogen, continued. Effects on Skin Health Increases Water/Moisture Content of Skin Increases Collagen Formation Thickens Skin Improves Vascularization, Elasticity & Firmness Maintains of Oral Health Maintains of Eye Health Effects on the Urogenital System Increases Vascularity Thickens the Vaginal Epithelium Increases Elasticity Prevents Atrophic Vaginitis Thickens the Blood Vessel Walls Increases Vaginal Discharge Decreases Frequency Urinary Tract Infections Effects on the Skeletal System Subdues Osteoclasts, Slowing Bone Resorption Actions with other Hormones Increases Growth Hormone Increases Thyroid Binding Globulin Up-Regulates Itself Makes More Insulin Available Increases Production of the Progesterone Receptors Competes with T3 at the Receptor Level Increases the Production of Cortisol Independent of ACTH Estriol Effects on the Breasts Protects Against Breast and Uterine Cancer Competes with E1 & E2 at Receptor Site Demonstrated to induce remission of metastastic tumors in 37% of women. Deficiencies thought by some researchers to be associated with breast cancer. Effects on the Urogenital System Helps with Urinary Tract Infections Helps Prevent Vaginal Dryness and Painful Intercourse Helps Reduce Vaginal Atrophy, Dysuria, & Urinary Frequency 9

19 Understanding The Role of Hormones Actions & Effects of Hormones: Female Estriol, continued. Effects on the Reproductive System Stays Fairly Steady throughout Cycle Rarely Triggers Uterine Bleeding Effects on Skeletal System Relieves Hot Flashes in Many Women Lower FHS and LH Increases Bone Mass 1-5 % in 12 months Effects on the Cardiovascular System Lowers Total Cholesterol, LDL, & Triglycerides Doesn t Increase Clotting Raises HDL Effects on the Skin Reverses Wrinkling & Pore Size Improves Elasticity & Firmness Improves Moisture Content Improves Acne Scars 100% Testosterone Effects on the Skeletal System Increases Bone Mineral Density Reduces Fractures Aids in Forming Strong Bones Closes Epiphyses During Puberty Effects on the Muscular System Increases Muscle Bulk and Tone Maintains Lean Body Mass/Strength/Stamina Increases Levator Ani Support and Control of Sphincters Effects on the Brain Increases Libido Increases Sense of Well Being Affects the Structural Increases Energy Improves Memory Integrity of the Brain Supports Self-Esteem Improves Mood 10

20 Understanding The Role of Hormones Actions & Effects of Hormones: Female Testosterone, continued. Effects on the Cardiovascular System Lowers Cholesterol Lowers all Fractions of Lipids Immune: Decreases IL-6 Increases Arterial Vasodilatation Increases red cell mass, viscosity and reduces platelet aggregation Effects on the Skin Thickens Stratum Corneum Increases Sebum Increases Body and Facial Hair Effects on Glucose Increases Insulin Sensitivity Hormone Interactions Recall that bio-identical hormones are metabolized exactly like endogenous hormones. They flow down the steroid pathway as normal. When utilizing BHRT one must consider hormone interactions between the thyroid, adrenal hormones, and sex steroids. The Hormone Cascade 11

21 Understanding The Role of Hormones Actions & Effects of Hormones: Male Testosterone Cardiovascular Health Seventy years of research has clearly demonstrated the role of testosterone in heart health. Testosterone is the androgen of greatest concentration in cardiac tissues. As early as 1940 it was used to successfully treat angina and more recently shown to reduce risk of death from cardiac arrhythmia. Can make heart muscle more resistant to death during ischemia. Research demonstrates that testosterone levels were lower in patients who had suffered MI s than in those who had not. Results also suggested that low testosterone levels predispose men to MIs. Has been found to inhibit clot formation by decreasing fibrinogen and decrease hardening of the arteries by increasing HDL and decreasing triglycerides. Testosterone levels are lower in men with severe coronary artery atherosclerotic disease than in controls. High blood levels of testosterone might protect against atherosclerosis, especially in men over age 60. Erectile Dysfunction Low testosterone levels negatively impact the structure and function of the penile tissue and erectile nerves, European men with low sex drive have been treated with Testosterone since It is now being suggested for use along with meds such vaiagra. Testosterone gel has been shown to improve orgasmic function. Prostate Health A study in the International Journal of Andrology, in 2002 on the use of testosterone found that testosterone caused reduction in prostate glands size, PSA numbers dropped, frequency, urgency, dribbling, and getting up a night to urinate all improved. Various Testosterone Affects Fat Distribution Affects Male Hair Growth Maintains Bone Mass Improves Mood and Wellbeing (due to effects of the neurotransmitters) Improves Cognitive Ability Increases Muscle Mass and Strength Increases Bone Density 12

22 BHRT Dosing Forms Bio-identical hormones come in a variety of forms. In addition to the familiar pills and capsules, transdermal (transported via the skin) forms such as creams, gels, and patches, additional forms are sublingual lozenges or drops, troches which are placed in the cheek, and intra-vaginal suppositories, which are useful in treating vaginal dryness and atrophy. Hormones can also be delivered in pellets implanted under the skin. Of interest is the increase over the last decade in the transdermal delivery of hormones has been found to be effective and well tolerated, with the additional benefit of bypassing the first-pass effect of the liver. This allows for reduced dosages as more of the hormone is directly available for its therapeutic effect rather metabolization via the digestive tract. ( see Hermann, in references section). Studies now confirm the effectiveness of topical progesterone preparations in protecting the endometrium from proliferation, which may otherwise lead to the development of uterine cancer, in women using estrogen therapy for menopausal symptoms. (See references in the section on progesterone and the uterus ) Compounding pharmacies provide bio-hormones in convenient pumps or metered measure syringes for precise dosing. Dosing Form Pros and Cons Understandably, many women prefer the convenience of oral forms of BHRT, but there are important disadvantages to swallowing hormones. During this first-pass metabolism, 90% of the hormones are converted to metabolites, leaving only 10 percent of the hormones in their original forms. These metabolites have different effects from those of the original hormones. For example, oral progesterone can be converted to metabolites that often cause very significant drowsiness, depression and bloating in some women. In order to achieve an effective dose, the BHRT practitioner has to prescribe as much as ten times the dose compared to transdermal dosing. Typical dosing of oral progesterone is mg compared to 10-20mg transdermal progesterone. The greatest risk appears to be with oral estrogen. When estrogen undergoes the first-pass metabolism, it stimulates the liver to produce triglycerides and blood-clotting factors as well as binding proteins that can increase the risk of blood clots. A recent study indicates that women who use estrogen patches have no higher risk of blood clots than women who take no hormones, and the researchers believe it is due to transdermal delivery avoidance of first pass metabolism. (See reference section for citation.) Additionally, oral estradiol rapidly converts to estrone. If the body does not process this form of estrogen correctly, there can be an increased the risk of breast cancer due to the potential increased levels of 4 OH estrone. Yet another issue is the fact that the increased binding proteins created from oral dosing reduce circulating thyroid hormones and testosterone. Therefore, the BHRT Advisors of The Hormone Treatment Centers of America will be suggesting Transdermal application in their recommendations submitted to our providers. 13

23 BHRT Dosing Forms A Comparative Analysis: Transdermal versus Oral Estrogen Transdermal Estrogen Lowers Triglycerides Lowers LDL Decreased Tendency Toward Clotting Less Likely to Raise Blood Pressure Has Minimal Impact on C-Reactive Protein Is Times More Efficiently Absorbed Oral Estrogen May Increase Triglycerides Converts Rapidly to Estrone: Body may convert into metabolites increasing breast cancer risk. Raises HDL, Lowers LDL Increase Risk of Blood Clots Increases Interleukin 6 Increases C-Reactive Protein Increases Sex Hormone Binding Globulin, Cortisol Binding Globulin, Thyroid Binding Globulin (which makes other hormones less available) Transdermal Dosing Forms PerQ Gel TM A once-a-day application of this percutaneous gel is all that is required to achieve a near constant 24- hour steady state of hormone levels. Two cc s of PerQ Gel TM are spread (not rubbed) onto the skin over a large surface slowly releasing hormones from its microcirculation into the main circulation continuously over a 24-hour period. This gel is extremely hypo-allergenic and compliance is very good due to once-a-day dosing. Cream Creams may be applied once-a-day or twice-a-day and rubbed into the skin. Cream delivery is also convenient and very well absorbed. 14

24 Testing & Monitoring Hormone Levels Hormone testing gives the practitioner and BHRT specialists the ability to accurately detect hormone imbalances, helps monitor dosing, and serves to increase patient compliance. The combination of lab results and a review of symptoms effectively direct the protocols necessary to reach the optimal outcome. Saliva testing is the preferred method of testing since it measures the amount of hormones that are available to the hormone-responsive tissue. Saliva testing makes it quite simple to measure the change in hormone levels when hormone supplementation is given and provides the practitioner with a very valuable clinical tool. To explain further, the steroid hormones deliver their message to cells by leaving the blood flow at the capillaries to enter cells and bond with their specific receptors. These are free or unbound hormones. As these hormones circulate through the liver, they become protein-bound with Sex Hormone Binding Globulin or albumin. Once hormones are bound by protein, they become water-soluble, thus facilitating their excretion in urine. Measuring the level of protein-bound hormones in the urine or serum is irrelevant since it does not measure the more clinically significant free, bio-available hormones. Saliva testing is accurate because the non-protein bound bioavailable hormones diffuse easily from the blood capillaries into the salivary gland and then into saliva. In contrast, the protein-bound, non-bio-available hormones do not pass into the salivary glands. Saliva testing is used at The Hormone Treatment Centers of America to evaluate the following hormone levels: Estradiol, Progesterone, Progesterone-to-Estradiol ratio, Testosterone, DHEA, and Cortisol. In most cases saliva testing is ordered before BHRT protocols are submitted to the provider. Follow up testing is typically recommended after 3 months of initiating therapy and then every 6 months afterward. 15

25 Hormone Treatment Centers of America Sample Treatment Plan Sample Treatment Plan (Detailed Version) Practice: Carolina Family Practice 100 Main Street Any Town, NC Ordering Physician: John Smith, MD Date: HTCA Contact: Jane Doe Patient: Jane Smith Status: 57 YO, 5 1, 161 lb, post-menopausal woman LMP: 1998 Presenting issues of note: Slightly Elevated BP: 134/90 Abnormal Pap: 1/08 Will follow up with GYN: 3/08 Supplements/Diet: Appropriate diet. Variety of appropriate supplements. Exercise/Activity: Exercises regularly. Medications: Intra-vaginal Estrace cream 3 x weekly for past 3 ½ years. Drug Allergies: Demerol, Darvon, Percaset, Percadan, Codine. Hormone Usage: Currently on OTC Progesterone cream 12mg 2 x daily for past 8-9 months. Comments: Patient indicates that it has helped some with sleep. Past Hormone Usage: FemHRT and Prempro. Both caused weight gain. Patient Concerns: Wants to lose weight, reduce vaginal dryness, reduce aches in knees/hips, improve sleep, and reduce sinus infections. Other Remarkable Sx: Night sweats, foggy thinking, sleep disturbances, water retention, low libido, fatigue, sugar cravings, cold body temperature with cold hands and feet, constipation. Preliminary Recommendations: Stop Estrace for 7 days prior to Saliva Test. 16

26 Sample Treatment Plan (Detailed Version) Saliva Test Summary Saliva panel, 2/18/06 on 12mg Progesterone Cream b.i.d. Hormone Results Reference Range Estradiol <0.5 LOW Progesterone 4630 HIGH Ratio 9260 HIGH Testosterone 20 OK (Low-Normal) DHEA 2.8 LOW 3-10 Cortisol 3.5 OK (Low-Normal) 3-8 Detail of Recommendations: Restore sex hormone deficiencies, address adrenal fatigue indicated by low DHEA and low-normal cortisol levels. BHRT: Biest Gel: 0.1mg 50/50 ratio with compounded with 20mg of Progesterone. Apply 1 ml qd. DHEA caps, 10mg po qd. Improving DHEA level should slightly augment low-normal Testosterone level. DHEA DHEA is precursor to hormones that are estrogenic and androgenic. If DHEA levels are deficient, deficiencies of other hormones usually ensues triggering effects through out the endocrine system. Replacing deficient DHEA can also serve to increase low levels of Estradiol and/or Testosterone; therefore therapy should be properly monitored. Specifically, a deficiency of DHEA has been found to correlate with: Chronic Inflammation Greater Risk for Certain Cancers Immune Dysfunction Excess Body Fat Depression Cognitive Decline Rheumatoid Arthritis Heart Disease (In Men) Type-II Diabetic Complications Osteoporosis 17

27 Sample Treatment Plan (Detailed Version) DHEA, continued. Signs of low DHEA levels may include the following symptoms. However, it is important to note that they are not exclusively linked to low DHEA as they could also be reflective of other hormone deficiencies or conditions. Stressed Lack of Stamina Intolerance to Loud Noises Constant Fatigue Moodiness Decreased Immunity Memory Loss Lack of Pubic Hair Poor Abdominal Muscle Support Dry Eyes & Skin Actions & Effects of DHEA Protects the Cardiovascular System Low Sex Drive Low levels are linked to ischemic heart disease. Prevents blood clots. Strengthens the heart muscle. Protects against atherosclerosis. Increases resistance of LDL to oxidation and decreases cholesterol. Is incorporated into the HDL and LDL molecule and acts as an antioxidant. Reduces amount of atherosclerotic plaque (animal studies). Decreases levels of lipid peroxides and platelet superoxide dismutase (SOD) (animal studies). Protects the Brain & Central Nervous System Protects against the deterioration of mental function with aging. Low levels are associated with neurodegenerative diseases. Shown to be protective from hippocampal damage due to stroke. Protective against amyloid-beta protein and excess glutamate. Protects brain form excess Cortisol possibly protecting form dementia. Low levels are found in depressed patients. 18

28 Sample Treatment Plan (Detailed Version) DHEA, continued. Actions & Effects of DHEA Effects on Chronic Inflammatory Diseases Low levels found in chronic inflammatory diseases such as rheumatoid arthritis. Inhibits pro-inflammatory cytokines (which signal immune system and further cellular destruction. Inhibits interleukin 6 (IL-6) and tumor necrosis factor (TNF); these are elevated in inflammatory diseases. IL-6 is implicated in bone loss, autoimmune conditions, atherosclerosis, osteoporosis, Alzheimer s and certain cancers. Has been found beneficial in Lupus patients. Low levels found in patients with chronic back pain, neck and shoulder pain along with low beta endorphins. Antibacterial/Antiviral Properties/Immune Function Possibly can protect against activation of HIV. Used with melatonin; possible adjunct therapy against retroviral infection and anthrax toxin. Protective against bacterial sepsis. Necessary for the development of mature immune cells and enhanced antibody production. Protects the thymus against atrophy induced by glucocorticoids. Additional Properties of DHEA Decreases Formation of Fatty Deposits Promotes Weight Loss Increases Sense of Well-Being Helps with Repair and Maintenance of Tissues Increases Bone Growth Increases Bbrain Function Decreases Allergic Reactions 19

29 Hormone Treatment Centers of America Sample Treatment Plan Adrenal Support Adrenal function appears to be sub-optimal indicated by the low DHEA and low-normal cortisol levels. Adrenal support is indicated. Adrenal Cortex glandular supplement at 1 capsule bid to tid, additional vitamin C w/bioflavonoids 500mg tid, and Ashwagandha at 1 capsule bid to tid. Vitamin C: Vitamin C is the most important nutrient for adrenal metabolism and is essential to the adrenal cascade. It is just as important to the health of the adrenal glands under producing cortisol as those that are over producing cortisol. Vitamin C is required to manufacture the adrenal hormones. Because C is water soluble and rapidly excreted it should be used several times per day. Pantothenic Acid is essential for the conversion of glucose into energy and is present in the highest quantities in the adrenal glands. This is because a lot of energy is required to produce the adrenal hormones. Combining magnesium (in current multi), Vitamin C and E with pantothenic acid increases energy levels and the stress on the adrenal glands is reduced. Recommended reading: Adrenal Fatigue, The 21st Century Stress Syndrome, by James Wilson, PhD, DC, ND. The action of adrenal glandulars is to support, fortify, and restore normal adrenal function. They are not replacement hormones but provide constituents for adrenal repair. As the cells of the adrenals recover they can begin to produce normal levels of adrenal hormones once again. Ashwaganda: Helps with inflammation and pain and normalizes cortisol levels. Supplements Maintain foundational nutrients of comprehensive multiple vitamin/mineral, pharmaceutical quality fish oil. Maintain low glycemic diet and exercise. Follow-Up Repeat Saliva Test in 3 months to evaluate BHRT dosing. 20

30 Hormone Treatment Centers of America Sample Treatment Plan Sample Treatment Plan Summary Practice: Carolina Family Practice 100 Main Street Any Town, NC Ordering Physician: John Smith, MD Date: HTCA Contact: Jane Doe Patient: Jane Smith Summary: 1. Bi-est Gel: 0.1mg 50/50 ratio with compounded with 20mg of Progesterone. Apply 1 ml qd. 2. DHEA capsules, 10mg po qd. 3. Adrenal Cortex glandular supplement at 1 capsule tid. 4. Vit C w/bioflavonoids 500mg tid. 5. Ashwagandha at 1 capsule tid. 6. Continue exercise and low glycemic index diet. 7. Repeat Saliva Test in 2 to 3 months. 8. Follow up visit with healthcare provider for review of results after repeat testing. Date: Approved Approved w/provider Changes Provider Signature: Provider Changes (list if needed): 21

31 Progesterone and Menstrual Health/PMS, Infertility, Pregnancy Overview Progesterone is named because of its essential functions in maintaining conditions in the uterus that sustain pregnancy (pro-gestation). During the menstrual cycle, estrogen causes the uterine lining to proliferate in preparation for implantation of a fertilized egg, and it stimulates a number of physiological events designed to prepare the body for pregnancy, such as stimulation of protein and lipid synthesis and mitotic activity. Progesterone levels rise after ovulation and act to complete the preparation of the uterus for implantation, resulting in increased secretory activity and strengthening of the blood vessels. When progesterone becomes the dominant hormone in the secretory (luteal) phase of the menstrual cycle, it reduces the estrogen-binding capacity of the endometrial tissue. If implantation does not occur, progesterone levels fall and the endometrial lining is shed in the menstrual flow. If implantation does occur and pregnancy results, progesterone levels remain high to support the conditions required by the developing fetus. The consistently high physiological progesterone levels seen in pregnancy would indicate that progesterone is safe as a therapeutic agent. Progesterone therapy is commonly used in women with infertility problems due to luteal progesterone insufficiency in order to raise circulating and endometrial progesterone levels to those of the normal luteal phase. Progesterone has also been used to treat premenstrual syndrome (PMS), because the falling levels of progesterone prior to menstruation are thought to contribute to symptoms in some patients. However, there are few published studies. Yonkers K. Review: progesterone or progestogens lead to a marginal reduction in premenstrual syndrome symptoms. Evid Based Ment Health May;5(2):56. The author conducted an analysis of randomized, double blind, placebo-controlled studies of progesterone or progestins in women diagnosed with PMS. Oral micronized progesterone and the progestogens MPA, norethisterone and dydrogesterone, all showed a marginal benefit over placebo in symptom reduction. Shantha S, Brooks-Gunn J, Locke RJ, Warren MP. Natural vaginal progesterone is associated with minimal psychological side effects: a preliminary study. J Women Health Gend Based Med 2001 Dec;10(10): This 3 month, multicenter randomized study evaluated the psychological side effects of a vaginally applied progesterone gel in reproductive aged women treated for hypothalamic amenorrhea or premature ovarian failure. No differences were noted in psychometric measures as evaluated by the Hopkins Symptom Checklist. Natural progesterone in a vaginal gel can be an effective treatment for women requiring hormone therapy. Wyatt K, Dimmock P, Jones P, Obhrai M, O Brien S. Efficacy of progesterone and progestogens in management of premenstrual syndrome: systematic review. BMJ Oct 6;323(7316): This systematic review of published studies of progesterone or progestogens for treatment of PMS found a small positive effect of oral micronized progesterone over placebo in the 3 trials that studied this. No published studies of progesterone cream were found. A statistically, but not clinically, significant improvement was seen with progestogen treatment. 22

32 Progesterone and Menstrual Health/PMS, Infertility, Pregnancy, continued. Sofuoglu M, Babb DA, Hatsukami DK. Progesterone treatment during the early follicular phase of the menstrual cycle: effects on smoking behavior in women. Pharmacol Biochem Behav 2001 May-Jun;69 (1-2): In this unique randomized controlled study, administration of progesterone (200 mg oral) demonstrated a decrease in craving for and subjective effects of cigarette smoking in female smokers. With progesterone treatment, there was a noted trend to decrease smoking. Fitzpatrick LA, Good A. Micronized progesterone: clinical indications and comparison with current treatments. Fertil Steril 1999 Sep;72(3): The literature reviewed in this tutorial indicates a potential use for oral micronized progesterone for the treatment of secondary amenorrhea, dysfunctional uterine bleeding, luteal phase disorders, premenopausal bleeding disorders, and as a component of hormone replacement therapy that may provide a better safety profile than commonly utilized synthetic progestins. Massai R, Miranda P, et al. Preregistration study on the safety and contraceptive efficacy of a progesterone-releasing vaginal ring in Chilean nursing women. Contraception 1999 Jul;60(1):9-14. In this long-term controlled study, the safety and efficacy of a progesterone-releasing vaginal contraceptive device was compared to that of the copper-t 380A IUD in nursing mothers. There was no difference in breastfeeding performance or infant growth between groups. The participants using the progesterone-releasing ring had a longer period of lactational amenorrhea than did the group using the copper T. Women were tracked for over 2000 women-months of exposure in both groups. The Chilean government found the progesterone-releasing ring to be a safe and effective contraceptive alternative. Hajek Z, Uhlir M. [Micronized progesterone in the treatment of imminent necrosis of a myoma during pregnancy. Ultrasound changes during treatment] Ceska Gynekol 1999 Jun;64(3): [Article in Czech] Progesterone has a role in increasing blood flow to the uterus during pregnancy. As such, these researchers studied the effect of progesterone treatment to resolve imminent necrosis of a myoma in two cases. Both resolved within several days following oral and vaginal doses of progesterone ( mg/day). Both women went on to deliver healthy, full-term infants. Pouly JL, Bassil S, Frydman R, et al. Luteal support after in-vitro fertilization: Crinone, a sustained release vaginal progesterone gel, versus Utrogestan, an oral micronized progesterone. Human Reprod 1996;11: mg of vaginal estrogen gel daily was compared to 300 mg oral progesterone daily in a randomized open-label trial of 283 IVF patients. Delivery rates, safety parameters, frequency of spontaneous abortion, ratio of newborn babies to embryo transfer were nearly identical for both groups. The oral progesterone group reported more drowsiness. 23

33 Progesterone and Menstrual Health/PMS, Infertility, Pregnancy, continued. Nappi C, Affinito P. Double-blind controlled trial of progesterone vaginal cream treatment for cyclical mastodynia in women with benign breast disease. J Endocrin Invest 1994;15(11): Eighty regularly menstruating women with mastodynia were studied to evaluate the clinical effectiveness of vaginally administered micronized progesterone. Subjects were randomly assigned to one of two groups, with all participating in a control cycle prior to treatment. One group received 4 grams of vaginal cream containing 2.5% natural progesterone for six cycles from day 19 to day 25 of the cycle. The other group was similarly treated with placebo. Both subjective reporting on a daily basis and clinical examination revealed a significant reduction in breast pain, defined as 50% reduction, in 64.9% of subjects receiving progesterone and 22.2% of subjects receiving placebo. Effects of breast nodularity were not significant. No side effects were detected. Martorano JT, Ahlgrimm M, Meyers D. Differentiating between natural progesterone and synthetic progestogens: clinical implications for PMS management. Comprehensive Therapy 1993; 19(3):96-8. Clinical observations demonstrate that patients suffering from PMS respond to treatment with natural progesterone, whereas synthetic progestins may exacerbate the condition. The authors review the differences between natural progesterone and synthetic progestins. Saarikoski S, Yliskoski M, Penttila I. Sequential use of norethisterone and natural progesterone in premenopausal bleeding disorders. Maturitas 1990 Jun;12(2): This randomized controlled study evaluated the effects of norethisterone (NET) and micronized progesterone (MP) on bleeding disorders in pre-menopausal women. 80 patients were randomized to the trial and all were found via endometrial morphology to need progestogen therapy. They were subsequently treated with NET or MP. In both treatment groups, hyperplastic changes disappeared during the first three cycles, with the duration of treatment being 6 months. NET decreased follicle-stimulating hormone, luteinizing hormone, estradiol and sex-hormone-binding globulin levels (P < 0.001) whereas no changes were seen during MP treatment. High-density-lipoprotein cholesterol and triglyceride levels were also lowered by NET (P< ) slightly decreased phospholipids. MP treatment had no effect on lipid profiles suggesting it may be a preferred progestogen for the treatment of bleeding disorders. Sitruk-Ware R, Bricaire C, De Lignieres B, Yaneva H, Mauvais-Jarvis P. Oral micronized progesterone. Bioavailability pharmacokinetics, pharmacological and therapeutic implications--a review. Contraception 1987 Oct; 36(4): This paper reviews the effects and benefits of oral micronized progesterone. Progesterone exhibits anti-estrogenic effects, anti-androgenic effects, and anti-mineralocorticoid effects in addition to its progestational effects. No side effects have been reported for micronized progesterone with respect to lipid profile, coagulation, or blood pressure, leading the authors to recommend micronized progesterone as suitable for treatment of PMS, menopause, irregular cycles, and pregnancy maintenance. 24

34 Progesterone and Menstrual Health/PMS, Infertility, Pregnancy, continued. Dennerstein L, Spencer-Gardner C, Gotts G, Brown JB, Smith MA, Burrows GD. Progesterone and the premenstrual syndrome: a double blind crossover trial. Br Med J (Clin Res Ed) 1985 Jun 1; 290(6482): In this double-blind, placebo-controlled, randomized, crossover trial, oral micronized progesterone demonstrated effectiveness in alleviating premenstrual complaints. Twenty-three women completed a Beck, et al depression inventory, Moos s menstrual distress questionnaire, Spielberger, et al state anxiety inventory, and daily symptom diary before and during each treatment. There was an overall benefit of treatment for all variables, except positive moods, restlessness, and interest in sex. For most parameters, maximum benefit was seen within the first month of treatment, demonstrating an effectiveness of progesterone as a viable treatment option for women with PMS. Ferre F, Uzan M, Janssens Y, Tanguy G, Jolivet A, Breuiller M, Sureau C, Cedard L. Oral administration of micronized natural progesterone in late human pregnancy. Effects on progesterone and estrogen concentrations in the plasma, placenta, and myometrium. Am J Obstet Gynecol 1984 Jan 1; 148(1): Levels of progesterone, 17 beta-estradiol, and estrone were measured in the plasma, in the placenta, and at different sites in myometrium following a single dose of micronized oral progesterone administered to 15 pregnant women immediately prior to elective cesarean section. In comparison to a control group, progesterone levels in the treated women increased in the plasma and myometrium 150 minutes after administration. Placenta progesterone levels did not demonstrate any change. No change was seen in 17 beta-estradiol levels in the plasma or the myometrium, however placental levels were increased. Estrone levels were decreased in the myometrium and in the placenta, and unchanged in the plasma. Dalton K. The effects of progesterone and progestogens on the foetus. Neuropharmacology 1981; 20: This article looks at the differing effects of progesterone and synthetic progestogens on the fetus. Of note in this article is evidence that progesterone supplementation may reduce episodes of pre-eclampsia. Synthetic progestogen supplementation during pregnancy may produce a variety of side effects. Several references are made to articles documenting cases of masculinization of external genitalia in female babies. There are two known cases of true hermaphroditism and several cases of behavioral problems developing in adolescent girls whose mothers took oral synthetic progestogens during pregnancy. More problematic may be administration of oral estrogen-progestogen preparations. Side effects may include spina bifida, esophageal anomalies, heart defects and limb reduction deformities. Dalton K. Prenatal progesterone and educational attainments. British Journal of Psychiatry 1976; 126: This study compares educational attainments of 34 children whose mothers received prenatal progesterone with 37 normal and 12 toxemic controls. Results at ages showed that progesterone children were more likely to continue schooling after 16 years, a higher number left school with O and A level grades and more obtained entrance to university. The best academic results were found for children whose mothers had received over 5 grams of progesterone for a minimum of eight weeks, with treatment beginning before week sixteen. 25

35 Progesterone & Breast Cancer Overview After the WHI trial was stopped it was widely reported that the patients receiving combined treatment with estrogen and progestin had a higher incidence of breast cancer than the group receiving estrogen alone, however bioidentical progesterone was not used in this study. Bioidentical progesterone has been found not to increase the risk of breast cancer when used in combined hormone therapy with estrogens, while synthetic progestins did increase the risk (Fournier et al. 2005). Endogenous progesterone levels were found not to increase breast cancer risk in the first study to investigate this in postmenopausal women; this was true even for progesterone receptor positive tumors, which were the most strongly affected by all circulating steroid hormones measured except for progesterone (Missmer et al. 2004). On the other hand, higher levels of endogenous estrogens and androgens were significantly correlated with increasing breast cancer incidence. Kaaks et al. (2005) also found a significant reduction in breast cancer risk in women with high progesterone levels. There is evidence that women with progesterone deficiency have a markedly increased incidence of breast and other cancers (Cowan et al. 1981), and also that the application of progesterone cream to the breasts decreases proliferative activity in breast tissue while estradiol cream increases such activity (Chang et al. 1995). In a study of women undergoing breast tumor excision, those who had higher levels of circulating progesterone at the time of excision had a significantly improved prognosis, the authors concluding that the tumor tissue was not in an actively proliferating state at the time of excision because of progesterone s antiproliferative effects (Mohr et al. 1996). The role of hormone balance in the development and prevention of breast cancer is still a subject of much controversy. More research is needed in this area. An interesting discussion that is on the edge of new thinking about the role of natural and synthetic hormones in the biochemistry of breast cancer can be found in the book What your doctor may not tell you about breast cancer how hormone balance can help save your life by JR Lee, D Zava and V Hopkins, Warner Books, New York Campagnoli C, Abba C, Ambroggio S, Peris C. Pregnancy, progesterone and progestins in relation to breast cancer risk. J Steroid Biochem Mol Biol 2005; 97(5): The authors review recent findings that show that the production of progesterone during pregnancy and the use of bioidentical progesterone in hormone therapy do not increase breast cancer risk, and can even protect against the development of breast cancer. Kaaks R, Berrino F, Key T, Rinaldi S, Dossus L, Biessy C, Secreto G, Amiano P, Bingham S, Boeing H, Bueno de Mesquita HB, Chang-Claude J, Clavel-Chapelon F, Fournier A, van Gils CH, Gonzalez CA, Barricarte Gurrea A, Critselis E, Khaw KT, Krogh V, Lahmann PH, Nagel G, Olsen A, Onland-Moret NC, Overvad K, Palli D, Panico S, Peeters P, Quirós JR, Roddam A, Thiebaut A, Tjønneland A, Chirlaque MD, Trichopoulou A, Trichopoulos D, Tumino R, Vineis P, Norat T, Ferrari P, Slimani N, Riboli E. Serum sex steroids in premenopausal women and breast cancer risk within the European Prospective Investigation into Cancer and Nutrition (EPIC). J Natl Cancer Inst 2005; 97: In this large multicenter study, higher serum progesterone levels were associated with a significant reduction in breast cancer risk. 26

36 Progesterone & Breast Cancer, continued. Fournier A, Berrino F, Riboli E, Avenel V, Clavel-Chapelon F. Breast cancer risk in relation to different types of hormone replacement therapy in the E3N-EPIC cohort. Int J Cancer 2005; 114(3): Combined HRT with estrogen (either oral or transdermal) and synthetic progestins was found to carry a significantly increased risk of breast cancer compared with estrogens plus oral micronized progesterone. In fact, no increase in breast cancer risk was seen in the estrogen plus oral micronized progesterone group compared with estrogen alone. This large multicenter study therefore suggests that there is a dramatic difference between the effects of bioidentical progesterone versus synthetic progestins on breast cancer risk. Missmer SA, Eliassen AH, Barbieri RL, Hankinson SE. Endogenous estrogen, androgen, and progesterone concentrations and breast cancer risk among postmenopausal women. J Natl Cancer Inst 2004; 96(24): Blood progesterone levels were found not to be related to breast cancer risk in this first study to investigate this in postmenopausal women. The occurrence of progesterone receptor positive tumors was the tumor type most strongly affected by all the circulating steroid hormones measured except for progesterone. Higher levels of endogenous estrogens and androgens were significantly correlated with increasing breast cancer incidence. This suggests that circulating natural progesterone does not increase breast cancer risk. Malet C, Spritzer P, Guillaumin D, Kuttenn F. Progesterone effect on cell growth, ultrastructural aspect and estradiol receptors of normal human breast epithelial (HBE) cells in culture. J Ster Biochem Mol Biol 2002; 73: In a culture system, progesterone was found to have an inhibitory effect on breast cell growth. When given following estradiol (E2), it limited the stimulatory effect of E2 on cell growth. Desreux J, Kebers F, Noel A, Francart D, Van Cauwenberge H, Heinen V, Thomas JL, Bernard AM, Paris J, Delansorne R, Foidart JM. Progesterone receptor activation- an alternative to SERMs in breast cancer. Eur J Cancer 2000 Sep;36 Suppl 4:S90-1. This review emphasizes progesterone s role in supporting healthy breast homeostasis and opposing the proliferative effects of estradiol in the breast, unlike synthetic progestins. Plu-Bureau G, Le MG, Thalabard JC, Sitruk-Ware R, Mauvais-Jarvis P. Percutaneous progesterone use and risk of breast cancer: results from a French cohort study of premenopausal women with benign breast disease. Cancer Detect Prev 1999;23(4): This cohort study followed 1150 premenopausal French women diagnosed with benign breast disease. Topical progesterone cream, a common treatment for mastalgia in Europe, had been prescribed to 58% of the women. Follow-up accumulated 12,462 person-years. There was no association noted between progesterone cream use and breast cancer risk. Furthermore, women who had used both progesterone cream and an oral progestogen had a significant decrease in breast cancer risk (RR= 0.5) as compared to women who did not use progesterone cream. There was no significant difference in the risk of breast cancer in percutaneous progesterone users versus nonusers among oral progestogen users. These results suggest there are no deleterious effects caused by percutaneous progesterone use in women with benign breast disease. 27

37 Progesterone & Breast Cancer, continued. Formby B, Wiley TS. Bcl-2, survivin and variant CD44 v7-v10 are downregulated and p53 is upregulated in breast cancer cells by progesterone: inhibition of cell growth and induction of apoptosis. Mol Cell Biochem 1999 Dec;202(1-2): This study sought to elucidate the mechanism by which progesterone inhibits the proliferation of breast cancer cells. Utilizing breast cancer cell lines with and without progesterone receptors (T47-D and MDA-231, respectively) in vitro, the authors looked at apoptosis (programmed cell death) in response to progesterone exposure as a possible mechanism. The genetic markers for apoptosis - p53, bcl-2 and surviving, were utilized to determine whether or not the cells underwent apoptosis. The results demonstrated that progesterone does produce a strong antiproliferative effect on breast cancer cell lines containing progesterone receptors, and induced apoptosis. The relatively high levels of progesterone utilized were similar to those seen during the third trimester of human pregnancy. Lin VC, Ng EH, Aw SE, Tan MG, Ng EH, Chan VS, Ho GH. Progestins inhibit the growth of MDA-MB-231 cells transfected with progesterone receptor complementary DNA. Clin Cancer Res 1999 Feb;5(2): Progesterone is mainly thought to exert its effects via the estrogen-dependent progesterone receptor (PR), the effects of which may be overshadowed by the presence of estrogen. In order to study the independent effects of progesterone on breast cancer cell lines, PR expression vectors were transfected into a PR and ER negative cell line (MDA-MB-231). The growth of these cells was then studied in response to progesterone and several progestins. Progesterone was found to significantly inhibit DNA synthesis and cell growth in a dose-dependant fashion. The results of this study indicate that progesterone and progestins independent of estrogen have an antiproliferative effect on breast cancer cells via the progesterone receptor. This suggests a possible role in the treatment of PR negative breast cancer via re-activation of the PR receptor. Formby B, Wiley TS. Progesterone inhibits growth and induces apoptosis in breast cancer cells: inverse effects on Bcl-2 and p53. Ann Clin Lab Sci 1998 Nov-Dec;28(6): This study explored the mechanism by which progesterone inhibits breast cancer cell proliferation (growth). In progesterone receptor positive T47-D breast cancer cells, the mechanism of apoptosis appeared to be through the regulation of the genes p53 and bcl-2 by progesterone. These genes control the apoptotic process. It was demonstrated that at progesterone levels that approximate the third trimester of pregnancy, there was a strong antiproliferative effect in at least 2 breast cancer cell lines. Foidart JM, Colin C, Denoo X, Desreux J, Beliard A, Fournier S, de Lignieres B. Estradiol and progesterone regulate the proliferation of human breast epithelial cells. Fertil Steril 1998 May;69(5): In this double-blind randomized study, to evaluate the effects of estrogen and progesterone on normal breast cells, 40 postmenopausal women received daily topical application of a gel containing either placebo, estradiol, progesterone, or estradiol + progesterone for two weeks prior to esthetic breast surgery or the excision of a benign breast lesion. The results showed that increased estrogen concentration increased the number of cycling epithelial cells, whereas exposure to progesterone for 14 days reduced the estrogen-induced proliferation of normal breast epithelial cells. 28

38 Progesterone & Breast Cancer, continued. Pasqualini JR, Paris J, Sitruk-Ware R, Chetrite G, Botella J. Progestins and breast cancer. J Steroid Biochem Mol Biol 1998 Apr;65(1-6): This review article outlines the many functions of progestogens in hormone-dependent and independent breast cancer and suggests new clinical applications for their use in the treatment of breast cancer. Mohr PE, Wang DY, Gregory WM, Richards MA, Fentiman IS. Serum progesterone and prognosis in operable breast cancer. British Journal of Cancer 1996;73: Higher blood levels of progesterone measured during surgical treatment of breast cancers were associated with significantly better survival, especially in women who were node-positive (P<0.01). There was no significant relationship between estradiol levels and survival. This study demonstrated that a higher level of progesterone at time of excision is associated with improved prognosis in women with operable breast cancer. Chang KJ, et al. Influences of percutaneous administration of estradiol and progesterone on human breast epithelial cell cycle in vivo. Fertil Steril 1995; 63(4): The effect of transdermal estradiol (1.5 mg), transdermal progesterone (25 mg), and combined transdermal estradiol and progesterone (1.5 mg and 25 mg) on human breast epithelial cell cycles was evaluated in vivo. Results demonstrated that estradiol significantly increases cell proliferation, while progesterone significantly decreases cell replication below that observed with placebo. Transdermal progesterone was also shown to reduce estradiol-induced proliferation. Laidlaw IJ, Clarke RB. The proliferation of normal breast tissue implanted into athymic nude mice is stimulated by estrogen, but not by progesterone. Endocrinology Jan 1995;136(1): Normal human breast tissue was implanted subcutaneously into athymic nude mice. The mice were then treated with estradiol or progesterone such that serum levels approximated those seen in normal menstruating women. Immunocytochemical measures were made of proliferative activity and steroid receptor expression of the tissue implants. It was found that physiologic levels of estradiol significantly stimulated the proliferation of human breast epithelial cells and increased progesterone receptor expression fold. Progesterone failed to affect proliferation alone or after estradiol priming. Nappi C, Affinito P. Double-blind controlled trial of progesterone vaginal cream treatment for cyclical mastodynia in women with benign breast disease. J Endocrin Invest 1994;15(11): Eighty regularly menstruating women with mastodynia were studied to evaluate the clinical effectiveness of vaginally administered micronized progesterone. Subjects were randomly assigned to one of two groups, with all participating in a control cycle prior to treatment. One group received 4 grams of vaginal cream containing 2.5% natural progesterone for six cycles from day 19 to day 25 of the cycle. The other group was similarly treated with placebo. Both subjective reporting on a daily basis and clinical examination revealed a significant reduction in breast pain, defined as 50% reduction, in 64.9% of subjects receiving progesterone and 22.2% of subjects receiving placebo. Effects of breast nodularity were not significant. No side effects were detected. 29

39 Progesterone & Breast Cancer, continued. Mauvais-Jarvis P, Kuttenn F, Gompel A. Antiestrogen action of progesterone in breast tissue. Horm Res 1987;28(2-4): In a review of international literature on the cellular effects of progesterone on both normal breast cells and breast cancer cell lines, the authors conclude that most data indicate progesterone and progestins have an antiestrogenic effect on the breast, as reflected in the decrease in estradiol receptor content, the decrease in cell proliferation, and an increase in a marker of cell differentiation, 17 beta-hydroxysteroid activity, which is mediated by the progesterone receptor. Cowan LD, Gordis L, Tonascia JA, et al. Breast cancer incidence in women with a history of progesterone deficiency. American Journal of Epidemiology 1981; 114:209.,083. Infertile women were followed for years. Those who were deficient in progesterone showed a fivefold greater incidence of premenopausal breast cancer. 30

40 Progesterone & Safety Overview Historically, progesterone and synthetic progestins have been lumped together with respect to their safety profiles, although they are very different in their molecular structure and effects. This is discussed in the section on Bioidentical (Natural) Progesterone vs Synthetic Progestins. There is no documented evidence in the scientific literature of any cases of cancer as a result of treatment with bioidentical progesterone. Unfortunately, progesterone has been implicated in the development of breast cancer because of the results of large trials in which an increase in the incidence of breast cancer was seen when synthetic progestins were used in combination with estrogens for postmenopausal hormone therapy. These studies, such as the Women s Health Initiative, DID NOT use bioidentical progesterone. A large European study, on the other hand, found that women using bioidentical progesterone plus estrogen had a lower risk of breast cancer than women using estrogen alone, whereas women using synthetic progestins plus estrogen had a significantly higher risk of breast cancer. See the section on Progesterone and Breast Health for the research on progesterone and breast cancer. The following studies particularly highlighted the lack of adverse findings with bioidentical progesterone. Stephenson K, Price C, Kurdowska A, Neuenschwander P, Stephenson J, Pinson B, Stephenson D, Alfred D, Krupa A, Mahoney D, Zava D, Bevan M. Topical progesterone cream does not increase thrombotic and inflammatory factors in postmenopausal women. Presented at the 46th Annual Meeting of the American Society of Hematology, San Diego, December 4-7, Blood 2004; 104(11): Abstract No change in any of the thrombotic or inflammatory markers studied (total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, PAI-1, CRP, TNFα, and IL-6) was observed, despite significant symptomatic improvement compared to placebo, in 30 women receiving 20 mg/day progesterone cream for 4 weeks. Bagis T, Gokcel A, Zeyneloglu HB, Tarim E, Kilicdag EB, Haydardedeoglu B. The effects of short-term medroxyprogesterone acetate and micronized progesterone on glucose metabolism and lipid profiles in patients with polycystic ovary syndrome: a prospective randomized study. J Clin Endocrinol Metab 2002 Oct;87(10): This randomized prospective study evaluated and compared the effects of ten days treatment with oral and vaginal progesterone (MP) and medroxyprogesterone acetate (MPA) on glucose metabolism, lipid profiles, and hormonal parameters in 28 patients with polycystic ovary syndrome (PCOS). Oral MPA and oral MP decreased LH (P = 0.028, P = 0.009, respectively) and total testosterone (P = 0.013, P = 0.037, respectively) levels. There was no change in hormonal parameters with vaginal MP. Basal insulin decreased (P = 0.021) and insulin sensitivity increased significantly in the oral MPA group. Low density lipoprotein cholesterol (LDL) and lipoprotein (a) levels decreased only in the MPA group. This study concluded that MPA and oral MP may reduce insulin sensitivity in patients with PCOS. Vaginal MP had no effect on glucose metabolism and lipid profiles. Shantha S, Brooks-Gunn J, Locke RJ, Warren MP. Natural vaginal progesterone is associated with minimal psychological side effects: a preliminary study. J Women Health Gend Based Med 2001 Dec;10(10): This 3 month, multicenter randomized study evaluated the psychological side effects of a vaginally applied progesterone gel in reproductive aged women treated for hypotha lamic amenorrhea or premature ovarian failure. No differences were noted in psychomet ric measures as evaluated by the Hopkins Symptom Checklist. Natural progesterone in a vaginal gel can be an effective treatment for women requiring hormone therapy. 31

41 Progesterone & Safety, continued. de Ziegler D, Fanchin R. Progesterone and progestins: applications in gynecology. Steroids 2000 Oct- Nov;65(10-11): This paper reviews the use of a transvaginal progesterone gel as a viable option to other routes of application of natural progesterone (intramuscular, oral micronized), and offered it as a viable option to synthetic progestins given the low incidence of side effects noted in existing studies. Sitruk-Ware R. Progestins and cardiovascular risk markers. Steroids 2000 Oct-Nov;65(10-11): This article reviews the effects of various synthetic progestins and progesterone on cardiovascular health. Many synthetic progestins, especially 19-nortestosterone and some 17-hyroxyprogesterones, have negative effects on cardiovascular risk factors, whereas natural progesterone does not. Further studies utilizing natural and other steroids should be considered. Fitzpatrick LA, Good A. Micronized progesterone: clinical indications and comparison with current treatments. Fertil Steril 1999 Sep;72(3): The literature reviewed in this tutorial indicates a potential use for oral micronized progesterone for the treatment of secondary amenorrhea, dysfunctional uterine bleeding, luteal phase disorders, premenopausal bleeding disorders, and as a component of hormone replacement therapy that may provide a better safety profile than commonly utilized synthetic progestins. Darj E, Axelsson O, et al. Liver Metabolism During Treatment with Estradiol and Natural Progesterone. Gynecological Endocrinology June 1993; 7(2): Thirty postmenopausal women were treated daily for four months with 2 mg micronized 17 beta-estradiol and micronized progesterone orally in doses of 50, 100 and 200 mg daily. Serum concentrations of sex hormonebinding globulin (SHBG), corticosteroid binding globulin (CBG), ceruloplasmin, lipoprotein A and liver enzymes were measured. Serum SHBG and CBG increased during treatment with a weak association shown between progesterone and serum CBG. Levels of lipoprotein A and liver enzymes did not change, concluding that natural progesterone supplementation in postmenopausal women does not appear to cause any side effects to the liver. Ottosson UB, Johansson BG, et al. Subfractions of high-density lipoprotein cholesterol during estrogen replacement therapy: A comparison between progestogens and natural progesterone. American Journal of Obstetrics and Gynecology 1993 Mar;151(6): Fifty-eight postmenopausal women were followed with respect to subfractions of high-density lipoprotein during 3 cycles of unopposed estrogen. The women received either levonorgestrel, medroxyprogesterone acetate, or natural progesterone during the last ten days of the treatment period. Both progestogens significantly lowered HDL cholesterol, whereas natural progesterone had no effect on HDL levels. 32

42 Progesterone & Cardiovascular Health Overview There is already significant evidence that progesterone, unlike synthetic progestins, has no adverse effects on cardiovascular risk factors. No change in any of the thrombotic or inflammatory markers studied was observed, despite significant symptomatic improvement compared to placebo, in 30 women receiving 20 mg/day progesterone cream for 4 weeks (Stephenson et al. 2004). When bioidentical progesterone (an oral micronized preparation) was used in one group in the PEPI study in place of medroxyprogesterone acetate (MPA), this group had a significantly higher HDL cholesterol levels than the MPA group, indicating a different pharmacological effect than the synthetic progestin with a more favorable effect on blood lipids (Writing Group for the PEPI Trial, 1995). Not only is there a lack of adverse effects of bioidentical progesterone on the cardiovascular system, but there is evidence of beneficial effects also. A progesterone vaginal gel produced an increase in exercise tolerance in postmenopausal women with coronary artery disease or previous myocardial infarction who were being treated with estradiol, while MPA did not, compared with estradiol alone (Rosano et al. 2000), suggesting an advantage for progesterone in women at risk for cardiovascular disease. Progesterone s long term hemostatic role is suggested by its ability to reduce coronary hyperreactivity even in the presence of atherosclerosis in oophorectomized rhesus monkeys (Hermsmeyer et al. 2004). Koh et al. (2004) reported that progesterone together with lower dose conjugated equine estrogens (CEE) had comparable beneficial effects to conventional high dose CEE on flow mediated dilation, high density lipoproteins, and triglycerides, which may suggest that peripheral vascular function in postmenopausal women is markedly improved by direct actions on the vascular wall. The reduction of the risk of heart attacks, angina pectoris, stroke, and other major heart and vascular disease by restoring hormone balance could delay the decline in cardiovascular function in women for decades. Stephenson K, Price C, Kurdowska A, Neuenschwander P, Stephenson J, Pinson B, Stephenson D, Alfred D, Krupa A, Mahoney D, Zava D, Bevan M. Topical progesterone cream does not increase thrombotic and inflammatory factors in postmenopausal women. Presented at the 46th Annual Meeting of the American Society of Hematology, San Diego, December 4-7, Blood 2004; 104(11): Abstract No change in any of the thrombotic or inflammatory markers studied (total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, PAI-1, CRP, TNFα, and IL-6) was observed, despite significant symptomatic improvement compared to placebo, in 30 women receiving 20 mg/day progesterone cream for 4 weeks. This finding indicates a lack of potential adverse effects of progesterone on the cardiovascular system, particularly with respect to risk of coronary artery disease and stroke. Menopause May-Jun;11(3): Chronic treatment with progesterone but not medroxyprogesterone acetate restores the endothelial control of vascular tone in the mesenteric artery of ovariectomized rats. Chataigneau T, Zerr M, Chataigneau M, Hudlett F, Hirn C, Pernot F, Schini-Kerth VB. This study helps explain the more beneficial effects on the cardiovascular system of progesterone compared with MPA because of its enhancement of the protective effects of endothelial cells on the arterial walls. 33

43 Progesterone & Cardiovascular Health, continued. Hermsmeyer RK, Mishra RG, Pavcnik D, Uchida B, Axthelm MK, Stanczyk FZ, Burry KA, Illingworth DR, Kaski JC, Nordt FJ. Prevention of coronary hyperreactivity in pre-atherogenic menopausal rhesus monkeys by transdermal progesterone. Arterioscler Thromb Vasc Biol May;24(5): Previous studies by Hermsmeyer, et al demonstrated a reduction of coronary reactivity in response to subphysiological levels of progesterone in non-atherogenic monkeys. In this study, the authors sought to determine if transdermal progesterone cream conferred coronary vascular protection in surgically menopausal pre-atherosclerotic rhesus monkeys. Compared with monkeys receiving placebo cream (n= 5), treated monkeys (n= 7) experienced reduced Lipoprotein (a) levels, and an attenuation of coronary vasoconstriction, which was artificially stimulated by intracoronary serotonin plus U Coronary hyperreactivity is a component of coronary artery disease and was demonstrated in this study to be prevented in pre-atherosclerotic primates by progesterone cream treatment. Koh KK, Shin M-S, Sakuma I, Ahn JY, Jin DK, Kim HS, Kim DS, Han SH, Chung W-J, Shin EK (2004). Effects of conventional or lower doses of hormone replacement therapy in postmenopausal women. Arterioscler Thromb Vasc Biol. 2004; 24: Rosano GM, Webb CM, Chierchia S, Morgani GL, Gabraele M, Sarrel PM, de Ziegler D, Collins P. Natural progesterone, but not medroxyprogesterone acetate, enhances the beneficial effect of Progesterone together with lower dose conjugated equine estrogens (CEE) had comparable beneficial effects to conventional high dose CEE on flow mediated dilation, high density lipoproteins, and triglycerides, which may suggest that peripheral vascular function in postmenopausal women is markedly improved by direct actions on the vascular wall. Bagis T, Gokcel A, Zeyneloglu HB, Tarim E, Kilicdag EB, Haydardedeoglu B. The effects of short-term medroxyprogesterone acetate and micronized progesterone on glucose metabolism and lipid profiles in patients with polycystic ovary syndrome: a prospective randomized study. J Clin Endocrinol Metab 2002 Oct;87(10): This randomized prospective study evaluated and compared the effects of ten days treatment with oral and vaginal micronized progesterone (MP) and medroxyprogesterone acetate (MPA) on glucose metabolism, lipid profiles, and hormonal parameters in 28 patients with polycystic ovary syndrome (PCOS). Oral MPA and oral MP decreased luteinizing hormone (P = 0.028, P = 0.009, respectively) and total testosterone (P = 0.013, P = 0.037, respectively) levels. There was no change in hormonal parameters with vaginal MP. Basal insulin decreased (P = 0.021) and insulin sensitivity increased significantly in the oral MPA group. Low density lipoprotein cholesterol (LDL) and lipoprotein (a) levels decreased only in the MPA group. This study concluded MPA and oral MP may reduce insulin sensitivity in patients with PCOS. Vaginal MP had no effect on glucose metabolism and lipid profiles. 34

44 Progesterone & Cardiovascular Health, continued. Mather KJ, Norman EG, Prior JC, Elliott TG. Preserved forearm endothelial responses with acute exposure to progesterone: A randomized cross-over trial of 17-beta estradiol, progesterone, and 17-beta estradiol with progesterone in healthy menopausal women. J Clin Endocrinol Metab 2000 Dec;85(12): Regularly menstruating women enjoy relative protection from cardiovascular disease. Until recently, this has been attributed to the function of estrogen, despite the fact that progesterone is also present. This study evaluated the differing acute effects of 17-beta estradiol with and without progesterone with progesterone alone on endothelial function in a randomized crossover trial. Endothelial function was evaluated via endothelium dependent and independent forearm blood flow (FBF) using venous occlusion plethysmography. Flow responses were measured during brachial artery infusions achieving physiological levels of E2, E2 + P4, or P4 respectively along with either acetylcholine (an endothelium-dependent vasodilator), or sodium nitroprusside (an endothelium-independent vasodilator) in 27 healthy menopausal women with no cardiovascular disease risk factors. Small, statistically non-significant increases in endothelium-dependent flow responses were seen with all treatments. No impairment in response was seen with P4 alone or in combination with E2. The authors concluded that progesterone does not have detrimental vascular effects in humans. Mather KJ, Norman EG, Prior JC, Elliott TG. Preserved forearm endothelial responses with acute exposure to progesterone: A randomized cross-over trial of 17-beta estradiol, progesterone, and 17-beta estradiol with progesterone in healthy menopausal women. J Clin Endocrinol Metab 2000 Dec;85(12): Regularly menstruating women enjoy relative protection from cardiovascular disease. Until recently, this has been attributed to the function of estrogen, despite the fact that progesterone is also present. This study evaluated the differing acute effects of 17-beta estradiol with and without progesterone with progesterone alone on endothelial function in a randomized crossover trial. Endothelial function was evaluated via endothelium dependent and independent forearm blood flow (FBF) using venous occlusion plethysmography. Flow responses were measured during brachial artery infusions achieving physiological levels of E2, E2 + P4, or P4 respectively along with either acetylcholine (an endothelium-dependent vasodilator), or sodium nitroprusside (an endothelium-independent vasodilator) in 27 healthy menopausal women with no cardiovascular disease risk factors. Small, statistically non-significant increases in endothelium-dependent flow responses were seen with all treatments. No impairment in response was seen with P4 alone or in combination with E2. The authors concluded that progesterone does not have detrimental vascular effects in humans. Rosano GM, Webb CM, Chierchia S, Morgani GL, Gabraele M, Sarrel PM, de Ziegler D, Collins P. Natural progesterone, but not medroxyprogesterone acetate, enhances the beneficial effect of estrogen on exercise-induced myocardial ischemia in postmenopausal women. J Am Coll Cardiol 2000 Dec;36(7): This randomized crossover study compared the effects of estradiol (E2) (2mg/day), estradiol + progesterone (P4) vaginal gel (2 mg/day + 90 mg on alternate days), and estradiol + medroxyprogesterone acetate (MPA) (2 mg/day + 10 mg/day) on exercise-induced myocardial ischemia in eighteen postmenopausal women with coronary artery disease (CAD) or previous myocardial infarction (MI). Utilizing treadmill testing, patients were evaluated for exercise tolerance after each estradiol phase and at day 10 of each progestogen phase. The results demonstrated an increase in exercise tolerance with both E2 alone and E2 + progesterone, but not by E2 + MPA as compared to baseline. Furthermore, E2 + P4 demonstrated a significant increase in exercise tolerance when compared to MPA. The results suggest that progesterone may be preferred to progestins for hormone replacement therapy in women at risk for cardiovascular disease. 35

45 Progesterone & Cardiovascular Health, continued. Sitruk-Ware R. Progestins and cardiovascular risk markers. Steroids 2000 Oct-Nov;65(10-11): This article reviews the effects of various synthetic progestins and progesterone on cardiovascular health. Many synthetic progestins, especially 19-nortestosterone and some 17-hyroxyprogesterones, have negative effects on cardiovascular risk factors, whereas natural progesterone does not. Further studies utilizing natural and other steroids should be considered. Minshall RD, Stanczyk FZ, Miyagawa K, Uchida B, Axthelm M, Novy M, Hermsmeyer K (1998). Ovarian steroid protection against coronary artery hyperreactivity in rhesus monkeys. J Clin Endocrinol Metab 1998; 83(2): Medroxyprogesterone acetate, but not natural progesterone, negated the protective effects of estradiol against coronary artery hyperreactivity. Miyagawa K, Rosch J, Stanczyk F, Hermsmeyer K. Medroxyprogesterone interferes with ovarian steroid protection against coronary vasospasm. Nature Medicine 1997;3(3): Ovariectomized rhesus monkeys were treated with physiological levels of 17-beta estradiol in combination with either medroxyprogesterone or progesterone (oral micronized) for four weeks. Following pathophysiological stimulation without injury to induce coronary vasospasm, it was shown that progesterone plus estradiol was protective against vasospasm, whereas estradiol plus medroxyprogesterone allowed vasospasm, concluding that medroxyprogesterone increases risk of coronary vasospasm, while progesterone does not. Writing Group for the PEPI Trial. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. The postmenopausal estrogen/progestin interventions (PEPI) trial. JAMA 1995; 273(3): Bioidentical progesterone (an oral micronized preparation) was used in one group in the PEPI study in place of MPA. Results showed that the progesterone group had significantly higher HDL cholesterol levels than the MPA group, indicating a different pharmacological effect than the synthetic progestin with a more favorable effect on blood lipids. Ottosson UB, Johansson BG, et al. Subfractions of high-density lipoprotein cholesterol during estrogen replacement therapy: A comparison between progestogens and natural progesterone. American Journal of Obstetrics and Gynecology 1993 Mar;151(6): Fifty-eight postmenopausal women were followed with respect to subfractions of high-density lipoprotein during 3 cycles of unopposed estrogen. The women received either levonorgestrel, medroxyprogesterone acetate, or natural progesterone during the last ten days of the treatment period. Both progestogens significantly lowered HDL cholesterol, whereas natural progesterone had no effect on HDL levels. 36

46 Progesterone & Cardiovascular Health, continued. Bolaji II, Grimes H, Mortimer G, Tallon DF, Fottrell PF, O Dwyer EM. Low-dose progesterone therapy in oestrogenised postmenopausal women: effects on plasma lipids, lipoproteins and liver function parameters. Eur J Obstet Gynecol Reprod Biol 1993 Jan;48(1):61-8. This 12 month prospective, open, non-comparative study measured the effects progesterone (oral micronized 100mg/day) paired with mg conjugated equine estrogens (CEE) and found progesterone had no adverse effects on the lipid profile when combined with CEE. This lack of effect differs from other studies that noted adverse effects on lipid profiles when synthetic progestins were utilized with CEE. Saarikoski S, Yliskoski M, Penttila I. Sequential use of norethisterone and natural progesterone in premenopausal bleeding disorders. Maturitas 1990 Jun;12(2): This randomized controlled study evaluated the effects of norethisterone (NET) and micronized progesterone (MP) on bleeding disorders in pre-menopausal women. 80 patients were randomized to the trial and all were found via endometrial morphology to need progestogen therapy. They were subsequently treated with NET or MP. In both treatment groups, hyperplastic changes disappeared during the first three cycles, with the duration of treatment being 6 months. NET decreased follicle-stimulating hormone, luteinizing hormone, estradiol and sex-hormone-binding globulin levels (P < 0.001) whereas no changes were seen during MP treatment. High-density-lipoprotein cholesterol and triglyceride levels were also lowered by NET (P< ) slightly decreased phospholipids. MP treatment had no effect on lipid profiles suggesting it may be a preferred progestogen for the treatment of bleeding disorders. Hargrove JT, Maxson WS, Wentz AC, Burnett LS. Menopausal hormone replacement therapy with continuous daily oral micronized estradiol and progesterone. Obstetrics & Gynecology April 1989; 73( 4): Fifteen menopausal subjects were studied to determine the efficacy and safety of hormone replacement therapy with micronized estradiol (E2) and progesterone. Ten subjects were given 0.7-E2 (1.05 mg daily) and progesterone ( mg daily) and evaluated over one year at month 0, 1, 3, 6 and 12. Five subjects were administered conjugated estrogens (0.625mg daily) and medroxyprogesterone acetate (10 mg daily) and evaluated at the same intervals. Results showed all 10 women on E2 and progesterone had a decrease in total cholesterol with an increase in HDLs and sustained amenorrhea with no endometrial hyperplasia or withdrawal bleeding after six months of observation. Four of five women in the conjugated estrogen group continued to have withdrawal bleeding without endometrial hyperplasia. HDLs also increased in this group but no significant change in total cholesterol was found. Rylance PB, Brincat M, Lafferty K, De Trafford JC, Brincat S, Parsons V, Studd JW. Natural progesterone and antihypertensive action. Br Med J (Clin Res Ed) 1985 Jan 5;290(6461):13-4. In a placebo controlled, double blind crossover study, increasing doses of natural progesterone was given orally to six men and four postmenopausal women with mild to moderate hypertension who were not receiving any other antihypertensive drugs. Compared to before treatment values and to placebo, progesterone caused a significant reduction in blood pressure, suggesting that progesterone has an antihypertensive action rather than a hypertensive one as has been previously thought. The authors suggest this protective effect of progesterone should be investigated further. 37

47 Progesterone & Bone Health Overview Bone loss, leading to reduced bone mineral density (BMD) and eventually osteoporosis, is the result when osteoclast-mediated bone resorption and osteoblast-mediated bone formation become unbalanced. In the normal state, the bone is in a state of homeostasis in which bone is formed at the same rate as it is resorbed (i.e., broken down to release calcium into the bloodstream, which is a mechanism for maintaining blood calcium levels) and this is referred to as bone turnover. Hormones play a significant role in these processes. Progesterone has bone-forming activity by binding to receptors on the osteoblasts. This explains the decreases in spinal bone density seen in premenopausal women with low progesterone levels (see Prior 1990, below). In the Michigan Bone Health Study, those premenopausal women with the lowest bone mass had the highest rates of progesterone deficiency (see Sowers 1998 below). The effects of progesterone and estrogen on bone are synergistic and complementary to each other, and some clinical trials have found greater increases in spinal BMD when the progestin medroxyprogesterone acetate (MPA) is added to estrogens than with estrogens alone. However, fracture risk data for conjugated equine estrogens (CEE) and CEE/MPA were not analyzed separately in the Women s Health Initiative study, which reported a reduction in fractures with hormone therapy. The wide variation in synthetic progestins and progesterone formulations has hindered the statistical power of meta-analyses to detect the effects on BMD of the progesterone/progestin component of hormone therapy, but tibolone (a synthetic progestin widely used in Europe but not approved in the US) was shown to have similar effects on BMD to any estrogen compound in one meta-analysis (see Dören 2003). At the time of the menopausal transition, the rate of bone turnover is still high and so progesterone s bone-forming effects are harder to see. However, longer term studies of progesterone treatment, currently in progress, may further confirm the benefit of this hormone for maintenance of bone density after the first few years following the onset of menopause. Dören M, Nilsson J-A, Johnell O. Effects of specific post-menopausal hormone therapies on bone mineral density in post-menopausal women: a meta-analysis. Human Reprod 2003; 18(8): Liang M, Liao EY, Xu X, Luo XH, Xiao XH. Effects of progesterone and 18-methyl levonorgestrel on osteoblastic cells. Endocr Res Nov;29(4): The authors evaluated in this study the effects of progesterone (P4) and levonorgestrel (LNG) on markers of bone growth, utilizing normal human osteoblasts as well as the osteosarcoma cell line, MG-63. Their study found that, compared with placebo, both P4 and LNG increased the proliferation and differentiation of human osteoblasts through osteocalcin gene transcription. Sowers M, Randolph JF Jr, Crutchfield M, Jannausch ML, Shapiro B, Zhang B, La Pietra M. Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass. J Bone Miner Res 1998; 13(7):

48 Progesterone & Bone Health, continued. Prior JC, Vigna Y, Alojado N. Progesterone and the prevention of osteoporosis. Canadian Journal of Obstetrics/Gynecology and Women s Health Care 1991; 3(4): In this review article, the authors propose that cyclic progesterone both prevents bone loss and acts as a bonebuilder. The studies discussed focus on abnormal menstrual cycles as an important risk factor for osteoporotic fractures. Their conclusion is that the first step in preventing osteoporosis is treating ovulation disorders. Prior JC, Vigna YM, Schecter MI, Burgess AE. Spinal bone loss and ovulatory disturbances. New England Journal of Medicine 1990; 323(18): A review of the available data indicates that progesterone acts to promote bone metabolism. It appears to be independent of estrogen by either acting directly at progesterone receptors, or indirectly through competition at glucocorticoid receptors in the osteoblasts. Lee JR. Osteoporosis reversal; the role of progesterone. International Clinical Nutrition Review 1990;10(3): Transdermal progesterone supplementation with and without conjugated estrogens was evaluated in a clinical setting using 100 women aged 38 to 83 years. The average time from onset of menopause was 16 years. 63 women were followed for three years with dual photon absorptiometry. Treatment also included dietary changes, nutritional supplements, and exercise. All individuals followed showed an increase in bone mineral density over the three years, with the greatest increase occurring in the first year. There was no difference noted between estrogen/progesterone and progesterone only groups. Subjective changes included increased libido, diminished hot flushes, reduced joint pain, and increased mobility and energy. No side effects were noted during treatment protocol. Progesterone & Relief of Menopausal Symptoms A number of hormonal changes occur as women enter menopause. The transition is characterized by erratic, but on average higher than premenopausal estrogen levels, while progesterone levels steadily decline as the number of menstrual cycles without ovulation increases, and remain extremely low thereafter. Few researchers have studied the relationship between longitudinal changes in hormones and menopausal symptoms, although a recent study (see Randolph 2005 below) looked at levels of reproductive hormones and found only serum FSH (follicle-stimulating hormone) was associated with the frequency of hot flashes and night sweats. Unfortunately the authors did not look at progesterone levels. Menopausal symptoms are therefore not simply a sign of estrogen deficiency. Progestins or progesterone have long been a component of postmenopausal hormone therapy, because they have been given to protect the uterus from the proliferative effects of estrogen that may otherwise lead to uterine cancer. There is a great need for more studies of progesterone alone, without estrogen, in women with menopausal symptoms, since those that have been done have shown significant relief. Dr. Jerilynn Prior is currently recruiting postmenopausal women for a study of micronized oral progesterone versus placebo, and examining its effects on vasomotor symptoms, forearm blood flow, lipid levels, and blood pressure in perimenopausal women experiencing vasomotor symptoms ( identifier: NCT ). Completion is expected in

49 Progesterone & Relief of Menopausal Symptoms, continued. Randolph JF, Sowers MF, Bondarenko I, Gold EB, Greendale GA, Bromberger JT, Brockwell SE, Matthews K. The relationship of longitudinal change in reproductive hormones and vasomotor symptoms during the menopausal transition. J Clin Endocrinol Metab Nov;90(11): Vasomotor symptoms are experienced by 65-76% of women going through menopause. This study examined longitudinal changes in estradiol, FSH, testosterone, DHEA, sex hormone binding globulin, free estrogen index and free testosterone index, and found that only FSH levels were associated with the prevalence and frequency of vasomotor symptoms. Stephenson K, Price C, Kurdowska A, Neuenschwander P, Stephenson J, Pinson B, Stephenson D, Alfred D, Krupa A, Mahoney D, Zava D, Bevan M. Topical progesterone cream does not increase thrombotic and inflammatory factors in postmenopausal women. Presented at the 46th Annual Meeting of the American Society of Hematology, San Diego, December 4-7, Blood 2004; 104(11): Abstract No change in any of the thrombotic or inflammatory markers studied (total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin, PAI-1, CRP, TNFα, and IL-6) was observed, despite significant relief of vasomotor symptoms compared to placebo, in 30 postmenopausal women receiving 20 mg/day progesterone cream for 4 weeks. From this study the authors conclude that administration of topical progesterone cream at a daily dose of 20 mg significantly relieves menopausal symptoms in postmenopausal women without adversely altering prothrombotic potential. Since the thrombotic complications that are typically observed with conventional hormone replacement therapy, and have led to an increase in stroke, do not seem to occur with topical progesterone, this treatment should be seriously considered as an effective and safe alternative clinical therapy for women suffering from menopausal symptoms. Montplaisir J, Lorrain J, Denesle R, Petit D. Sleep in menopause: differential effects of two forms of hormone replacement therapy. Menopause 2001; 8(1): This randomized clinical trial compared the effects of conjugated equine estrogen (CEE) and medroxyprogesterone acetate to CEE and oral micronized progesterone. Twenty-one postmenopausal women were studied in a sleep lab, with results demonstrating an improvement in subjective measures of menopausal symptoms and sleep in both groups. The group receiving natural progesterone had significantly improved sleep efficiency, whereas the medroxyprogesterone acetate group did not, suggesting that the former might better improve sleep in postmenopausal women. Ryan N, Rosner A. Quality of life (QOL) and costs associated with micronized progesterone and medroxyprogesterone acetate in hormone replacement therapy for non-hysterectomized, postmenopausal women. Clin Ther 2001 Jul;23(7): This prospective, multicenter, randomized, parallel-group study enrolled 182 postmenopausal women 45 to 65 years of age and evaluated the quality of life and menopausal symptoms associated with the use of medroxyprogesterone acetate vs oral micronized progesterone when used as a part of a regular hormone replacement therapy. Menopausal symptoms improved in both groups from baseline to 9 months, as did QOL measures. In addition, patients using micronized progesterone had specific improvements in the areas of cognition and menstrual problems whereas the patients using MPA did not. Micronized progesterone was seen as an effective, cost-comparable alternative to MPA as well as being better tolerated. 40

50 Progesterone & Relief of Menopausal Symptoms, continued. Fitzpatrick LA, Pace C, Wiita B. Comparison of regimens containing oral micronized progesterone or medroxyprogesterone acetate on quality of life in postmenopausal women: a cross-sectional survey. J Women Health Gend Based Med 2000 May;9(4): A cross-sectional survey was conducted to examine quality of life (QOL) related to physiological, somatic, and vasomotor effects of switching progestogen treatment from medroxyprogesterone acetate (MPA) to micronized progesterone in postmenopausal women already using hormone replacement therapy (HRT). One hundred seventy-six women who were currently using hormone replacement therapy (HRT) containing micronized progesterone for 1-6 months and had previously received HRT containing MPA were surveyed to assess QOL. Women using micronized progesterone-containing HRT experienced significant improvement in vasomotor symptoms, anxiety, somatic complaints and depressive symptoms. Women reported improved control of menopausal symptoms and perceptions of their vaginal bleeding patterns while on the micronized progesterone-containing regimen. Approximately 80% of women reported satisfaction with the progesterone-containing therapy. A micronized progesterone-containing HRT therapy offers the potential for improved QOL with respect to menopausal symptoms. Leonetti HB, Longo S, Anasti JN. Transdermal progesterone cream for vasomotor symptoms and postmenopausal bone loss. Obstet Gynecol 1999 Aug;94(2): In this randomized controlled trial, 102 menopausal women were treated with topical progesterone (Pro-gest, 20 mg daily) or placebo and monitored for 1 year. Improvement in vasomotor symptoms was seen in 83% of the women in the treatment group who had experienced hot flashes, compared to 19% in the placebo group (p<.001). There was no difference noted in bone mineral densities between groups after one year. All women studied received a daily multivitamin and 1200 mg calcium. Wetzel W. Micronized progesterone: a new option for women s health care. Nurse Pract 1999 May;24(5):62-6, 71, This paper discusses the use of micronized progesterone as a safe, effective, and well-tolerated therapy and reviews indications for use. It also includes case studies and issues of patient compliance and the need for an individualized treatment plan for women receiving hormone therapy. Sherwin BB. Progestogens used in menopause. Side effects, mood and quality of life. J Reprod Med 1999 Feb;44(2 Suppl): This review summarizes the effects of progesterone on mood and other brain functions. Progesterone receptors are present in many of the same areas of the brain as estrogen receptors, including the limbic system and hypothalamus. The limbic system plays a prominent role in regulating mood and emotion. As a comparison, progesterone decreases brain excitability, while estrogens increase it. This relates to why women with epilepsy have a higher frequency of seizures during the part of the cycle when estrogen levels are high, and a reduced frequency when progesterone levels are high. Estrogen and progesterone may also have differing effects on MAO, thereby affecting concentration of serotonin (a mood elevator) in the brain. 41

51 Progesterone & Nervous System/Brain In this emerging area of progesterone research, several research studies attest to the neuroprotective effects of progesterone, an absence of neurological side effects, and a benefit for cognitive function. Wright DW, Kellermann AL, Hertzberg VS, Clark PL, Frankel M, Goldstein FC, Salomone JP, Dent LL, Harris OA, Ander DS, Lowery DW, Patel MM, Denson DD, Gordon AB, Wald MM, Gupta S, Hoffman SW, Stein DG. ProTECT: a randomized clinical trial of progesterone for acute traumatic brain injury. Ann Emerg Med 2006 Sep 28; [Epub ahead of print]. A neuroprotective effect of progesterone was observed in this randomized, placebo-controlled trial of very high dose, intravenous progesterone therapy given for 3 days after acute traumatic brain injury. Only 13% of the patients died within 30 days after injury in the progesterone group, compared with 30% of the placebo group, and the progesterone group was more likely to have a moderate to good functional outcome after 30 days than the placebo group. Even at the extremely high dose used, no serious adverse events were seen with progesterone. Gibson CL, Murphy SP. Progesterone enhances functional recovery after middle cerebral artery occlusion in male mice. J Cereb Blood Flow Metab Jul;24(7): Differences in outcomes following ischemia have been noted between men and women, and this is thought to be attributed to sex steroids. This study investigated the potential benefits of progesterone administration after focal cerebral ischemia of the middle cerebral artery of male mice. Male mice undergoing 60-minute middle cerebral artery occlusion (MCAO) received either progesterone or vehicle following occlusion. The mice receiving progesterone had significantly reduced lesion volume (p< 0.05) when compared with the vehicle treated mice (control). Progesterone treatment also improved survival rate, weight recovery, and motor ability when compared to the control group. In addition, mice treated with progesterone demonstrated motor ability comparable to mice that did not undergo MCAO. The authors suggest the need to further investigate the mechanisms of progesterone action on recovery from cerebral injury. Schumacher M, Guennoun R, Robert F, Carelli C, Gago N, Ghoumari A, Gonzalez Deniselle MC, Gonzalez SL, Ibanez C, Labombarda F, Coirini H, Baulieu EE, De Nicola AF. Local synthesis and dual actions of progesterone in the nervous system: neuroprotection and myelination. Growth Horm IGF Res Jun;14 Suppl A:S This paper reviews of the effects of progesterone as an autocrine/paracrine hormone in the brain. The brain, spinal cord and peripheral nerves all synthesize progesterone from the precursor, pregnenolone. Macroglial cells, including astrocytes, oligodendroglial cells and Schwann cells, also have the capacity to synthesize progesterone. This production is regulated by cellular interactions. Recent research has suggested the role progesterone plays in the brain is likely a significant one, supporting the viability of neurons and the formation of myelin sheaths. In mice and rat studies, progesterone also demonstrated a neuroprotective effect. These actions of progesterone suggest viable therapeutic possibilities for the prevention and treatment of neurodegenerative diseases, as well as for repair processes and for preserving cognitive functions with age. Mahesh VB, Brann DW, and Hendry LB. Diverse modes of action of progesterone and its metabolites. J Steroid Biochem Molec Biol 1996;56(1-6): A review of the actions of progesterone and its metabolites demonstrates physiological significance in such biological activities as may have importance in the regulation of stress, post-partum depression, memory, cognition, PMS, and depression, to name a few. 42

52 Progesterone & Nervous System/Brain, continued. Grossman KJ, Goss CW, Stein DG. Effects of progesterone on the inflammatory response to brain injury in the rat. Brain Res May 15;1008(1): Progesterone has a known anti-inflammatory effect. In this study, male rats treated with progesterone (4 mg/kg) and/or vehicle, were examined with respect to cellular inflammatory response to frontal cortex injury on postsurgical days 1, 3, 5, 7 and 9. The treated mice suffered significantly less edema than untreated mice, as well as showed an increase in the accumulation of activated microglia, demonstrating a neuroprotective effect on the rat brain. Cummings JA, Brizendine L. Comparison of physical and emotional side effects of progesterone or medroxyprogesterone in early postmenopausal women. Menopause 2002 Jul-Aug;9(4): Twenty-three early postmenopausal women were randomized to either medroxyprogesterone acetate (MPA) or oral micronized progesterone combined with conjugated equine estrogens (CEE) and followed for 91 days in a sequence of treatments. None of the hormone treatments had any noticeable effect on mood. Participants using MPA experienced more breast tenderness and bleeding than those using progesterone. This study debunks the belief that progesterone depresses mood in healthy individuals. de Wit H, Schmitt L, Purdy R, Hauger R. Effects of acute progesterone administration in healthy postmenopausal women and normally-cycling women. Psychoneuroendocrinology 2001 Oct;26(7): This randomized controlled study investigated the effects of acute progesterone administration (25, 50, 100 mg, intramuscularly, 1 dose/wk) on mood. Contrary to the investigators expectations, very few unwanted behavioral effects were noted, and only in the highest dose (100 mg) did women slightly increase their self-rating of sluggishness. Baulieu E, Schumacher M. Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination. Steroids 2000 Oct-Nov;65(10-11): This paper reviews the effects of progesterone on the brain, with special focus on its role in the formation of the myelin sheath surrounding nerve fibers. Other roles of progesterone in the brain include activating GABA receptors, which induces a calming effect. Arafat ES, Hargrove JT, Maxson WS, Desiderio DM, Wentz AC, Andersen RN. Sedative and hypnotic effects of oral administration of micronized progesterone may be mediated through its metabolites. Am J Obstet Gynecol 1988 Nov; 159(5): This small pilot study evaluated progesterone and its metabolites following administration of oral micronized progesterone in eight postmenopausal women. Progesterone and its metabolites were measured in serum extracts by radioimmunoassay and gas chromatography-mass spectrometry. Evaluation of serial blood samples showed elevated levels of serum progesterone and its metabolites from baseline, reaching a peak between 2 and 6 hours after oral administration. The following compounds: progesterone, 5 beta-pregnan-3 alpha, 5 alphapregnan-3 alpha-ol-20-one, 5 beta-pregnan-3 alpha-ol-20-one, 20 beta-diol, and 5 beta-pregnan-3 alpha-ol-11-,20-dione, were identified. These compounds have reported anesthetic qualities, which may contribute to the sedative and hypnotic effects seen with oral administration of progesterone. The authors reported that, in one subject, 400 mg of oral micronized progesterone induced a hypnotic state lasting approximately 2 hours. 43

53 Progesterone & The Uterus Overview Progesterone or progestins are a necessary adjunct to estrogen therapy in women who have a uterus (i.e., who have not had a hysterectomy) because of the risk of endometrial (uterine) cancer when estrogen is given alone. Progesterone has an antiproliferative effect and therefore protects the uterus. Leonetti HB, Landes J, Steinberg D, Anasti JN. Topical progesterone cream as an alternative progestin in hormone therapy. Altern Ther Health Med 2005; 11(6): This study evaluated the endometrial effects and determined patients acceptance of transdermal progesterone cream compared to standard hormone therapy. Healthy menopausal women were recruited and received a pretreatment endometrial biopsy (EMB). They were randomized to mg conjugated equine estrogen (CEE) daily and 2.5 mg medroxyprogesterone acetate (MPA) (PremproTM) or daily mg CEE and twice daily 20 mg transdermal PC (Pro-gest ). At the end of 6 months, a repeat EMB was obtained, and the women were crossed over to other treatment. A final EMB was performed after the final 6 months. Twenty-six women completed both arms of the study. Seventy-seven percent of women preferred the CEE/PC to the CEE/MPA (P<.001). Of the 52 post-treatment endometrial biopsies: 40 revealed atrophic endometrium and 12 proliferative endometrium (7 in the oral progestin group and 5 in the PC group). There was no evidence of endometrial hyperplasia in any of the specimens. The incidence of vaginal spotting was similar in both groups. Conclusion: Patients preferred transdermal PC over oral MPA. These preliminary data indicate that CEE/PC has a similar effect on the endometrium as standard oral HT over a 6-month period. Anasti JN, Leonetti HB, Wilson KJ. Topical progesterone cream has antiproliferative effect on estrogenstimulated endometrium. Obstet & Gynecol 2001; 97(4 Suppl.):10S and Fertil Steril 2003;79(1): This randomized, controlled study involving 58 postmenopausal women demonstrated that topically applied progesterone cream (Pro-gest ) had an antiproliferative effect in postmenopausal women who had been given oral estrogens x 14 days prior to progesterone treatment. Treatment with topical progesterone did not differ in effects from vaginally applied progesterone (Crinone ), and both progesterone applications demonstrated a significant effect over placebo. Patients preferred the topical application of progesterone cream. Leonetti HB, Anasti JN, Landes J. Topical progesterone cream: an alternative progestin in hormone replacement therapy. Obstet & Gynecol 2003; 101(4 Suppl.): women completed a 1 year randomized, controlled, cross-over study comparing conjugated equine estrogen (Premarin, mg) paired with progesterone cream (Pro-gest, 20 mg) vs. conjugated equine estrogen paired with medroxyprogesterone acetate (PremproTM). Endometrial biopsies were performed at the end of each 6-month arm of the study. No hyperplasia was found in either group. Incidence of spotting was similar in both groups. Participants preferred the progesterone cream formulation (76% vs 5%, p<0.001). Cicinelli E, de Ziegler D, Galantino P, Pinto V, Barba B, Morgese S, Schonauer S. Twice-weekly transdermal estradiol and vaginal progesterone as continuous combined hormone replacement therapy in postmenopausal women: a 1-year prospective study. Am J Obstet Gynecol 2002 Sep;187(3): In this study of 35 postmenopausal women, twice-weekly administration of a progesterone vaginal gel (45 mg P4/day) sufficiently protected the endometrium in women receiving transdermal estradiol (0.05 mg/d) as revealed by endometrial thickness and histology. The authors present vaginally applied progesterone as a viable option for hormone replacement therapy at menopause. 44

54 Progesterone & The Uterus, continued. Montz FJ, Bristow RE, Bovicelli A, Tomacruz R, Kurman RJ. Intrauterine progesterone treatment of early endometrial cancer. Am J Obstet Gynecol 2002 Apr;186(4): This study evaluated the use of a progesterone-releasing IUD as a feasible treatment for early stage endometrial cancer (IA, grade 1). Twelve subjects were followed for 36 months. Results suggested IUD progesterone appeared to resolve some cases of early endometrial cancer. Ferrero S, Gerbaldo D, Fulcheri E, Cristoforoni P. Vaginal micronized progesterone in continuous hormone replacement therapy. A prospective randomized study. Minerva Ginecol 2002; 54(6): Transvaginal micronized progesterone (100 mg/day for 12 days/month) effectively promoted a functional, secretory endometrium, while cyclic oral MPA or transdermal norethisterone acetate more often produced endometrial atrophy, in women receiving continuous transdermal estradiol. Fanchin R, De Ziegler D, Bergeron C, et al. Transvaginal administration of progesterone. Obstet Gynecol 1997;90: Three different doses of transvaginal progesterone gel were administered to 40 estrogen-deprived women aged years. Estradiol was administered orally for 28 days, with progesterone added vaginally on alternate days from days Plasma gonadotropins, E1, E2 and progesterone were measured, and an endometrial biopsy was obtained to assess endometrial status and estrogen and progesterone receptor determinations. Transvaginal progesterone induced normal secretory transformation despite low serum progesterone levels, suggesting a direct transit of progesterone into the uterus, or first uterine pass effect. Casanas-Roux F, Nisolle M, Marbaix E, et al. Morphometric, immunohistological and three-dimensional evaluation of the endometrium of menopausal women treated by oestrogen and Crinone, a new slowrelease vaginal progesterone. Human Reprod 1996;11: Twenty estrogen-deprived women were given oral estrogen for 12 days followed by oral estrogen- vaginal progesterone gel for 12 days. Endometrial evaluation occurred before treatment, after the estrogen-only phase and after estrogen-progesterone gel treatment. Atrophy was present before treatment in all patients. Typical proliferative changes occurred after estrogen-only treatment, and secretory transformation occurred after estrogen-progesterone treatment, indicating that sustained-release progesterone gel can effectively counteract the proliferative effects of estrogen treatment in postmenopausal women. Whitehead MI, Fraser D, Schenkel L, et al. Transdermal administration of oestrogen/progestogen hormone replacement therapy. The Lancet 1990; 335: Sixteen estrogen-deficient women were evaluated on a course of transdermal estradiol and transdermal progestogen for five cycles. Regular withdrawal bleeding was noted in all but one patient. Fourteen endometrial biopsies were performed after the fifth cycle, with no evidence of endometrial hyperplasia. 45

55 Progesterone & The Uterus, continued. Moyer DL, de Lignieres B, Driguez P, Pez JP. Prevention of endometrial hyperplasia by progesterone during long-term estradiol replacement: influence of bleeding pattern and secretory changes. Fertil Steril 1993 May;59(5): It is often presumed that progesterone levels must be high enough to induce endometrial bleeding by withdrawal in order to convey protection during estrogen replacement therapy. In this expanded observational study, the authors sought to determine the influence of withdrawal bleedings, secretory transformation, and reduction of mitosis on the prevention of endometrial hyperplasia during long-term estrogen-replacement therapy. Hysteroscopy and endometrial biopsies were utilized to establish maturation patterns, glandular epithelial mitosis rates, and macroscopic endometrial appearance. The results showed an increase in withdrawal bleeding with higher levels of progesterone, with those levels producing distinct secretory responses. However, incidence of endometrial hyperplasia after 5 years of E2/P therapy was independent of secretory changes and withdrawal bleeding, and was more related to the control of mitosis, which was seen even with low doses of progesterone. The authors conclude that a relatively low dose of P may be offered to women seeking hormone replacement therapy with similar levels of endometrial safety. 46

56 Progesterone & Dosing Forms Overview Historically dosed via injection, suppository, or troche, progesterone is now available in several additional delivery modes. Of interest is the increase over the last decade in the delivery of hormones through the skin, with various effective modes of application including patches, creams, and gels. Transdermal delivery of hormones has been found to be effective and well tolerated, with the additional benefit of bypassing the first-pass effect of the liver, allowing for reduced dosages as more of the hormone is directly available for its therapeutic effect rather than being mostly metabolized (see Hermann, below). Micronization of progesterone in oral formulations has also benefited absorption, and some doctors prefer to prescribe oral micronized progesterone because of its tendency to induce drowsiness and thus aid sleep, which may help women who are troubled with night-time menopausal symptoms. Further references in the section on progesterone and the uterus confirm the effectiveness of topical progesterone preparations in protecting the endometrium from proliferation, which may otherwise lead to the development of uterine cancer, in women using estrogen therapy for menopausal symptoms. Hermann AC, Nafziger AN, Victory J, Kulawy R, Rocci ML Jr, Bertino JS Jr. Over-the-counter progesterone cream produces significant drug exposure compared to a food and drug administration-approved oral progesterone product. J Clin Pharmacol 2005; 5(6): In 12 healthy, postmenopausal women, steady state levels of progesterone in the blood were measured in a randomized crossover study comparing topical progesterone cream (Pro-gest, 40 mg twice daily x 12 days ) with oral micronized progesterone (Prometrium, 200 mg daily x 12 days). There was no significant difference noted in mean steady state levels between the two products as measured by area under the curve (AUC) of progesterone levels in whole blood. This indicates that equivalent blood levels of progesterone can be achieved with substantially lower doses of topical progesterone cream than with oral micronized progesterone. Waddell BJ, O Leary PC. Distribution and metabolism of topically applied progesterone in a rat model. J Steroid Biochem Mol Biol 2002 Apr; 80(4-5): Following topical application of a commercially available progesterone cream, concentrations of fat and watersoluble metabolites of progesterone were measured in various tissues (uterus, kidney, salivary gland, liver) as well as plasma and urine. The topically applied progesterone was demonstrated to be well absorbed and had distribution and metabolism patterns similar to that seen with intravascular progesterone delivery. Kleinstein J, Schlegelmilch R, Mazur D, Vens-Cappell B. [Pharmacokinetic comparison of progesterone capsules with a progesterone gel after vaginal administration] Arzneimittelforschung 2002; 52(8): [Article in German] Twenty-four women participated in this randomized controlled, crossover study comparing the bioavailability and pharmacokinetics of a vaginal progesterone capsule (200 mg/dose) vs a progesterone vaginal gel (90 mg/dose). Both were well tolerated, and no differences were noted with respect to safety. The vaginal capsule delivered more progesterone, however peak concentrations between the two preparations didn t differ. 47

57 Progesterone & Dosing Forms, continued. de Ziegler D, Fanchin R. Progesterone and progestins: applications in gynecology. Steroids 2000 Oct- Nov;65(10-11): This paper reviews the use of a transvaginal progesterone gel as a viable option to other routes of application of natural progesterone (intramuscular, oral micronized), and offered it as a viable option to synthetic progestins given the low incidence of side effects noted in existing studies. O Leary P, Feddema 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. Clin Endocrinol (Oxf) 2000 Nov;53(5): Absorption of progesterone as provided in a topical preparation of natural progesterone cream to 6 premenopausal and 6 postmenopausal women was demonstrated via salivary hormone levels. Salivary progesterone concentrations reached their peak 1-4 hrs after application. A five-fold increase in mean levels was seen in the premenopausal group. Serum progesterone levels were not significantly different from baseline in either group, and serum progesterone was not seen as an effective measure of absorption of topically applied progesterone. Burry KA, Patton PE, Hermsmeyer K. Percutaneous absorption of progesterone in postmenopausal women treated with transdermal estrogen. Am J Obstet Gynecol 1999 Jun;180(6 Pt 1): This pilot study demonstrated a significant increase in serum progesterone levels in 6 women receiving topical progesterone cream (Pro-gest ; mg P4/day) and 17beta estradiol (0.05mg patch). The absorption of progesterone via a topical cream correlated well with estrogen absorption (p< 0.001). They concluded that progesterone cream appeared to be a safe and effective route of application. Fanchin R, De Ziegler D, Bergeron C, et al. Transvaginal administration of progesterone. Obstet Gynecol 1997;90: Three different doses of transvaginal progesterone gel were administered to 40 estrogen-deprived women aged years. Estradiol was administered orally for 28 days, with progesterone added vaginally on alternate days from days Plasma gonadotropins, estriol, estradiol and progesterone were measured, and an endometrial biopsy was obtained to assess endometrial status and estrogen and progesterone receptor determinations. Transvaginal progesterone induced normal secretory transformation despite low serum progesterone levels, suggesting a direct transit of progesterone into the uterus, or first uterine pass effect. Scarabin PY, Alhenc-Gelas M, Plu-Bureau G, Taisne P, Agher R, Aiach M. Effects of oral and transdermal estrogen/progesterone regimens on blood coagulation and fibrinolysis in postmenopausal women. A randomized controlled trial. Arterioscler Thromb Vasc Biol 1997 Nov; 17(11): Oral hormone replacement therapy postmenopausally has been associated with an increased risk of stoke due to thromboembolism. This randomized, placebo-controlled study evaluated the differing effects of oral and transdermal estrogen/progesterone therapy or placebo on hemostasis. Oral, but not transdermal therapy was seen to increase the susceptibility of clotting in healthy post-menopausal women years. The authors concluded that route of administration of hormones can affect the incidence of clotting, with oral hormone replacement increasing risk, and transdermal hormone replacement demonstrating no negative effect on clotting. 48

58 Progesterone & Dosing Forms Kimzey LM, et al. Absorption of micronized progesterone from a nonliquefying vaginal cream. Fertility and Sterility 1991; 56: The pharmacokinetics are compared between orally administered micronized progesterone, and that administered through a vaginal cream. Oral progesterone is extensively metabolized prior to reaching the target tissues, and progesterone metabolites may comprise a significant amount of progesterone measured in the serum. When compared, vaginal application sustained progesterone levels over a longer period of time than orally administered progesterone. Hargrove JH, Maxson WS, Wentz AC. Absorption of oral progesterone is influenced by vehicle and particle size. Am J Obstet Gynecol 1989;161: This small sample study shows that significant serum progesterone levels can be achieved by oral administration of progesterone. Efficacy of absorption is improved using micronization in oil formulations. Sitruk-Ware R. Transdermal delivery of steroids. Contraception 1989 Jan; 39(1): This review summarizes the advantages of delivering steroids through the skin, as well as reviews skin biology. The authors make a strong case for the choice of transdermal delivery of hormones (especially estrogen, progesterone, and testosterone) for both male and female patients with respect to safety, efficacy, and ease of use and predict this delivery method to make a significant impact on the quality of care for both male and female patients. Nillius SV, Johansson EDB. Plasma levels of progesterone after vaginal, rectal or intramuscular administration of progesterone. Am J Obstet Gynecol 1971;110: Plasma levels of progesterone equivalent to normal luteal phase levels were obtained using 25 mg of injected progesterone or 100 mg via rectal or vaginal administration at 8 hours after administration. 49

59 The Difference Between Bioidentical Progesterone & Progestin Confusion exists, even among leading experts in the field of hormone research, between the terms progesterone, progestin, and progestogen. Although these terms are often used interchangeably, they are not synonymous. Until an authoritative definition is formally adopted, the following reflects the current culture of the usage of these terms, and clarifies how they are used by Women in Balance: progesterone refers to the hormone produced in the body, or produced from a plant source but still chemically and structurally identical to human progesterone, and it is therefore referred to as bioidentical or natural. In contrast, progestin refers to a hormone that is synthetically produced and differs in structure from progesterone. There are numerous synthetic progestins used in hormone therapy, in contrast to only one molecule referred to as progesterone. Progestogen (sometimes spelled progestagen ) is a general term for hormones that act like progesterone in the uterus, and therefore includes progestins. There is increasing evidence that, by virtue of their different chemical structures, synthetic progestins do not always act as progesterone would at the same target tissues. This has long been understood with respect to treatment of pregnancy and fertility issues, when progesterone is effectively prescribed, yet synthetic progestins are contraindicated. While synthetic progestins may mimic some of progesterone s effects, progestins may react differently with progesterone receptors in the body. A significant consequence of the additional side effects seen with synthetic progestins has been the recent observation that natural progesterone in hormone therapy regimens does not affect the risk of developing breast cancer, while the use of synthetic progestins increases this risk (see Fournier, below). Clinical trials such as the Women s Health Initiative, in which more breast cancer was seen in the group taking progestins, did not study natural progesterone. Campagnoli C, Clavel-Chapelon F, Kaaks R, Peris C, Berrino F. Progestins and progesterone in hormone replacement therapy and the risk of breast cancer. J Steroid Biochem Mol Biol 2005; 96(2): The authors discuss the non-progesterone-like effects of synthetic progestins, which can contribute to the increased risk of breast cancer when these are used as part of a combined hormone therapy regimen. In contrast, bioidentical progesterone does not increase the risk of breast cancer, consistent with experimental in vivo data that shows progesterone has no adverse effect on breast tissue. Fournier A, Berrino F, Riboli E, Avenel V, Clavel-Chapelon F. Breast cancer risk in relation to different types of hormone replacement therapy in the E3N-EPIC cohort. Int J Cancer 2005; 114(3): Combined HRT with estrogen (either oral or transdermal) and synthetic progestins was found to carry a significantly increased risk of breast cancer compared with estrogens plus oral micronized progesterone. In fact, no increase in breast cancer risk was seen in the estrogen plus oral micronized progesterone group compared with estrogen alone. This large multicenter study therefore suggests that there is a dramatic difference between the effects of bioidentical progesterone versus synthetic progestins on breast cancer risk. 50

60 The Difference Between Bioidentical Progesterone & Progestin, continued. Leonetti HB, Landes J, Steinberg D, Anasti JN. Topical progesterone cream as an alternative progestin in hormone therapy. Altern Ther Health Med 2005; 11(6): This study evaluated the endometrial effects and determined patients acceptance of transdermal progesterone cream (PC) compared to standard hormone therapy. Healthy menopausal women were recruited and received a pretreatment endometrial biopsy (EMB). They were randomized to mg conjugated equine estrogen (CEE) daily and 2.5 mg medroxyprogesterone acetate (MPA) PremproTM or daily mg CEE and twice daily 20 mg transdermal PC (Pro-gest ). At the end of 6 months, a repeat EMB was obtained, and the women were crossed over to other treatment. A final EMB was performed after the final 6 months. Twenty-six women completed both arms of the study. Seventy-seven percent of women preferred the CEE/PC to the CEE/MPA (P<.001). Of the 52 post-treatment endometrial biopsies: 40 revealed atrophic endometrium and 12 proliferative endometrium (7 in the oral progestin group and 5 in the PC group). There was no evidence of endometrial hyperplasia in any of the specimens. The incidence of vaginal spotting was similar in both groups. Conclusion: Patients preferred transdermal PC over oral MPA. These preliminary data indicate that CEE/PC has a similar effect on the endometrium as standard (CEE/MPA) oral HT over a 6-month period. Stanczyk FZ. All progestins are not created equal. Steroids 2003; 68: This paper describes the molecular and pharmacokinetic differences between various progestins and progesterone. Orally administered progestins require relatively high doses for therapeutic use because of extensive first pass metabolism in the liver. Also, the various molecules show profound differences in progestational activity between human and animal tissues, particularly with respect to androgenicity, which has led to erroneous conclusions being drawn from animal studies regarding androgenicity in humans. The author classifies the various molecules by chemical structure and concludes that they differ widely in metabolism, pharmacokinetics, and potency. Greendale GA, Reboussin BA, Slone S, Wasilauskas C, Pike MC, Ursin G. Postmenopausal hormone therapy and change in mammographic density. J Natl Cancer Inst 2003 Jan 1;95(1):30-7. Breast cancer risk independently increases with mammographic density. Use of hormone replacement therapy (HRT) postmenopausally is associated with an increase in mammographic density, but the extent of the density increase is unknown. This study evaluated mammograms from 571 of the 875 women enrolled in the PEPI trial at baseline and after 12 months HRT. The women had been randomized to receive placebo, conjugated equine estrogens (CEE) + medroxyprogesterone acetate (MPA) in a continuous or cyclic fashion, or CEE + micronized progesterone (MP). Mammograms were analyzed digitally and a linear regression analysis was utilized to quantify breast density change in all four treatment arms. The adjusted absolute mean changes in mammographic percent density over 12 months were 4.76% (95% confidence interval [CI] = 3.29% to 6.23%), 4.58% (95% CI = 3.19% to 5.97%), and 3.08% (95% CI = 1.65% to 4.51%) for women in the CEE+MPA-cyclic, CEE+MPA-continuous, and CEE-MP groups, respectively. Each of those absolute mean changes was statistically significantly different from the adjusted absolute mean change in mammographic percent density for women in the placebo group, which was -0.07% (95% CI = -1.50% to 1.38%). Greater mammographic density was associated with the use of estrogen/progestin combination therapy, although the micronized progesterone containing arm appeared to induce a smaller increase that that with MPA. 51

61 The Difference Between Bioidentical Progesterone & Progestin, continued. Ferrero S, Gerbaldo D, Fulcheri E, Cristoforoni P. Vaginal micronized progesterone in continuous hormone replacement therapy. A prospective randomized study. Minerva Ginecol 2002; 54(6): Transvaginal micronized progesterone (100 mg/day for 12 days/month) effectively promoted a functional, secretory endometrium, while cyclic oral medroxyprogesterone acetate or transdermal norethisterone acetate more often produced endometrial atrophy, in women receiving continuous transdermal estradiol. Bagis T, Gokcel A, Zeyneloglu HB, Tarim E, Kilicdag EB, Haydardedeoglu B. The effects of short-term medroxyprogesterone acetate and micronized progesterone on glucose metabolism and lipid profiles in patients with polycystic ovary syndrome: a prospective randomized study. J Clin Endocrinol Metab 2002 Oct;87(10): This randomized prospective study evaluated and compared the effects of 10 days treatment with oral and vaginal micronized progesterone (MP) and medroxyprogesterone acetate (MPA) on glucose metabolism, lipid profiles, and hormonal parameters in 28 patients with polycystic ovary syndrome (PCOS). Oral MPA and oral MP decreased LH (P = 0.028, P = 0.009, respectively) and total testosterone (P = 0.013, P = 0.037, respectively) levels. There was no change in hormonal parameters with vaginal MP. Basal insulin decreased (P = 0.021) and insulin sensitivity increased significantly in the oral MPA group. Low density lipoprotein cholesterol (LDL) and lipoprotein (a) levels decreased only in the MPA group. This study concluded that MPA and oral MP may reduce insulin sensitivity in patients with PCOS. Vaginal MP had no effect on glucose metabolism and lipid profiles. De Lignieres B. Effects of progestogens on the postmenopausal breast. Climacteric 2002; 5(3): In this review, the author highlights the differences between progesterone and synthetic progestins in the breast and cautions that progestogens not be all put in the same bag with respect to safety. A strong case is made for the protective effect of progesterone on the breast. Sitruk-Ware R. Progestogens in hormonal replacement therapy: new molecules, risks, and benefits. Menopause 2002;9(1):6-15. The classifications of various progestogens (natural and synthetic) are reviewed in terms of their risks and benefits. This review clearly elucidates the differences in the mode of action of various synthetic progestins as well as progesterone. Montplaisir J, Lorrain J, Denesle R, Petit D. Sleep in menopause: differential effects of two forms of hormone replacement therapy. Menopause 2001; 8(1): This randomized clinical trial compared the effects of conjugated equine estrogen (CEE) and medroxyprogesterone acetate to CEE and oral micronized progesterone. Twenty-one postmenopausal women were studied in a sleep lab, with results demonstrating an improvement in subjective measures of menopausal symptoms and sleep in both groups. The group receiving natural progesterone had significantly improved sleep efficiency, whereas the medroxyprogesterone acetate group did not, suggesting that the former might better improve sleep in postmenopausal women. 52

62 The Difference Between Bioidentical Progesterone & Progestin, continued. Otsuki M, Saito H, Xu X, Sumitani S, Kouhara H, Kishimoto T, Kasayama S. Progesterone, but not medroxyprogesterone, inhibits vascular cell adhesion molecule-1 expression in human vascular endothelial cells. Arterioscler Thromb Vasc Biol 2001 Feb;21(2): This study utilizing human umbilical vein endothelial cells (HUVEC s) demonstrated that progesterone, but not medroxyprogesterone acetate (MPA) inhibited expression of vascular cell adhesion molecule-1 (VCAM-1), demonstrating a role for progesterone in the prevention of atherosclerosis. The differing effects of progesterone and MPA are clinically important, as MPA is widely used in hormone replacement therapy, when, as this research suggests, progesterone might be a more appropriate option. Ryan N, Rosner A. Quality of life (QOL) and costs associated with micronized progesterone and medroxyprogesterone acetate in hormone replacement therapy for non-hysterectomized, postmenopausal women. Clin Ther 2001 Jul;23(7): This prospective, multicenter, randomized, parallel-group study enrolled 182 postmenopausal women 45 to 65 years of age and evaluated the quality of life (QOL) and menopausal symptoms associated with the use of medroxyprogesterone acetate vs oral micronized progesterone when used as a part of a regular hormone replacement therapy. Menopausal symptoms improved in both groups from baseline to 9 months, as did QOL measures. In addition, patients using micronized progesterone had specific improvements in the areas of cognition and menstrual problems whereas the patients using MPA did not. Micronized progesterone was seen as an effective, cost-comparable alternative to MPA as well as being better tolerated. Rosano GM, Webb CM, Chierchia S, Morgani GL, Gabraele M, Sarrel PM, de Ziegler D, Collins P. Natural progesterone, but not medroxyprogesterone acetate, enhances the beneficial effect of estrogen on exerciseinduced myocardial ischemia in postmenopausal women. J Am Coll Cardiol 2000 Dec;36(7): Eighteen postmenopausal women were randomized to receive 17-beta estradiol with a synthetic progestin (medroxyprogesterone acetate) or a progesterone vaginal gel for 4 weeks, then crossed over to the alternate treatment. Researchers found through treadmill testing that estrogen plus progesterone significantly increased exercise time before myocardial ischemia, when compared to estradiol plus synthetic progestin. In addition, 2 patients on the synthetic progestin arm had to discontinue due to unstable angina. This research suggests that women at risk for cardiovascular disease need to consider progesterone as a safer alternative to synthetic progestins as a part of their hormone replacement therapy regime. Sitruk-Ware R. Progestins and cardiovascular risk markers. Steroids 2000 Oct-Nov;65(10-11): This article reviews the effects of various synthetic progestins and progesterone on cardiovascular health. Many synthetic progestins, especially 19-nortestosterone and some 17-hyroxyprogesterones, have negative effects on cardiovascular risk factors, whereas natural progesterone does not. Further studies utilizing natural and other steroids should be considered. 53

63 The Difference Between Bioidentical Progesterone & Progestin, continued. Fitzpatrick LA, Pace C, Wiita B. Comparison of regimens containing oral micronized progesterone or medroxyprogesterone acetate on quality of life in postmenopausal women: a cross-sectional survey. J Women Health Gend Based Med 2000 May;9(4): A cross-sectional survey was conducted to examine quality of life (QOL) related to physiological, somatic, and vasomotor effects of switching progestogen treatment from medroxyprogesterone acetate (MPA) to micronized progesterone in postmenopausal women already using hormone replacement therapy (HRT). One hundred seventy-six women who were currently using hormone replacement therapy (HRT) containing micronized progesterone for 1-6 months and had previously received HRT containing MPA were surveyed to assess QOL. Women using micronized progesterone-containing HRT experienced significant improvement in vasomotor symptoms, anxiety, somatic complaints and depressive symptoms. Women reported improved control of menopausal symptoms and perceptions of their vaginal bleeding patterns while on the micronized progesterone-containing regimen. Approximately 80% of women reported satisfaction with the progesterone-containing therapy. A micronized progesterone-containing HRT therapy offers the potential for improved QOL with respect to menopausal symptoms. De Lignieres B. Oral micronized progesterone. Clin Ther 1999; 21(1): This review article examines the rationale for selecting oral micronized progesterone over synthetic progestins. It reviews research regarding efficacy and safety and concludes that oral micronized progesterone has fewer side effects than synthetic progestins and is a convenient way to deliver natural progesterone. Lobo RA. Progestogen metabolism. J Reprod Med 1999 Feb;44(2 Suppl): This review clearly elucidates what s known about the differences in metabolism of various progestins as compared with endogenous or natural progesterone. Not only are there different pathways for metabolism, but the route of administration also has a significant effect. The physiologic and pathologic state of the patient further influences the metabolism, and there are measurable variations between patients. The authors also review the differences expressed by various tissues in metabolizing progestogens as well as the different biologic potencies of the various progestogens. Most importantly, the authors state the lack of knowledge about the synthetic progestins as compared to natural progesterone, which has a much better understood effect in the body. Miyagawa K, Rosch J, Stanczyk F, Hermsmeyer K. Medroxyprogesterone interferes with ovarian steroid protection against coronary vasospasm. Nature Medicine 1997;3(3): Ovariectomized rhesus monkeys were treated with physiological levels of 17-beta estradiol in combination with either medroxyprogesterone or progesterone (oral micronized) for four weeks. Following pathophysiological stimulation without injury to induce coronary vasospasm, it was shown that progesterone plus estradiol was protective against vasospasm, whereas estradiol plus medroxyprogesterone allowed vasospasm, concluding that medroxyprogesterone increases risk of coronary vasospasm, while progesterone does not. 54

64 The Difference Between Bioidentical Progesterone & Progestin, continued. Writing Group for the PEPI Trial. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. The postmenopausal estrogen/progestin interventions (PEPI) trial. JAMA 1995; 273(3): Bioidentical progesterone (an oral micronized preparation) was used in one group in the PEPI study in place of medroxyprogesterone acetate (MPA). Results showed that the progesterone group had significantly higher HDL cholesterol levels than the MPA group, indicating a different pharmacological effect than the synthetic progestin with a more favorable effect on blood lipids. Ojasoo T. Multivariate preclinical evaluation of progestins. Menopause: The Journal of the North American Menopause Society 1995; 2(2): Specificity profiles of numerous progestins were evaluated by multivariate analysis. Twenty steroid hormones, including natural progesterone, were tested for anti-estrogenic activity and for binding to the androgen, progesterone, and glucocorticoid receptors. Martorano JT, Ahlgrimm M, Meyers D. Differentiating between natural progesterone and synthetic progestogens: clinical implications for PMS management. Comprehensive Therapy 1993; 19(3):96-8. Clinical observations demonstrate that patients suffering from PMS respond to treatment with natural progesterone, whereas synthetic progestins may exacerbate the condition. The authors review the differences between natural progesterone and synthetic progestins. Ottosson UB, Johansson BG, et al. Subfractions of high-density lipoprotein cholesterol during estrogen replacement therapy: A comparison between progestogens and natural progesterone. American Journal of Obstetrics and Gynecology 1993 Mar;151(6): Fifty-eight postmenopausal women were followed with respect to subfractions of high-density lipoprotein during 3 cycles of unopposed estrogen. The women received either levonorgestrel, medroxyprogesterone acetate, or natural progesterone during the last ten days of the treatment period. Both progestogens significantly lowered HDL cholesterol, whereas natural progesterone had no effect on HDL levels. Saarikoski S, Yliskoski M, Penttila I. Sequential use of norethisterone and natural progesterone in premenopausal bleeding disorders. Maturitas 1990 Jun;12(2): This randomized controlled study evaluated the effects of norethisterone (NET) and micronized progesterone (MP) on bleeding disorders in pre-menopausal women. 80 patients were randomized to the trial and all were found via endometrial morphology to need progestogen therapy. They were subsequently treated with NET or MP. In both treatment groups, hyperplastic changes disappeared during the first three cycles, with the duration of treatment being 6 months. NET decreased follicle-stimulating hormone, luteinizing hormone, estradiol and sex-hormone-binding globulin levels (P < 0.001) whereas no changes were seen during MP treatment. High-density-lipoprotein cholesterol and triglyceride levels were also lowered by NET (P< ) slightly decreased phospholipids. MP treatment had no effect on lipid profiles suggesting it may be a preferred progestogen for the treatment of bleeding disorders. 55

65 The Difference Between Bioidentical Progesterone & Progestin, continued. Dalton K. The effects of progesterone and progestogens on the foetus. Neuropharmacology 1981; 20: This article looks at the differing effects of progesterone and synthetic progestogens on the fetus. Of note in this article is evidence that progesterone supplementation may reduce episodes of pre-eclampsia. Synthetic progestogen supplementation during pregnancy may produce a variety of side effects. Several references are made to articles documenting cases of masculinization of external genitalia in female babies. There are two known cases of true hermaphroditism and several cases of behavioral problems developing in adolescent girls whose mothers took oral synthetic progestogens during pregnancy. More problematic may be administration of oral estrogen-progestogen preparations. Side effects may include spina bifida, esophageal anomalies, heart defects and limb reduction deformities. Bioidentical Estrogens Estradiol and Estriol Overview Estrogen therapy is still widely recommended for short-term treatment of troublesome menopausal symptoms such as hot flashes and vaginal atrophy, although non-estrogenic vaginal lubricants can also be effective for vaginal symptoms. Estrogen therapy is no longer advised for long-term protection against cardiovascular disease because of the negative outcome of the Women s Health Initiative study. However, some researchers suggest that much lower doses of estrogen given to women very soon after the onset of menopause could help protect against later cardiovascular disease, and this is being tested in clinical trials such as the Kronos Early Estrogen Prevention Study (KEEPS). This trial is currently enrolling 720 women early in menopause for the evaluation of lower estrogen doses (0.45 mg/day oral conjugated equine estrogens or 0.05 mg/day transdermal estradiol) in combination with cyclic oral micronized progesterone (Prometrium, 200 mg/day for 12 days per month) compared with placebo. The researchers are looking at measures of cardiovascular health. Completion of the study is expected in Also, the Early Versus Late Intervention Trial With Estradiol (ELITE) is enrolling 504 women either less than 6 years or 10 years or more after menopause. They will be randomized to receive either 1 mg/day oral estradiol or placebo (with 4% vaginal progesterone gel or placebo respectively for 10 days each month in women with a uterus) and cardiovascular outcomes measured. Estrogens have also been prescribed for the prevention of bone loss because of the known effect of estrogen in inhibiting bone resorption. Again, the long-term adverse effects with estrogen therapy seen in clinical trials have led to recommendations for a lower estrogen dosage. Bioidentical estrogens given transdermally (patches or creams) can be used in much lower doses than oral estrogens. This is because they avoid the first-pass metabolism by the liver when drugs are given orally. The recent study by Ho and colleagues below also demonstrates a cardiovascular protective effect of transdermal bioidentical estradiol without the damaging inflammatory effects of oral conjugated estrogens. Atherosclerotic vascular disease is now widely thought to be a result of inflammation. 56

66 Bioidentical Estrogens Estradiol & Estriol, continued. Ho JY, Chen MJ, Sheu WH, Yi YC, Tsai AC, Guu HF, Ho ES. Differential effects of oral conjugated equine estrogen and transdermal estrogen on atherosclerotic vascular disease risk markers and endothelial function in healthy postmenopausal women. Hum Reprod 2006; 21(10): In this comparison of mg/day oral conjugated equine estrogen (CEE) versus 0.6 mg/day 17β-estradiol transdermal gel for 6 months or no treatment, the oral CEE group showed significantly increased levels of C- reactive protein (CRP), a marker of inflammation, while the transdermal and control groups showed no increase in CRP. The transdermal estradiol group showed a similar beneficial effect on flow-mediated vasodilation in the brachial artery to the CEE group, indicating a comparative therapeutic benefit but without increasing the risk of atherosclerosis. Ouyang P, Michos ED, Karas RH. Hormone replacement therapy and the cardiovascular system: lessons learned and unanswered questions. J Am Coll Cardiol 2006; 49(9): This review discusses the lack of benefit of estrogen therapy in older women for the prevention of cardiovascular problems. Friel PN, Hinchcliffe C, Wright JV. Hormone replacement with estradiol: conventional oral doses result in excessive exposure to estrone. Altern Med Rev Mar;10(1): Barrett-Connor E, Grady D, Stefanik ML. The rise and fall of menopausal hormone therapy. Ann Rev Public Health 2005; 26: This important review by leading epidemiologists looks at the results of major clinical trials of hormone therapy and suggests that any long term benefits of estrogen therapy for the prevention of fractures could be achieved with much lower doses of estrogen. Ettinger B. Rationale for use of lower estrogen doses for post-menopausal hormone therapy. Business Briefing: Women s Healthcare 2004; [accessed 6/27/06]. Prestwood KM, Kenny AM, Kleppinger A, Kulldorff M. Ultralow-dose micronized 17β-estradiol and bone density and bone metabolism in older women: a randomized controlled trial. JAMA 2003; 290(8): This study of 0.25 mg/day oral estradiol versus placebo for 3 years in women over 65 years old (with 100 mg/ day oral micronized progesterone in women with a uterus in both groups) found an increase in bone density in the estradiol group compared with placebo. Archer DF; EstroGel Study Group. Percutaneous 17beta-estradiol gel for the treatment of vasomotor symptoms in postmenopausal women. Menopause Nov-Dec; 10(6):

67 Bioidentical Estrogens Estradiol & Estriol, continued. Cohen, L, Soares, C, Poitras, J, Prouty, J, Alexander, A, Shifren, J. Short-term use of estradiol for depression in perimenopausal and postmenopausal women: a preliminary report. Am J Psychiatry Aug;160(8): Twenty-two peri- or post-menopausal women with median age of 50 years experiencing moderate severity depression (DSM-IV major depression, minor depression, or dysthymia) were enrolled in a 4 week open-label clinical trial of 100 micrograms of transdermal 17B estradiol. Results showed decreased score on Montgomery- Asberg Depression Rating Scale (20 to 11.50) and Beck Depression Inventory. Greene-Climacteric Scale scores showed measured improvement during the 4 week study. Changes in depression scales and climacteric scales were not significantly correlated. Perimenopausal (6) women showed greater improvement in depression scales than postmenopausal women (2). Authors suggested this study supports previous results showing that the effect of estrogen therapy on mood may be independent of antidepressant effects mediated by alleviation of vasomotor symptoms and that estrogen therapy may be of benefit to perimenopausal women experiencing moderately severe depression. Jarupanich T, Lamlertkittikul S, Chandeying V. Efficacy, safety and acceptability of a seven-day, transdermal estradiol patch for estrogen replacement therapy. J Med Assoc Thai Sep;86(9): Wren BG, Day RO, McLachlan AJ, Williams KM. Pharmacokinetics of estradiol, progesterone, testosterone and dehydroepiandrosterone after transbuccal administration to postmenopausal women. Climacteric Jun;6(2): Collette J, Viethel P, Dethor M, Chevallier T, Micheletti MC, Foidart JM, Reginster JY. [Comparison of changes in biochemical markers of bone turnover after 6 months of hormone replacement therapy with either transdermal 17 beta-estradiol or conjugated equine estrogen plus nomegestrol acetate] Gynecol Obstet Fertil May;31(5): Zegura B, Keber I, Sebestjen M, Koenig W. Double blind, randomized study of estradiol replacement therapy on markers of inflammation, coagulation and fibrinolysis. Atherosclerosis May;168(1): Zegura B, Keber I, Sebestjen M, Borko E. Orally and transdermally replaced estradiol improves endothelial function equally in middle-aged women after surgical menopause. American Journal of Obstetrics and Gynecology May;188(5): Forty-three surgically induced (6 weeks postop) menopausal women were randomly assigned in a double-blind study to 28 weeks of 2.0mg oral or 50mg transdermal estradiol. Looking at blood flow through the brachial artery, flow-mediated dilation (ultrasound) in the oral group increased 6.0 to 13.2% and in the transdermal group increased 7.0 to 14.9% Results indicate that both oral and transdermal administration had equal effect on arterial endothelium independent of lipid profiles and increased vasodilation. 58

68 Bioidentical Estrogens Estradiol & Estriol, continued. Arrenbrecht S, Boermans AJ. Effects of transdermal estradiol delivered by a matrix patch on bone density in hysterectomized, postmenopausal women: a 2-year placebo-controlled trial. Osteoporos Int 2002;13(2): Granberg S, Eurenius K, Lindgren R, Wilhelmsson L. The effects of oral estriol on the endometrium in postmenopausal women. Maturitas 2002 Jun 25;42(2): This study conducted endometrial evaluation using both transvaginal ultrasound (TVS) and histologic biopsy by Pipelle in postmenopausal women taking a low-dose oral estriol (1 or 2 mg daily) for a mean duration of 4.3 years. Mean endothelial thickness in the study group after one year was 3.0mm and in the control group was 2.4mm. There was a noted increase in atrophic vaginal epithelium in the control group. There was a noted increased incidence of endometrial polyps in the study group (14.1%) compared to the control group (2.9%) although this was not determined to be clinically significant. Yoshimura T, Okamura H. Short term oral estriol treatment restores normal premenopausal vaginal flora to elderly women. Maturitas 2001 Sep 28;39(3): This study looked at short term (14 days) oral estriol (2.0mg/day) treatment for atrophic vaginitis in 59 postmenopausal women aged years. The results showed that in the majority of women in the study group the oral estriol restored normal vaginal flora by the end of the treatment period. Perera M, Sattar N, Petrie JR, Hillier C, Small M, Connell JM, Lowe GD, Lumsden MA. The effects of transdermal estradiol in combination with oral norethisterone on lipoproteins, coagulation, and endothelial markers in postmenopausal women with Type 2 diabetes: a randomized, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism 2001 Mar;86(3): This study showed that transdermal estradiol and oral norethisterone reduce plasma triglyceride and total cholesterol levels, factor VII activity and von Willebrand factor antigen levels in women with Type 2 diabetes without a concurrent change in adiposity or glycemic control. The authors suggest that this protocol might be of benefit for women at high risk of cardiovascular disease. Manonai J, Theppisai U. Effect of oral estriol on urogenital symptoms, vaginal cytology, and plasma hormone level in postmenopausal women. J Med Assoc Thai 2001 Apr;84(4): Prestwood KM, Kenny AM, Unson C, Kulldorff M. The effect of low dose micronized 17b-estradiol on bone turnover, sex hormone levels, and side effects in older women: a randomized, double blind, placebo-controlled study. Journal of Clinical Endocrinology and Metabolism 2000 Dec;85(12): This study determined that oral low-dose estrogen (0.25mg/day) had similar beneficial effects on bone health in elderly (mean age 75 years) postmenopausal women without the breast tenderness and bleeding associated with higher doses. Authors recommended the use of serum E2 levels as the guide for therapeutic effect at a range of pg/l. 59

69 Bioidentical Estrogens Estradiol & Estriol, continued. Davis SR, Walker KZ, Strauss BJ. Effects of estradiol with and without testosterone on body composition and relationships with lipids in postmenopausal women. Menopause 2000 Nov-Dec;7(6): Bruce-Keller AJ, Keeling JL, Keller JN, Huang FF, Camondola S, Mattson MP. Antiinflammatory effects of estrogen on microglial activation. Endocrinology 2000 Oct;141(10): This study identified new pathways for the estrogenic anti-inflammatory effects on brain function, potentially leading to identification of new methods for improving neurodegenerative disease, specifically involving the microglial cells. Itoi H, Minakami H, Iwasaki R, Sato I. Comparison of the long-term effects of oral estriol with the effects of conjugated estrogen on serum lipid profile in early menopausal women. Maturitas 2000 Oct 31;36(3): This was a randomized comparison study with three arms: 2.0mg estriol + 2.5mg medroxyprogesterone (E3), 0.625mg conjugated estrogen + 2.5mg medroxyprogesterone (CE) and a vitamin D and Calcium combination (control), looking at changes in serum lipid profiles in early menopausal women. The sample size was 67 women. After 48 months on the randomized protocol, the serum lipid profiles showed that those in the E3 group decreased total cholesterol and triglycerides (-4.9 and 6.7) compared to the control of (+5.4 and +6.1) and CE group of (-1.9 and +17.6). The E3 group showed less significant changes in HDL cholesterol and LDL cholesterol when compared to the CE protocol: E3 (+3.8 and -5.2), CE (+10.7 and -11.4), control (-3.6 and +11.8). The results show the improvement of serum lipid profiles in response to estrogen. The authors suggested that in women where bleeding has been a problem response to certain estrogen protocols, the low-dose estriol may be an alternative treatment for those at risk of cardiovascular disease. Miller BE, De Souza MJ, Slade K, Luciano AA. Sublingual administration of micronized estradiol and progesterone, with and without micronized testosterone: effect on biochemical markers of bone metabolism and bone mineral density. Menopause 2000 Sep-Oct;7(5): Lose G, Englev E. Oestradiol-releasing vaginal ring versus oestriol vaginal pessaries in the treatment of bothersome lower urinary tract symptoms. BJOG 2000 Aug;107(8): Takahashi K, Okada M, Ozaki T, Kurioka H, Manabe A, Kanasaki H, Miyazaki K. Safety and efficacy of oestriol for symptoms of natural or surgically induced menopause. Hum Reprod 2000 May;15(5): de Lignieres B, Silberstein S. Pharmacodynamics of oestrogens and progestogens. Cephalalgia 2000 Apr;20(3): Hayashi T, Ito I, Kano H, Endo H, Iguchi A. Estriol (E3) replacement improves endothelial function and bone mineral density in very elderly women. J Gerontol A Biol Sci Med Sci 2000 Apr;55(4):B183-90; discussion B

70 Bioidentical Estrogens Estradiol & Estriol, continued. Takahashi K, Manabe A, Okada M, Kurioka H, Kanasaki H, Miyazaki K. Efficacy and safety of oral estriol for managing postmenopausal symptoms. Maturitas 2000 Feb 15;34(2): Head KA. Estriol: safety and efficacy. Altern Med Rev Apr;3(2): Scarabin PY, Alhenc-Gelas M, Plu-Bureau G, Taisne P, Agher R, Aiach M. Effects of oral and transdermal estrogen/progesterone regimens on blood coagulation and fibrinolysis in postmenopausal women. A randomized controlled trial. Arterioscler Thromb Vasc Biol 1997 Nov;17(11): Snabes, M, Payne, J, Kopelen, H, Dunn, JK, Young, R, Zoghbi, W. Physiologic estradiol replacement therapy and cardiac structure and function in normal postmenopausal women: a randomized, double-blind, placebo-controlled crossover trial. Obstetrics and Gynecology, 1997;89(3): In a randomized, double-blind, placebo-controlled, crossover clinical trial of 31 postmenopausal women, average age 59.7 years, using 2.0 mg of oral estradiol (E2) daily, the authors investigated the effects of estradiol on cardiac function and structure. This study did not include the use of progestins with estrogen. 12 weeks of E2 therapy showed no change in left ventricular thickness or mass, left atrial size or aortic size. There was a small but significant increase in left ventricular end-diastolic volume but it was not associated with change in end-systolic volume or ejection fraction changes. Heart rate and systolic and diastolic pressures were unchanged after 3 months of treatment. Time-velocity integral of flow and peak flow velocities were unaffected by E2 treatment. Authors concluded that estrogen replacement therapy did not affect cardiac structure or size in normal postmenopausal women (after 12 weeks of treatment). Suvanto-Luukkonen E, Sundstrom H, Penttinen J, Laara E, Pramila S, Kauppila A. Percutaneous estradiol gel with an intrauterine levonorgestrel releasing device or natural progesterone in hormone replacement therapy. Maturitas 1997 Apr;26(3): Haines, C, Chung, T, Chang, A, Masarei, J, Tomlinson, B, Wong, E. Effect of oral estradiol on Lp(a) and other lipoproteins in postmenopausal women. A randomized, double-blind, placebo-controlled, crossover study.arch Intern Med 1996 Apr 22;156(8): In a randomized, double-blind, placebo-controlled, crossover study, 91 surgically postmenopausal women received either 6 months of 2mg daily oral estradiol followed by 6 months of placebo or the opposite regimen. During treatment phase, Group One showed decreased Lipoprotein (a) concentration (10.78 to 6.44 mg/dl) and LDL-C with increase in HDL-C and TG while Group Two showed a less pronounced decrease (12.74 to 10.75). 53 women continued oral estrogen therapy for an additional 12 months. Lipoprotein (a) levels were essentially unchanged from previous measures at the end of the treatment phase after 12 months of additional therapy. Authors suggested that reduced Lipoprotein (a) levels with extended oral estrogen therapy support a cardioprotective effect of HRT in postmenopausal women. Lindoff C, Peterson F, Lecander I, Martinsson G, Astedt B. Transdermal estrogen replacement therapy: beneficial effects on hemostatic risk factors for cardiovascular disease. Maturitas 1996 May;24(1-2):

71 Bioidentical Estrogens Estradiol & Estriol, continued. Nozaki M, Hashimoto K, Inoue Y, Sano M, Nakano H. [Usefulness of estriol for the treatment of bone loss in postmenopausal women] Nippon Sanka Fujinka Gakkai Zasshi Feb;48(2):83-8. Evans SF, Davie MW. Low and conventional dose transdermal oestradiol are equally effective at preventing bone loss in spine and femur at all post-menopausal ages. Clin Endocrinol (Oxf) 1996 Jan;44(1): Koloszar S, Kovacs L. [Treatment of climacteric urogenital disorders with an estriol-containing ointment] Orv Hetil 1995 Feb 12;136(7): Riedel M, Oeltermann A, Mugge A, Creutzig A, Rafflenbeul W, Lichtlen P. Vascular responses to 17 betaoestradiol in postmenopausal women. Eur J Clin Invest 1995 Jan;25(1):44-7. Yang TS, Tsan SH, Chang SP, Ng HT. Efficacy and safety of estriol replacement therapy for climacteric women. Zhonghua Yi Xue Za Zhi ( Taipei ) May;55(5): Darj E, Axelsson O, Carlstrom K, Nilsson S, von Schoultz B. Liver metabolism during treatment with estradiol and natural progesterone. Gynecol Endocrinol 1993 Jun;7(2): Kainz C, Gitsch G, Stani J, Breitenecker G, Binder M, Schmidt JB. When applied to facial skin, does estrogen ointment have systemic effects? Arch Gynecol Obstet 1993;253(2):71-4. Raul, R, Stamm, W. A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infections. The New England Journal of Medicine. 1993;329(11): This randomized, double blind, placebo-controlled trial looked at the incidence of urinary tract infections (UTI) in 93 postmenopausal women using 0.5 mg estriol vaginal cream once nightly for two weeks followed by twice weekly application or placebo. Results showed significantly lower UTI rates in treatment group (0.5 infections per patient-year vs. 5.9 for placebo group). The mean vaginal ph fell from to for treatment group and /2 to in placebo group and there was an increase in vaginal colonization with lactobacilli in the treatment group. Authors recommend use of topical vaginal estriol in preventive treatment of women with frequent UTI as possible replacement for long-term use of nitrofurantoin, co-trioxazole, trimethoprim, cehpalexin or fluoroquinolones. 62

72 Bioidentical Estrogens Estradiol & Estriol, continued. Ettinger B, Genant HK, Steiger P, Madvig P. Low-dosage micronized 17 beta-estradiol prevents bone loss in postmenopausal women. American Journal of Obstetrics and Gynecology Feb;166(2): The authors evaluated the effects of 17 beta-estradiol (E2) in a randomized, double-blind, dose ranging study of 41 postmenopausal women conducted in 2 phases. Phase one included phased E2 doses (0.5mg, 1.0mg, 2.0mg) plus calcium supplementation (to serum value of 1500mg). Phase two included E2 doses plus random cessation of calcium supplementation. Progestins were added during phase two (total study time of 18 months). Results showed very little change in bone density results for placebo group ( %) whereas treatment group showed significant increases from baseline bone density. In phase two the treatment groups showed an annual change in bone density of 2.0%. There was a positive correlation between total calcium intake and the change in bone density results. The study showed a continuous dose-response effect on bone density results. Authors concluded that low dose (1.0mg) beta-estradiol and 1000mg of calcium prevented bone loss in postmenopausal women. Iosif CS. Effects of protracted administration of estriol on the lower genitourinary tract in postmenopausal women. Arch Gynecol Obstet 1992;251(3): Chetkowski RJ, Meldrum DR, Steingold KA, Randle D, Lu JK, Eggena P, Hershman JM, Alkjaersig NK, Fletcher AP, Judd HL. Biologic effects of transdermal estradiol. N Engl J Med 1986 Jun 19;314(25): Twenty-three postmenopausal women were randomly assigned to use of transdermal estradiol in four increasing doses (25, 50, 100, 100 micrograms per 24 hours) followed by daily oral dose of conjugated equine estrogens in two doses (0.625 mg, 1.25 mg) or to use of oral conjugated equine estrogens followed by transdermal estradiol. Results showed a dose-response relationship between the amount of estradiol delivered and the serum measure of the hormone. Estrone concentrations also rose with transdermal application. At the 50 and 100 microgram transdermal dose levels, results were comparative to the and 1.25 mg conjugated equine estrogen results. Non-hepatic markers (serum gonadotropin, vaginal cytologic studies, urinary calcium levels and urinary calcium/creatinine ratios all increased in dose-dependent fashion. Hepatic markers (hepatic protein level, lipid metabolism, clotting factors, renin substrate) were not affected by transdermal doses of estradiol. Transdermal estradiol provided benefit of increased serum hormone levels without hepatic protein effects of oral conjugated equine estrogens. Haspels AA, Luisi M, Kicovic PM. Endocrinological and clinical investigations in post-menopausal women following administration of vaginal cream containing oestriol. Maturitas 1981 Dec;3(3-4): Tzingounis VA, Aksu MF, Greenblatt RB. Estriol in the management of the menopause. JAMA 1978 Apr 21;239(16): Callantine MR, Martin PL, Bolding OT, Warner PO, Greaney MO Jr. Micronized 17 beta-estradiol for oral estrogen therapy in menopausal women. Obstet Gynecol 1975 Jul;46(1):

73 Andropause & Androgens Overview An increasing number of researchers is looking at the phenomenon of androgen deficiency in women (Miller 2001), as levels of dehydroepiandrosterone (DHEA) and testosterone fall steadily with age and contribute to many of the symptoms seen in postmenopause. Androgen therapy with DHEA or testosterone has been found to restore sexual function, particularly in women who have experienced a sudden drop in androgen levels, such as younger women undergoing surgical menopause, or women with adrenal insufficiency (Arlt 2006). DHEA has been found to successfully restore sexual function in older women and has had a good safety profile, even after 50 mg/day oral DHEA was administered for one year, as found in the DHEAge study, which also found an increase in bone mineral density with DHEA treatment in women over 70 years old (Baulieu et al. 2000). Labrie et al. (2005) have suggested the use of DHEA for physiological hormone replacement because of its nature as a hormone precursor, producing estrogens and testosterone in the hormone-dependent target tissues precisely according to local needs, and thus alleviating many of the symptoms of hormone deficiency while avoiding the systemic side effects of exogenous hormone therapy. They call this new concept intracrinology as opposed to endocrinology and suggest that it offers new options for menopausal women. Arlt (2006) has reviewed the use of androgen therapy in women and concluded that this should be reserved for women with severe androgen insufficiency. Arlt W. Androgen therapy in women. Eur J Endocrinol 2006; 154:1-11. This is a current review of the use of androgen therapy in women by a leading specialist in this area. It concludes that treatment with androgens should be reserved for younger women undergoing surgical menopause, or women with severe adrenal insufficiency. Androgen therapy with DHEA or testosterone has been found to restore sexual function, particularly in women who have experienced a sudden drop in androgen levels after surgical menopause (removal of the ovaries). Labrie F, Luu-The V, Bélanger A, Lin S-X, Simard J, Pelletier G. Is dehydroepiandrosterone a hormone? J Endocrinol 2005; 187: These researchers suggest the use of DHEA for physiological hormone replacement because of its nature as a hormone precursor, producing estrogens and testosterone in the hormone-dependent target tissues precisely according to local needs. It could thus alleviate many of the symptoms of hormone deficiency while avoiding the systemic side effects of too much direct hormone therapy with estrogens or testosterone. They call this new concept intracrinology as opposed to endocrinology and suggest that it offers new options for menopausal women. Schneider HP. Androgens and antiandrogens. Ann N Y Acad Sci Nov;997: Lobo RA. Androgens in postmenopausal women: production, possible role, and replacement options. Obstet Gynecol Surv Jun;56(6): Miller KK. Androgen deficiency in women. J Clin Endocrinol Metab 2001 Jun;86(6):

74 Andropause & Androgens, continued. Simon JA. Safety of estrogen/androgen regimens. J Reprod Med 2001 Mar;46(3 Suppl): Baulieu E-E, Thomas G, Legrain S, Lahlou N, Roger M, Debuire B, Faucounau V, Girard L, Hervy M-P, Latour F, Leaud M-C, Mokrane A, Pitti-Ferrandi H, Trivalle C, de Lacharriere O, Nouveau S, Rakoto-Arison B, Souberbielle J-C, Raison J, Le Bouc Y, Raynaud A, Girerd X, Forette F. Dehydroepiandrosterone (DHEA), DHEA sulfate, and aging: contribution of the DHEAge study to a sociobiomedical issue. Proc Natl Acad Sci 2000; 97(8): DHEA successfully restored sexual function in older women and had a good safety profile, even after 50 mg/day oral DHEA was administered for one year, in the DHEAge study, which also found an increase in bone mineral density with DHEA treatment in women over 70 years old. Stoll BA. Dietary supplements of dehydroepiandrosterone in relation to breast cancer risk. Eur J Clin Nutr 1999 Oct;53(10): Wiebke, A, Callies, F, Van Vlijmen, J, Koehler, I, Reincke, M, Bidlingmaier, M, Huebler, D, Oettel, M, Ernst, M, Schulte, H, Allolio, B. Dehydroepiandrosterone replacement in women with adrenal insufficiency. The New England Journal of Medicine, 1999;341(14): This double-blind crossover study reviewed alternately the effects of 50mg of oral dehydroepiandrosterone (DHEA) daily with placebo in 24 women with adrenal insufficiency. Participants were evaluated using established well-being (depression and anxiety scores) and sexuality (thoughts, interest, satisfaction) scales and serum profiles. Results showed that serum DHEA, DHEA-S and active androgen increased to normal or lownormal levels during treatment. SHBG levels were significantly lower following treatment. IGF-I concentrations increased after treatment (only in women with primary adrenal insufficiency), but IGF-binding protein 3 levels did not change. Serum total and HDL lipoprotein cholesterol levels decreased significantly during treatment. LDL and triglyceride concentrations did not change significantly. Psychological testing scores for well-being and sexuality both improved significantly during treatment. These effects were noticed after treatment for four months, but not after treatment for one month. Authors recommended that treatment with DHEA should be part of hormone replacement therapy for women with adrenal insufficiency. Arlt W, Callies F, van Vlijmen JC, Koehler I, Reincke M, Bidlingmaier M, Huebler D, Oettel M, Ernst M, Schulte HM, Allolio B. Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl J Med 1999 Sep 30;341(14): Davis SR.The therapeutic use of androgens in women. J Steroid Biochem Mol Biol Apr-Jun;69(1-6- ): Rako S.Testosterone deficiency: a key factor in the increased cardiovascular risk to women following hysterectomy or with natural aging? J Womens Health Sep;7(7):

75 Saliva Testing Research Aardal E, Holm AC. Cortisol in saliva-reference ranges and relation to cortisol in serum. Eur J Clin Chem Clin Biochem 1995;33: Parallel serum and saliva samples were acquired in 197 individuals, in the morning and evening. A slight decrease in morning cortisol was seen in the oldest individuals tested (61-70 years). As has been demonstrated in other work, once serum cortisol exceeded» 450 nmol/l, a marked increase in salivary cortisol was noted. This is attributed to a rapid increase in free plasma cortisol once the available sites on cortisol binding globulin are filled. The authors conclude by enumerating the advantages of saliva over serum (simplicity, decreased stress, convenience, sample mail-in, temperature stability). Aardal-Eriksson E, Karlberg BE, Holm AC. Salivary cortisol- and alternative to serum cortisol determinations in dynamic function tests. Clin Chem Lab Med 1998;36: Serum and salivary cortisol responses were compared in various dynamic tests of HPA axis function including insulin tolerance, CRH stimulation, and ACTH challenge. In 42 of 45 tests performed, consideration of salivary cortisol led to the same conclusion as serum cortisol. The authors conclude that due to a more pronounced cortisol response in saliva, and a closer correlation between salivary cortisol and serum ACTH, salivary cortisol may be used as an alternative parameter in dynamic endocrine tests. Allolio B, Hoffmann J, Linton EA, Winkelmann W, Kusche M, Schulte HM. Diurnal salivary cortisol patterns during pregnancy and after delivery: relationship to plasma corticotrophin-releasing hormone. Clin Endocrinol 1990;33: The circadian rhythm of salivary cortisol levels was studied in 10 pregnant women every 4 weeks throughout pregnancy. In 12 women, diurnal variation of salivary cortisol, serum cortisol, plasma ACTH, plasma CRH and serum progesterone was examined late in the third trimester and again 3-5 days after delivery. Mean salivary cortisol levels increased throughout pregnancy, reaching levels almost twice that of controls by late pregnancy, and falling rapidly with delivery. There was no correlation between plasma CRH, plasma ACTH, serum and saliva cortisol in late pregnancy. The authors conclude that although CRH levels increase in late pregnancy (due to placental production), this CRH has very little effect on baseline adrenocortical function. Instead, they hypothesize that the high progesterone levels in late pregnancy induce a state of glucocorticoid resistance, due to the antiglucocorticoid effect of progesterone. Barrou Z, Guiban D, Maroufi A, Fournier C, Dugue MA, Luton JP, Thomopoulos P. Overnight dexamethasone suppression test: comparison of plasma and salivary cortisol measurement for the screening of Cushing s syndrome. Eur J Endocrin 1996;134: Salivary cortisol was compared to plasma cortisol for screening of Cushing s Syndrome via overnight dexamethasone suppression test. Compared to plasma, salivary cortisol demonstrated superior performance with 100% specificity and 94% sensitivity (no overlap between controls and patients with Cushing s Syndrome). In addition, salivary cortisol levels in women taking oral contraceptives were normal whereas the corresponding plasma levels were elevated (false positive) in some cases. 66

76 Saliva Testing Research, continued. Belkien LD, Bordt J, Moller P, Hano R, Nieschlag E. Estradiol in saliva for monitoring follicular stimulation in an in vitro fertilization program. Fertil Steril 1985;44:322. Serial serum and saliva measurements were compared in 23 patients undergoing ovulation induction in an in vitro fertilization program. Serum and saliva were well correlated (r=0.77) throughout, except for salivary estradiol levels less than 4 pg/ml. (Note that estradiol can easily be detected below this level with modern methods.) The authors conclude that, assessment of ovarian function in clomiphene citrate (CC) or CC/hMG stimulated cycles can be performed precisely with the saliva estradiol assay. Bolaji II, Tallon DF, O Dwyer E, Fottrell PF. Assessment of bioavailability of oral micronized progesterone using a salivary progesterone enzymeimmunoassay. Gynecol Endocrinol 1993;7: These authors looked at salivary progesterone levels in 40 postmenopausal women taking 100 mg oral micronized progesterone in cyclical fashion (23 days/month) for one year. These levels fell into the range observed in the luteal phases of a control group of 40 normally cycling women. Five patients had simultaneous serum and saliva levels of progesterone measured for 12 hours after ingestion of a 100 mg dose of progesterone. Levels in both cases followed the same time curve, with both peaking at 2 hours. This supports the notion that progesterone diffuses rapidly from serum to saliva. Booth A, Johnson D, Granger D, Crouter A, McHale S. Testosterone and child and adolescent adjustment: the moderating role of parent-child relationships. October Unpublished manuscript. This study examined the relationship of salivary testosterone to self-reported risk behavior or depressive symptoms in adolescents. It was found that correlations between testosterone level and behavior were modulated by the quality of the parent-child relationship. The authors note, Salivary measures seem to be especially suited to studies of youth because sampling is painless, acceptance rates are higher, and bias caused by self-selection may thus be reduced. Campbell BC, Ellison PT. Menstrual variation in salivary testosterone among regularly cycling women. Horm Res 1992;37: Daily first morning saliva samples were collected from 20 regularly cycling women over the course of an entire menstrual cycle and assayed for testosterone and progesterone. Cycles were classified as ovulatory if salivary progesterone exceeded 100 pg/ml on any day in the cycle. Average testosterone level was significantly greater in anovulatory cycles. In both types of cycle, the same general features were observed: a luteal trough, a midcycle peak, and an early follicular peak, although in non-ovulatory cycles, the midcycle peak was longer, and the luteal trough shorter. These findings are consistent with what is known about changes in ovarian steroid metabolism throughout the menstrual cycle. Salivary assay is particularly well-suited to studies of this nature, which involve multiple measurements, since samples can be acquired with minimal inconvenience. 67

77 Saliva Testing Research, continued. Castro M, Elias PC, Quidute AR, Halah FP, Moreira AC. Out-patient screening for Cushing s Syndrome: the sensitivity of the combination of circadian rhythm and overnight dexamethasone suppression salivary cortisol tests. J Clin Endocrinol Metab 1999;84: These authors measured salivary cortisol levels in normal individuals and patients with Cushing s Syndrome. A single measurement at 23:00 hours produced a sensitivity and specificity of 93% for detection of Cushing s Syndrome. When a 23:00 hour measurement was combined with an overnight 1 mg dexamethasone suppression test, 100% sensitivity was achieved. The authors conclude, the collection of saliva rather than blood is a practical, simple and noninvasive out-patient alternative to screen for hypercortisolism. Choe JK, Khan-Dawood FS, Dawood MY. Progesterone and estradiol in the saliva and plasma during the menstrual cycle. Am J Obstet Gynecol 1983;147: Daily saliva and blood samples were obtained from 9 women throughout an entire menstrual cycle. Saliva and plasma levels of progesterone correlated reasonably well (r=0.58) although the saliva and blood samples were not acquired at the same time. The percentage of protein binding of progesterone in serum and saliva was also measured by equilibrium dialysis. Forty to fifty percent of the progesterone in saliva was protein bound, compared to three to six percent in plasma. However, unbound progesterone levels in plasma and saliva were comparable, supporting the contention that salivary progesterone represents unbound progesterone in the blood. The authors also measured salivary and plasma estradiol but did not display or mention the plasma data, commenting only that there was no correlation between levels in the two matrices. This casts suspicion on the validity of their plasma estradiol data. Christiansen K, Knussmann R. Androgen levels and components of aggressive behaviour in men. Horm Behav 1987;21: Saliva and serum testosterone as well as serum dihydrotestosterone were measured in normal males and correlated to measures of aggression. Weak but significant correlations (r»0.2) were seen between self-rated spontaneous aggressive tendency and both saliva and serum testosterone levels. Christiansen K, Knussmann R. Sex hormones and cognitive functioning in men. Neuropsychobiol 1987;18: Parallel blood and saliva samples were obtained from 117 men. Cognitive function (verbal and spatial ability ) was then assessed and correlated to androgenic hormone levels. Serum testosterone and dihydrotestosterone levels, as well as salivary testosterone levels correlated positively, though weakly to spatial ability scores, and negatively with verbal measures, in keeping with the known sex differences in cognitive function. Clements AD, Parker CR. The relationship between salivary cortisol concentrations in frozen versus mailed samples. Psychoneuroendocrinol 1998;23: Saliva specimens were obtained from 17 volunteers. Half of each specimen was immediately frozen. The other half was subjected to varying temperatures and physical handling intended to mimic conditions experienced during mailing. Both sample halves were ultimately assayed for cortisol, and a correlation coefficient of 0.92 was derived between the quick-frozen and mailed specimens. The authors conclude that mailing saliva samples intended for cortisol assay is acceptable. 68

78 Saliva Testing Research, continued. Dabbs JM. Salivary testosterone measurements: collecting, storing and mailing saliva samples. Phys Behav 1991;49: The authors assessed the effect of saliva storage time and storage temperature, the use of stimulants to collect saliva, as well as the effect of cotton swabs for sample collection in salivary testosterone assays. Cotton swabs were found to contribute an apparent testosterone concentration ranging from 20 to 70 pg/ml. Sugar-containing gums elevated apparent testosterone levels by» 30 pg/ml. Saliva samples could be stored unrefrigerated for up to 1 week with no effect on apparent testosterone level (specimens from women experienced an increase in apparent testosterone concentration if stored for more than a week at room temperature). The effect of storage time and temperature on apparent testosterone concentration was only studied on 2 men and 2 women, however. The authors conclude, Salivary measurements provide a robust indicator of subjects testosterone levels. Dabbs JM. Salivary testosterone measurements: reliability across hours, days and weeks. Phys Behav 1990;48: In this study, correlations between sequential measurements of salivary testosterone in men and women across intervals of hours, 2 days, 2 weeks, 5 weeks and 9 weeks were studied. The expected circadian variation, with highest levels in the a.m. was observed. Not surprisingly, correlations between measurements taken weeks apart were only modest, and worsened as the sampling interval increased. Random salivary testosterone levels were obtained from 126 cycling women and plotted as a function of time since last menstruation. No tendency toward a midcycle peak was observed, leading the authors to conclude that in behavioural studies, saliva samples could be acquired without regard to timing in the menstrual cycle. Dabbs JM, Campbell BC, Gladue BA, Midgley AR, Navarro MA, Read GF, Susman EJ, Swinkels LM, Worthman CM. Reliability of salivary testosterone measurements: a multicenter evaluation. Clin Chem 1995;41: Results of a multicenter evaluation (8 University laboratories, 1 commercial laboratory) of the reliability of salivary testosterone assay are presented in this paper. Each laboratory assayed aliquots of saliva specimens collected from 100 male and 100 female subjects. The intraclass correlation coefficient, which measures overall agreement on individual scores was r=0.87 for men and r=0.78 for women, Mean agreement between each laboratory and the combined set of all other laboratories was r=0.6 (both sexes). The authors concluded that the level of expertise in immunoassay of saliva was gradually increasing. Davies RH, Harris B, Thomas DR, Cook N, Read G, Riad-Fahmy D. Salivary testosterone levels and major depressive illness in men. Br J Psych 1992;161: Salivary testosterone levels were obtained on 11 men admitted to hospital for major depression. Mean levels in depressive were lower than controls, although the difference was not significant due to wide variances. There was significant negative correlation between salivary testosterone and various self-rated scales of anxiety and depression (Montgomery and Asberg, Leeds Anxiety and Depression Scale, Spielberger State Anxiety Inventory). 69

79 Saliva Testing Research, continued. Davis EP, Donzella B, Krueger WK, Gunnar MR. The start of a new school year: individual differences in salivary cortisol response in relation to child temperament. Dev Psychobiol 1999;35: Twice-daily (noon and evening) salivary cortisol levels were measured in 70 elementary school children during the first week of a new school year, in order to study the relationship between temperament and HPA axis functioning. Considering the group as a whole, there was no significant increase in cortisol during the first week of school. Individual changes in cortisol were examined, and there was no evidence that shy/anxious children produced more cortisol during the first week of school. In fact, consistent with studies on preschool children, more exuberant, extroverted children tended to exhibit the largest increases in cortisol. As in other studies of this type, particularly where small changes in cortisol might be seen, a non-invasive sampling method is critical for the proper interpretation of results, and avoids ethical issues of multiple venipunctures in children. Delfs TM, Klein S, Fottrell P, Naether OG, Leidenberger FA, Zimmermann RC. 24-Hour profiles of salivary progesterone. Fertil Steril 1994;62: Three groups of seven women were studied on days 5, 7 and 8 after urinary LH surge. Blood and saliva specimens were obtained at regular intervals for the ensuing 24 hours, and salivary progesterone levels were compared to plasma (total) levels. On average, salivary levels were» 1% of plasma levels. The authors report a correlation of r=0.6 between group mean plasma total progesterone and salivary progesterone, and also report that for a given individual, saliva progesterone levels fluctuate by at least a factor of 3 over 24 hours. They conclude that salivary progesterone is not suitable for diagnosing luteal insufficiency due to the poor correlation between total plasma and salivary progesterone. Duclos M, Corcuff JB, Arsac L, Moreau-Gaudry F, Rashedi M, Roger P, Tabarin A, Manier G. Corticotroph axis sensitivity after exercise in endurance-trained athletes. Clin Endocrin 1998;48: ACTH or CRH-stimulated saliva and plasma (total) cortisol were measured before and after 2 hours of strenuous exercise in 8 trained athletes. Both matrices gave the same basic information, although salivary cortisol responded more dramatically. The authors commented, the greater ability of saliva assays to detect a cortisol increase strongly supports its use to study HPA axis physiology, especially when studies are made during the morning when plasma cortisol levels and the CBG (cortisol binding globulin) buffer capacity are high and might blunt the plasma cortisol response the need to minimize sampling stress favours the use of saliva as opposed to venous samples. Filaire E, Duche P, Lac G, Robert A. Saliva cortisol, physical exercise and training: influences of swimming and handball on cortisol concentrations in women. Eur J Appl Physiol 1996;74: This study examined the effect of physical training in general and the type of exercise in particular, on salivary cortisol levels in elite women athletes. Multiple samples were acquired for cortisol assay over a 24 hour period which included a training workout (swimming or handball). Circadian variation was seen in all groups. Salivary cortisol increased significantly in handball players after exercise, but not in swimmers. These differences were explained with reference to differential rates of water loss during exercise, the aerobic or anaerobic nature of the workout, thermal stress, gravitational factors and body position during exercise, and psychological factors (handball players must continually adapt to rapidly changing conditions, and this may be more stressful). Salivary cortisol levels therefore differed in ways which correlated to various physiologic factors known to affect cortisol secretion in general. 70

80 Saliva Testing Research, continued. Filaire E, Lac G. Dehydroepiandrosterone (DHEA) rather than testosterone shows saliva androgen responses to exercise in elite female handball players. Int J Sports Med 2000;21: Saliva testosterone and DHEA levels were measured in elite female athletes upon arising, as well as before, and after a simulated handball match, and again the following morning. These same measurements were made in a group of sedentary controls. Resting salivary testosterone and DHEA levels were significantly lower in the trained women, in keeping with findings of other researchers studying other steroid hormones. Levels did not change with exercise. Saliva testosterone and DHEA levels were tightly correlated, and the authors conclude that DHEA could serve as a surrogate for testosterone in sports medicine studies of androgen status. Finn MM, Gosling JP, Tallon DF, Baynes S, Meehan FP, Fottrell PF. The frequency of salivary progesterone sampling and the diagnosis of luteal phase insufficiency. Gynecol Endocrinol 1992;6: The study sought to determine the minimum number of salivary progesterone determinations which would adequately describe luteal function and properly diagnose luteal phase insufficiency. Daily saliva samples were collected from 83 women, for 3 consecutive cycles, and analyzed for progesterone. It was determined that a frequency of one sample every three days during the luteal phase (5-6 samples total) was necessary to pick up problems such as a short luteal phase (delayed rise in progesterone) or poor progesterone surge (early termination of progesterone elevation). Gibson EL, Checkley S, Papadopoulos A, Poon L, Daley S, Wardle J. Increased salivary cortisol reliably induced by a protein-rich midday meal. Psychosom Med 1999;61: This study looked at the effect of a protein-rich midday meal on salivary cortisol in 10 men. When the subjects fasted at midday, cortisol levels declined steadily over the 2 hour period. When the subjects consumed a proteinrich meal, cortisol levels were significantly increased by 1 hour after eating (compared to fasting). In a second experiment, the effect of protein composition was studied. Increased protein content of the midday meal significantly increased the post-prandial rise in cortisol, and significantly improved subjective wellbeing. Note that a non-stress-provoking means of assaying cortisol was important for the design and interpretation of this study. Herbert J, Goodyer IM, Altham PM, Pearson J, Secher SM, Shiers HM. Adrenal secretion during major depression in 8- to 16-year-olds, I. Altered diurnal rhythms in salivary cortisol and dehydroepiandrosterone (DHEA) at presentation. Psychol Med 1996;26: Saliva cortisol, DHEA, and DHEAS levels were measured in 88 children (8-16 years old) determined to be suffering from major depression, and 25 children free of psychiatric illness. Serum was collected in parallel to saliva in the control subjects, and significant correlations between serum and saliva were seen for all 3 analytes. Higher evening cortisol levels and lower morning DHEA levels were found in the saliva of depressed children. Note that potential ethical problems related to frequent venipuncture in children were avoided by the use of saliva hormone testing. 71

81 Saliva Testing Research, continued. Granger DA, Schwartz EB, Booth A, Arentz M. Salivary testosterone determination in studies of child health and development. Horm Behav 1999;35: The characteristics of a salivary testosterone assay developed by the authors are described. Correlation of saliva values to serum is performed and r values of 0.96 and 0.78 are obtained for males and females, respectively. Morning levels correlated to pubertal status ratings for both males and females. Consistent with other studies reported by these authors, their assay values are artifactually elevated by the use of cotton or polyester swabs as well as the acidity of the saliva. The results of the assays were not affected by storage at -20 C or colder for up to 1 year. Some of the artifactual results reported are likely due to failure to extract the specimens before analysis. Granger DA, Schwartz EB, Booth A, Curran M, Zakaria D. Assessing dehydroepiandrosterone in saliva: a simple radioimmunoassay for use in studies of children, adolescents and adults. Psychoneuroendocrinology 1999;24: Performance of a radioimmunoassay for DHEA in saliva is reported. The authors note, Levels in matched serum and saliva samples showed strong linear relationships for adult males and females. Reference ranges are provided for children, adolescents and adults. Use of cotton swabs or sugared drinks to aid sample collection resulted in % overestimation of DHEA content. Failure to extract and preconcentrate specimens likely contributed to the susceptibility of this assay to outside factors. Granger DA, Weisz JR, Kauneckis D. Neuroendocrine reactivity, internalizing behavior problems, and control-related cognitions in clinic-referred children and adolescents. J Abn Psychology 1994;103: Salivary cortisol samples were taken from children and adolescents before and after a stressful interaction with a parent. Subjects who experienced the largest increases in cortisol had lower baseline levels, were more anxious and withdrawn, had more social problems and perceived themselves to have less control over their lives. Note that a non-stress-provoking method of cortisol assay was important for this study. Granger DA, Weisz JR, McCracken JT, Ikeda SC, Douglas P. Reciprocal influences among adrenocortical activation, psychosocial processes, and the behavioral adjustment of clinic-referred children. Child Dev 1996;67: Salivary cortisol measurements were made on a group of children before and after conflict-provoking parentchild interactions. The change in cortisol induced by conflict was stable over time and correlated to children s perceptions of the locus of control over their successes and failures. A criticism of this study is the use of cotton swabs and drink crystals to collect and stimulate saliva flow; these sample collection techniques can perturb the results in some saliva assays, particularly those in which the samples are not extracted prior to analysis. 72

82 Saliva Testing Research, continued. Harris B, Lovett L, Newcombe RG, Read GF, Walker R, Riad-Fahmy D. Maternity blues and major endocrine changes:cardiff puerperal mood and hormone study II. BMJ 1994 Apr;308: These authors studied the relationship of salivary progesterone and cortisol to mood in postpartum women. Salivary progesterone levels peaked just before delivery at roughly 1100 pg/ml, falling to 30 pg/ml by 10 days postpartum. Evening salivary cortisol levels peaked at delivery at around 2400 pg/ml, falling to approximately 600 pg/ml by 15 days postpartum. There were modest, statistically significant correlations between self-rated postpartum depressive symptoms, immediate antepartum progesterone levels and also the rate at which salivary progesterone increased in the 2 weeks preceding delivery. Interestingly, no correlation between postpartum blues and total plasma progesterone ( or changes thereof) was seen, demonstrating the utility of saliva as a vehicle to measure clinically significant parameters. Heim C, Ehlert U, Hanker JP, Hellhammer DH. Abuse-related posttraumatic stress disorder and alterations of the hypothalamic-pituitary-adrenal axis in women with chronic pelvic pain. Psychosom Med 1998;60: Women suffering from chronic pelvic pain (CPP) underwent CRF stimulation tests, with parallel measurement of salivary cortisol and serum ACTH levels. They also underwent an overnight dexamethasone suppression test with next-day measurement of salivary cortisol levels at 2 hour intervals over 16 hours. Compared to controls, CPP patients exhibited lower cortisol response to CRF in the face of normal ACTH output, and cortisol output was suppressed to a significantly greater extent by dexamethasone. Note that in this study, a non-invasive, nonstressful assay technique was mandatory for proper interpretation of the findings. Heine RP, McGregor JA, Dullien VK. Accuracy of salivary estriol testing compared to traditional risk factor assessment in predicting preterm birth. Am J Obstet Gynecol 1999;180:S Baseline risk of preterm labor was assessed in 601 pregnant women at weeks gestation via conventional multifactorial risk assessment (Creasy score). Weekly salivary estriol assays were performed until delivery, and were deemed positive if the result exceeded a threshold of 2100 pg/ml. Salivary estriol screening correctly predicted preterm birth in 91% of cases versus 75% with Creasy scoring. In patients deemed high risk at baseline, saliva estriol screening was vastly superior, correctly predicting outcome in 87% of cases versus only 7% using Creasy scoring. Johansson A, Henriksson A, Olofsson BO, Olsson T. Adrenal steroid dysregulation in dystrophia myotonica. J Int Med 1999;245: In this study, salivary cortisol levels were measured in 15 patients with dystrophia myotonica (DyM) (Clinical features of DyM include myotonia, skeletal, cardiac and smooth muscle atrophy, cardiac rhythm disturbances, insulin resistance and hyperinsulinemia, as well as hypogonadism.) Diurnal variation of cortisol, and response to dexamethasone were measured. First morning cortisol in DyM patients was lower than controls, but decline throughout the day was less. Consequently, end of day cortisols were higher in the DyM patients. There was no difference in response to dexamethasone suppression between Dym subjects and controls. Serum hormone levels (androstenedione, DHEAS, 17-hydoxyprogesterone, testosterone) levels were lower in DyM patients. 73

83 Saliva Testing Research, continued. Khan-Dawood FS, Choe JK, Dawood MY. Salivary and plasma bound and free testosterone in men and women. Am J Obstet Gynecol 1984;148: Testosterone was measured in matched saliva and plasma samples in a group of men and women. Correlation coefficient for salivary on plasma testosterone was r=0.71. Diurnal variation of testosterone levels was exhibited. Hourly variation of measured values was on the order of 10-15%. On average, dialyzable testosterone in saliva was approximately 60% of that measured in plasma. The authors conclude, The present investigation showed that salivary testosterone correlates significantly with plasma testosterone and reflects the response of the latter to some of the physiologic variables examined. Kiess W, Meidert A, Dressendorfer RA, Schriever K, Kessler U, Konig A, Schwarz HP, Strasburger CJ. Salivary cortisol levels throughout childhood and adolescence: relation with age, pubertal stage and weight. Pediatr Res 1995;37: Salivary cortisol was correlated to age, pubertal status and weight in this study of 138 infants, children and adolescents. The expected circadian variation was observed in subjects older than 1 year. The lack of such variation observed in subjects less than 1 year old agreed with the findings of other researchers. Salivary cortisol concentrations also correlated with pubertal stage and weight. The authors note that, The measurement of salivary free cortisol levels is a reliable, stress-free, and attractive way of testing adrenal function in infants and children. Kudielka BM, Schmidt-Reinwald AK, Hellhammer DH, Kirschbaum C. Psychological and endocrine responses to psychosocial stress and dexamethasone/corticotropin-releasing hormone in healthy postmenopausal women and young controls: the impact of age and a two-week estradiol treatment. Neuroendocrinol 1999;70: The response of serum and salivary cortisol to a stressful experience, and to a CRH challenge after dexamethasone suppression was measured in estradiol and placebo-treated post-menopausal women, and in untreated younger controls. Estradiol treatment reduced the serum and saliva cortisol response in a 2 hour period subsequent to CRH administration, although the cortisol response was still greater than that registered for younger controls. There was no difference in cortisol response to stress however, among the groups. These results point to a role for estradiol as an important modulator of HPA axis reactivity. Lac G, Lac N, Robert A. Steroid assays in saliva: a method to detect plasmatic contaminations. Arch Int Physiol Biochim Biophys 1993;101: The authors measure concurrent saliva and plasma (total) steroid hormone levels (DHEAS, Testosterone, DHEA, cortisol and androstenedione) and report correlation coefficients of 0.51, 0.77, 0.73, 0.74 and 0.92 respectively. They calculate that between microliters of plasma per ml of whole saliva must be present to perturb salivary hormone results by 20%. (Note: This is a substantial volume of exudate and would not be seen under normal circumstances such as avoidance of toothbrushing before sample acquisition, and absence of periodontal disease.) 74

84 Saliva Testing Research, continued. Lac G, Marquet P, Chassain P, Galen F. Dexamethasone in resting and exercising men. II. Effects on adrenocortical hormones. Matched serum and saliva specimens were obtained from men who performed a cycle ergometer workout after 4 days of dexamethasone suppression or placebo treatment. DHEA, DHEAS, cortisol and androstenedione were measured in both sample types, and testosterone, ACTH and aldosterone were measured in plasma. Dexamethasone prior to exercise suppressed the increase of all steroids except testosterone. In all cases, salivary gave the same information as plasma, and the authors conclude, the consistent results obtained from the 2 matrices allow us to consider salivary assays as a useful tool for steroid abuse detection. Lachelin GC, McGarrigle HH. A comparison of saliva, plasma unconjugated and plasma total oestriol levels throughout normal pregnancy. Brit J Obstet Gyn 1984;91: Parallel plasma and salivary estriol levels were measured weekly in 25 women throughout their second and third trimesters of pregnancy. Levels increased throughout pregnancy and correlation coefficients between results in the two matrices were in excess of 0.9. The percentage of protein binding of unconjugated estriol in plasma was measured by equilibrium dialysis and found to be roughly 12%. This allowed comparison of salivary estriol to free estriol in plasma. Salivary estriol ranged from 85% (early pregnancy) to 120% (late pregnancy) of the plasma free estriol. Hence, salivary estriol levels are very similar, but not identical to free estriol levels in plasma. The authors conclude that, salivary estriol concentrations parallel those of plasma unconjugated estriol throughout pregnancy and could probably replace them in the assessment of fetal wellbeing. Lechner W, Marth C, Daxenbichler G. Correlation of oestriol levels in saliva, plasma and urine of pregnant women. Acta Endocrinol 1985;109: The authors measured conjugated and unconjugated estriol in plasma, urine and saliva in 50 women in their last trimester of pregnancy. They reported significant correlations between unconjugated salivary and total plasma estriol, and between unconjugated plasma and total urinary estriol, but only weak correlation between saliva and urinary estriol. They concluded that no definite conclusion could be drawn regarding the biological significance and clinical importance of salivary estriol. Lipson SF, Ellison PT. Normative study of age variation in salivary progesterone profiles. J Biosoc Sci 1992;24: Luteal phase salivary progesterone levels were measured serially in 124 women ages Results for six groups of 20 women were reported. The expected variation of progesterone level according to menstrual cycle day was observed. Average luteal progesterone was lowest in the year old group (» 60 pg/ml), increased to a maximum of» 80 pg/ml in the year old group, and returned almost to 18 year old levels in the year old group. The authors commented, This study contributes to an increased understanding of human reproductive ecology by demonstrating that luteal progesterone production varies with age, and by showing that the pattern of age variation in ovarian function corresponds closely with the observed pattern of age variation in fecundity. 75

85 Saliva Testing Research, continued. Lipson SF, Ellison PT. Development of protocols for the application of salivary steroid analyses to field conditions. Am J Human Biol 1989;1: Practical parameters of interest for the performance of salivary hormone assays (testosterone, progesterone, androstenedione and cortisol) were examined in this study. Samples were centrifuged and extracted prior to analysis. Samples collected in polystyrene tubes gave the same results as samples collected in glass. Storage at room temperature for 1-6 months did not affect results. Freezing introduced a constant negative offset. Stimulants to saliva flow including lemon juice and sugared gum skewed results, as did coffee or milk, although these effects varied widely between individuals. Lo MS, Ng ML, Azmy BS, Khalid BA. Clinical applications of salivary cortisol measurements. Sing Med J 1992;33:170. The circadian variation of cortisol in plasma (total cortisol) and saliva was measured in 108 normal individuals. The correlation coefficient for cortisol in the two matrices was Comparison was also made between salivary and plasma cortisol levels in normal pregnant females, hyperthyroid patients, hypertensive patients and diabetics. The marked elevations in total plasma cortisol in these instances were not observed with salivary cortisol. The authors conclude that salivary cortisol is a better measure of adrenal status than plasma. Lu Y, Bentley GR, Gann PH, Hodges KR, Chatterton RT. Salivary estradiol and progesterone levels in conception and nonconception cycles in women: evaluation of a new assay for salivary estradiol. Fertil Steril 1999; 71: This study attempted to replicate earlier work by Lipson and Ellison. Daily salivary estradiol and progesterone levels were measured in consecutive menstrual cycles of 11 women, until conception occurred. The authors report that mean estradiol level in the preovulatory period in conception cycles (averaged across the group and over an unspecified number of days) was not statistically different (p=0.16) from non-conception cycles. There appeared to be a clear difference between groups when the data were graphed. Paired analysis (more appropriate for small n) was not performed on the preovulatory data, although such analysis on post-ovulatory data did reveal a difference between conception and nonconception cycles. Overall, the conclusion reached by the authors appears to be inconsistent with the data they present. Correlations between serum and saliva estradiol levels were reported in 7 other women. Unfortunately, serum samples were acquired in the morning, and saliva was acquired in the evening. Not surprisingly, individual correlation coefficients varied widely. Lu YC, Chatterton RT, Vogelsong KM, May LK. Direct radioimmunoassay of progesterone in saliva. J Immunoassay 1997;18: This study looked at various aspects of a salivary progesterone assay. The influence of the composition of the specimen collection tubes, and the composition of the analytical standards on final results was studied. Progesterone was found to adsorb on to the walls of some types of collection tubes, and standards made up in stripped saliva gave more accurate results than standards made up in a buffer. Daily salivary progesterone levels were measured in 10 women throughout the menstrual cycle, and the expected luteal phase increase was seen. Finally, matched serum and saliva specimens were collected from 48 women in the luteal phase of their cycle, and the correlation coefficient between the two sample types was found to be r=

86 Saliva Testing Research, continued. Martel FL, Hayward C, Lyons DM, Sanborn K, Varady S, Schatzberg AF. Salivary cortisol levels in socially phobic adolescent girls. Depression Anxiety 1999;10: Saliva samples were collected from 27 adolescent girls diagnosed with social phobia (SP) and 21 controls, before and after a standardized anxiety-provoking situation (Trier Social Stress Test or TSST). There was no difference between SP subjects and controls in baseline cortisol levels, or in the circadian variation of these levels. No difference was seen between the groups either before or after the TSST, and this is consistent with studies of adults with SP. Cortisol levels for both groups were higher in anticipation of the TSST. The authors comment that, salivary cortisol levels appear to be a sensitive measure of anticipatory anxiety. McGregor JA, Hastings C, Roberts T, Barrett J. Diurnal variation in saliva estriol level during pregnancy: a pilot study. Am J Obstet Gynecol 1999; 180:S The circadian variation of estriol level in saliva was studied in 14 pregnant women. A marked nocturnal surge in estriol occurred, with a peak between 2 and 4 a.m. These findings agree with other human and animal research on plasma estriol levels. The study demonstrates that salivary estriol correlates to plasma estriol. The authors conclude that if salivary estriol is measured for pregnancy screening purposes, samples should be acquired during daylight hours to avoid false positives. McGregor JA, Jackson GM, Lachelin GC, Goodwin TM, Artal R, Hastings C, Dullien V. Salivary estriol as risk assessment for preterm labor: a prospective trial. Am J Obstet Gynecol 1995;173: Serial salivary estriol monitoring in pregnancy was shown to be a better predictor of pre term labor and delivery compared to conventional risk assessment based on history. When a threshold of 2300 pg/ml was used, salivary estriol correctly predicted the occurrence of pre term labor in 77% of cases versus 37% for clinical risk score. The ability of salivary estriol to predict pre term delivery was lower (50%) but still better than risk score prediction (33%). The authors conclude, Serial collection and measurement of salivary estriol was easy to perform and relatively effective as a means to identify women at heightened risk of pre term labor and delivery. Metcalf MG, Evans JJ, Mackenzie JA. Indices of ovulation: comparison of plasma and salivary levels of progesterone with urinary pregnanediol. J Endocr 1984;100: This is an early study comparing salivary progesterone with plasma progesterone and with 24 hour urinary excretion of pregnanediol in the follicular and luteal phases of the menstrual cycles of 20 women. (One sample was collected in each half of the cycle, per woman.) Saliva progesterone levels in the luteal phase were seen to increase by a factor of 3, less than the increases seen for plasma progesterone and 24 hour urinary pregnanediol. The authors question the integrity of their assay however, and note that falsely elevated progesterone results tended to occur in samples acquired in the a.m. This suggests their antibody might have been crossreacting with other hormones including cortisol. Nevertheless, they concluded that salivary progesterone was not as reliable an indicator of the occurrence of ovulation compared to 24 hour urinary pregnanediol or plasma progesterone. 77

87 Saliva Testing Research, continued. Meulenberg PM, Hofman JA. Salivary progesterone excellently reflects free and total progesterone in plasma during pregnancy. Clin Chem 1989;35: Salivary progesterone was compared to free plasma progesterone (via equilibrium dialysis) and total serum progesterone in 36 pregnant women, sampled four times throughout pregnancy. Overall, salivary progesterone was well correlated to both free (r=0.88) and total serum progesterone (r+0.9), although salivary progesterone was on average» 50% of free progesterone in serum. The authors felt that centrifugation of saliva prior to analysis may have sequestered a fraction of the progesterone in saliva. Both free serum progesterone and salivary progesterone represented» 0.5-1% of total serum progesterone throughout pregnancy. In the postpartum period, binding of progesterone in serum decreased markedly (% free progesterone increased to 12% from» 1%), and overall, levels of progesterone fell drastically. The authors conclude, Despite the dramatic increase in concentrations of total progesterone and binding proteins in plasma during pregnancy, we found highly significant correlations between total and free progesterone in plasma and salivary progesterone in the group as a whole as well as individuals. Moran DJ, McGarrigle HH, Lachelin GC. Lack of normal increase in saliva estriol/progesterone ratio in women with labor induced at 42 weeks gestation. Am J Obstet Gynecol 1992;167: Salivary estriol and progesterone levels were measured in 16 pregnant women from gestational day 280 to day 294. The ratio of estriol to progesterone increased steadily (due to increasing estriol) over the 14 days in 6 women who went into labour spontaneously. The ratio did not change in 10 women whose labour was induced at 294 days. These findings are in agreement with other studies regarding the change in hormone levels and the initiation of labour. Moss HB, Vanyukov MM, Martin CS. Salivary cortisol responses and the risk for substance abuse in prepubertal boys. Biol Psych 1995;38: Salivary cortisol in prepubertal boys anticipating a stressful task was measured. Sons of fathers who used drugs or exhibited antisocial behavior had lower levels of cortisol while anticipating the stressor, compared to controls, despite similar professed levels of anxiety in each group. Note that the results of this study would likely have been skewed significantly if serum cortisol measurements had been made: anticipation of acquisition of the blood sample would likely have equaled or superseded anticipation of the stressful task. Hence saliva sampling was a critical aspect of this study. Nahoul K, Rao LV, Scholler R. Saliva testosterone time-course response to hcg in adult normal men, comparison with plasma levels. J Steroid Biochem 1986;24: Testosterone levels were measured in blood and saliva specimens collected for 96 hours in 13 men after injection of hcg. Salivary testosterone, on average, was approximately 2% of serum testosterone. Testosterone levels in both fluids traced out the same response curves over time, and were significantly correlated (r=0.65). The authors suggest that saliva could be employed instead of blood to perform this standard assay of testicular function. 78

88 Saliva Testing Research, continued. Nahoul K, Scholler R. Comparison of saliva and plasma 17-hydroxyprogesterone time-course response to hcg administration in normal men. J Steroid Biochem 1987;26: The response of salivary and plasma 17-hydroxyprogesterone to an injection of hcg was measured in 12 men, and very similar time behavior was noted in both cases. The hormone level in saliva was approximately 2.5% of that measured in plasma. Navarro MA, Nolla JM, Machuca MI, Gonzalez A, Mateo L, Bonnin RM, Roig-Escofet D. Salivary testosterone in postmenopausal women with rheumatoid arthritis. J Rheumatol 1998;25: Salivary testosterone, as well as serum free and total testosterone and SHBG levels were measured in 44 postmenopausal women with rheumatoid arthritis (RA), and compared to levels in controls. The correlation coefficient between saliva and serum free testosterone was There was no difference in levels between controls and RA patients not treated with glucocorticoids. In glucocorticoid-treated RA patients, salivary testosterone, total testosterone and SHBG were significantly lowered. Navarro MA, Villabona CM, Blanco A, Gomez JM, Bonnin RM, Soler J. Salivary excretory pattern of testosterone in substitutive therapy with testosterone enanthate. Fertil Steril 1994;61: Serial saliva and serum testosterone levels were measured in 16 men after a single intramuscular injection of testosterone enanthate. Testosterone levels in both fluids followed a similar time course, with an initial peak or plateau, then a gradual decline. Levels remained above the low end of the eugonadal range for» 2 weeks. The authors conclude that, Salivary testosterone concentration may be applied to assess the effectiveness of testosterone substitutive therapy. The simplicity of the salivary sampling technique means that it could replace serum in the monitoring of this type of therapy. O Leary P, Feddema 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. Clin Endocrinol 2000;53: This study measures the response of serum, salivary and urine progesterone and progesterone metabolites in premenopausal (n=6) and postmenopausal (n=6) women after a single application of a cream containing 64 mg of micronized progesterone. Serum levels rose marginally after cream application. Urine progesterone metabolites did not change. Saliva progesterone levels increase substantially within the first 3 hours of application. There was an approximately 7-fold greater increase in salivary progesterone levels in premenopausal women compared to postmenopausal women. On this basis, the authors conclude that no useful relationship between saliva progesterone levels and transdermal progesterone dose exists. This disparity between pre and postmenopausal transdermal progesterone levels has not been seen at ZRT Laboratory. Measurements taken after several cycles of progesterone use (2-3 weeks per cycle) might have been more relevant, giving postmenopausal women time to adapt to higher progesterone exposure. 79

89 Saliva Testing Research, continued. O Rourke MT, Ellison PT. Salivary estradiol levels decrease with age in healthy, regularly-cycling women. End J 1993;1: Daily salivary estradiol and progesterone levels were measured over one full menstrual cycle in 53 women ranging in age from 25 to 48 years. The average saliva estradiol profiles corresponded closely to published serum and plasma estradiol profiles. Average salivary follicular and luteal estradiol levels declined with age, although the mid-cycle peak level did not. These findings are consistent with the literature on fertility and aging. Petsos P, Ratcliffe WA, Heath DF, Anderson DC. Comparison of blood spot, salivary and serum progesterone assays in the normal menstrual cycle. Clin Endocrin 1986;24: This 1986 study compares serum, blood spot and salivary progesterone levels in 6 patients. Salivary progesterone displays the expected luteal phase increase and tracks serum progesterone well. Salivary levels correlated well (r=0.89) with serum. Despite the low sensitivity (detection limit» 3000 pg/ml) and high CV (12-14%), this study demonstrates the congruence of saliva and serum progesterone measurements. Pruessner JC, Hellhammer DH, Kirschbaum C. Burnout, perceived stress, and cortisol responses to awakening. Psychosom Med 1999;61: The salivary cortisol response to awakening was studied in 66 teachers, to explore the relationship of stress, HPA axis function and burnout. On 3 consecutive days, subjects submitted saliva specimens on awakening, and 15, 30 and 60 minutes thereafter. All subjects took 0.5 mg of dexamethasone the night before acquisition of the third set of saliva specimens. These data were correlated to data gathered from questionnaires pertaining to psychological state. On days 1 and 2, saliva cortisol peaked minutes after waking, then declined. On day 3, salivary cortisols increased throughout the hour, rather than peaking. Subjects who reported higher levels of stress and burnout had significantly lower cortisol secretion on all 3 days. Lower cortisol output correlated well with feelings of exhaustion, detachment from work, and a loss of drive, increased somatic complaints and low self esteem. Note that a non-stress-inducing method of cortisol assay was crucial to the design of this study. Quissell D. Steroid hormone analysis in human saliva. Ann N Y Acad Sci. 1993;694: In this very brief overview of salivary steroid hormone analysis, the author mentions several possible pitfalls to analysis including: metabolization of hormones by cells of the salivary gland or oral flora, contamination of saliva by plasma from traumatized oral tissue or gingival crevicular fluid. Nevertheless, the author concludes, Saliva collection has provided the medical and research community with an excellent medium for the monitoring of plasma steroid levels. Raff H, Raff JL, Findling JW. Late-night salivary cortisol as a screening test for Cushing s Syndrome. J Clin Endocrinol Metab 1998;83: Salivary cortisol levels were studied in 73 normal individuals and 78 patients suspected of having Cushing s Syndrome. Patients with spontaneous Cushing s Syndrome were found to have a markedly elevated late-night salivary cortisol, and could be identified with a sensitivity of 97%. The authors conclude, the measurement of late-night salivary cortisol is a simple, convenient, and reliable way to screen patients for Cushing s Syndrome. 80

90 Saliva Testing Research, continued. Read GF. Report on measurement of salivary estrogens & androgens. Ann NY Acad Sci 1993: This paper reviews the status of salivary hormone testing. Some of the tentative conclusions have since been validated. This paper is primarily of historical interest. Read GF, Harper ME, Peeling WB, Griffiths K. Changes in male salivary testosterone concentrations with age. Int J Androl 1981;4: Salivary testosterone levels were measured in males ranging in age from 20 to 80 years. Mean salivary testosterone decreased gradually with age, falling by a factor of approximately 2.4 between age 25 and 75. These findings agree with the age related decline also measured in plasma free testosterone. Read G, Fahmy D, Wilson D, Griffiths K. A new approach for breast cancer research, assays for steroids in saliva. This paper outlines studies validating some of the basic analytical characteristics of salivary steroid hormone assays. Levels of 17-hydroxyprogesterone and cortisol in mixed saliva correlated well (r= 0.98) with fluid collected directly from the parotid duct outlet. Serial simultaneous measurements of 17-hydroxyprogesterone in plasma, saliva and parotid fluid correlated well and displayed circadian variation. Levels of 17-hydroxyprogesterone and cortisol were equal in unstimulated and stimulated saliva samples. Plasma and salivary progesterone levels correlated well over a menstrual cycle, with salivary progesterone exhibiting the expected luteal phase increase. Women with suspected luteal phase deficiency were seen to have low or erratic levels of salivary progesterone. Read GF, Walker RF, Wilson DW, Griffiths K. Steroid analysis in saliva for the assessment of endocrine function. Ann NY Acad Sci 1993: A number of facets of salivary steroid hormone analysis are discussed in this paper. Salivary hormone levels (progesterone, 17-hydroxyprogesterone, cortisol and estriol) were shown to be independent of rate of saliva production. Saliva responded rapidly to change in serum levels; following IV administration of cortisol to dexamethasone-suppressed subjects, salivary cortisol levels peaked within 3 minutes of injection (serum levels peaked within 1 minute). The utility of salivary 17-hydroxyprogesterone levels for monitoring children with Congenital Adrenal Hyperplasia was discussed, as was the use of salivary progesterone for studying induction of ovulation in infertility. An excellent correlation between free plasma estriol and salivary estriol (r=0.95) was presented, and the role of salivary estriol for pregnancy monitoring discussed. Data on the correlation of salivary testosterone and progesterone to stage of sexual maturity were also presented. Read GF, Wilson DW, Campbell FC, Holliday HW, Blamey RW, Griffiths K. Salivary cortisol and dehydroepiandrosterone sulphate levels in postmenopausal women with primary breast cancer. Eur J Cancer Clin Oncol 1983;19: Serial salivary DHEAS and cortisol measurements were taken for 2 consecutive days in women with breast cancer, and age-matched controls. Circadian variation, with an a.m. peak was seen in both groups of women, for both hormones. Women with cancer appeared to have marginally elevated cortisol and marginally lower DHEAS levels compared to controls, but due to within-group variance, the differences were not significant. The authors comment on the advantage of using saliva specimens when large numbers of samples have to be acquired. 81

91 Saliva Testing Research, continued. Riad-Fahmy D, Read GF, Walker RF. Salivary steroid assays for assessing variation in endocrine activity. J Steroid Biochem 1983;19: The correlation between plasma and saliva hormone levels in a variety of situations are presented in this paper. Plasma and salivary cortisol levels after an injection of ACTH in a normal subject, measured at 15 minute intervals were well-correlated. Cortisol measurements in saliva and plasma were also acquired every 15 minutes over a 12 hour period in 3 individuals, and again the values were very well correlated. Other data validating salivary hormone assay are also presented, including serial measurements of salivary testosterone levels illustrating the expected circadian variation over 12 hours, and serial salivary progesterone and estradiol measurements over the course of menstrual cycles, once again demonstrating expected variation. The authors note, Studies like these illustrate the usefulness of salivary sampling regimens for monitoring short term changes in secretory activity. Since these samples unlike plasma, are easily collected by stress-free, non-invasive techniques, volunteers are more readily recruited, and undue disturbance of endocrine systems is avoided. Rosmond R, Bjorntorp P. The hypothalamic-pituitary-adrenal axis activity as a predictor or cardiovascular disease, type 2 diabetes and stroke. J Int Med 2000;247: Circadian variation in salivary cortisol, and salivary cortisol response to dexamethasone were measured in a group of men, along with blood lipids, insulin, blood pressure, body mass index, and waist-hip ratio. Preliminary work with the cortisol assay indicated excellent correlation (r=0.92) between plasma and saliva cortisol levels. The study demonstrated that HPA axis dysfunction (typified by low a.m. cortisol, low circadian variability, blunted suppression of cortisol by dexamethasone, and poor postprandial cortisol rise) was associated with a tight clustering of other established risk factors for diabetes, coronary artery disease and stroke. Ruutiainen K, Sannikka E, Santti R, Erkkola R, Adlercreutz H. Salivary testosterone in hirsutism:correlations with serum testosterone and the degree of hair growth. J Clin Endocrinol Metab 1987;64: Testosterone was measured in saliva and serum samples in 53 females attending a hirsutism clinic. Testosterone in saliva was significantly correlated ( r=0.41) to facial hirsutism whereas serum free testosterone was not correlated. The authors conclude, On the basis of the results, salivary testosterone seems to relate to the bioavailable fraction of the hormone and thus appears to be an optimal method for studying hirsute females. Sannikka E, Terho P, Suominen J, Santti R. Testosterone concentrations in human seminal plasma and saliva and its correlation with non-protein-bound and total testosterone levels in serum. Int J Andrology 1983;6: This is an early paper on salivary hormone analysis. Salivary testosterone in males was shown to be independent of saliva flowrate (correlation between testosterone concentration in stimulated and unstimulated saliva samples was 0.96.), and whole saliva values correlated well (r=0.86) with saliva collected directly from the parotid duct. The correlation coefficient for free serum testosterone on salivary testosterone was The salivary testosterone level correlated well with Tanner stage in adolescent boys. The authors conclude, Determination of testosterone in saliva could thus provide a convenient and accurate index of the non-protein-bound concentration of testosterone in serum i.e. the availability of hormone to tissues. 82

92 Saliva Testing Research, continued. Scheer FA, Buijs RM. Light affects morning salivary cortisol in humans. J Clin Endocrinol 1999;84: This study examined the effect of light on early morning salivary cortisol levels. In 14 males, levels were measured every 20 minutes for 1 hour after awakening, under 2 sets of conditions: 0 lux (for which subjects wore a dark cap over their eyes) and 800 lux (for which subjects wore an illuminated visor). Cortisol levels were significantly higher in the light exposed subjects, at least for the first 40 minutes after waking. Sixteen hours after waking,, the presence or absence of light did not influence salivary cortisol. These findings agree with the results of animal experiments. The authors chose saliva as a measurement vehicle because it was deemed to be, a convenient and reliable measure for the unbound, and therefore active, plasma cortisol levels. Schramm W, Smith RH, Craig PA, Grates HE. Testosterone concentration is increased in whole saliva but not in ultrafiltrate after toothbrushing. Clin Chem 1993;39: Testosterone levels were measured in whole saliva and an ultrafiltrate collected via an osmotically-pumped collection device before and after toothbrushing, in a group of males. On average, the concentration of testosterone in whole saliva was increased 14% by toothbrushing, compared to 5% for the ultrafiltrate, and these differences were statistically significant despite wide individual variation. It is evident that toothbrushing releases additional protein-bound testosterone into saliva in some individuals; saliva samples should be collected before toothbrushing to ensure highest possible accuracy. Schramm W, Smith RH, Craig PA, Paek SH, Kuo HH. Determination of free progesterojne in an ultrafiltrate of saliva collected in situ. Clin Chem 1990;36: This paper details the use of a saliva collection device consisting of a semipermeable membrane enclosing an osmotically-active material (sucrose). An ultrafiltrate of saliva is drawn into the collector over the course of 15 minutes. Progesterone levels in whole saliva correlated well with that in the ultrafiltrate (r=0.95), although progesterone in ultrafiltrate was»84% of that in whole saliva. The progesterone level in the ultrafiltrate also correlated well (r=0.86) to serum progesterone in 2 women over the course of two menstrual cycles. The authors felt that analysis of ultrafiltrate obviated the need for centrifugation and extraction of specimens prior to analysis. Schurmeyer T, Nieschlag E. Effect of ketoconazole and other imidazole fungicides on testosterone biosynthesis. Acta Endocrinol 1984;105: Serial serum and saliva testosterone levels in 5 men were measured for 24 hours after ingestion of ketoconazole. Testosterone levels in serum and saliva began to decline significantly within 1 hour, reached a minimum between 4 and 6 hours later, and had returned to pre-ingestion levels at 24 hours. The effect was determined to be exerted at the level of testosterone synthesis by Leydig cells, rather than through an effect on gonadotrophins. The study indicates that saliva testosterone was reflective of changes in serum testosterone in this unusual setting. 83

93 Saliva Testing Research, continued. Schwartz EB, Granger DA, Susman EJ, Gunnar MR, Laird B. Assessing salivary cortisol in studies of child development. Child Dev 1998;69: This study assessed the effect of a salivary flow stimulant (powdered drink crystals) on salivary cortisol levels. Cortisol levels in unstimulated saliva samples spiked with drink crystals after collection (to simulate concentrations achieved in saliva with field use of the stimulant) resulted in stimulant concentration-dependent overestimation of cortisol levels. Saliva specimens obtained under field conditions (drink crystals and cotton swab) returned cortisol levels which were significantly higher than expected. This study illustrates the pitfalls of salivary hormone assays which do not employ extraction methods. Shirtcliff EA, Granger DA, Schwartz EB, Curran MJ, Booth A, Overman WH. Assessing estradiol in biobehavioral studies using saliva and blood spots: simple radioimmunoassay protocols, reliability, and comparative validity. Horm Behav 2000;38: The authors describe a method for RIA of estradiol in saliva and blood spots, and compare both to serum. Salivary values correlated to serum values with r=0.68 for females and for males there was no correlation due to poor detection limits. Average salivary levels for males and females were 0.5 pg/ml and 1.0 pg/ml respectively (significantly lower than the normal ranges at ZRT Laboratory). Use of cotton swabs or chewing gum resulted in falsely elevated readings. Serial assays throughout the menstrual cycle revealed the expected fluctuations in estradiol levels. The low sensitivity and susceptibility to error of the asay is due to the small sample volume (300 microliters) and failure to extract or preconcentrate the specimens. In general however, the results validate the use of saliva for estradiol measurement. Shirtcliff EA, Granger DA, Schwartz E, Curran MJ. Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results. Psychoneuroendocrinol 2001;26: This paper outlines the performance of a commercially available salivary hormone assay (Salimetrics). Results for DHEA were seen to be artificially elevated when samples were collected via cotton swabs or plugs. Saliva DHEA levels correlated well to serum (r=0.73) when no cotton was used, but the correlation was lost when cotton was used, due to the spurious elevation. This study demonstrates one of the potential problems introduced when saliva is analyzed directly, without extraction. Steptoe A, Cropley M, Griffith J, Kirschbaum C. Job strain and anger expression predict early morning elevation in salivary cortisol. Psychosom Med 2000;62: Salivary cortisol levels at intervals throughout the day were measured in teachers and correlated to perception of job stress level and patterns of anger expression. Individuals experiencing high levels of job strain and exhibiting high levels of expressed anger had almost 22% higher salivary cortisol in the early part of the working day. Saliva sampling facilitated the acquisition of multiple samples with little or no disruption of work routines and minimal additional stress. 84

94 Saliva Testing Research, continued. Stones A, Groome D, Perry D, Hucklebridge F, Evans P. The effect of stress on salivary cortisol in panic disorder patients. J Affect Disorders 1999;52: Salivary cortisol measurements were used to assess the response to stress in patients suffering with panic disorders, and controls. Compared to control subjects, salivary cortisol levels before a stressful situation were lower, and did not change, indicating a difference in HPA axis regulation in these patients. (In controls, levels were higher before stress, and dropped afterward.). The authors note that a non-stressful sampling technique was mandatory in this work, to avoid perturbation of the findings. Sumiala S, Tuominen J, Huhtaniemi I, Maenpaa J. Salivary progesterone concentrations after tubal sterilization. Obstet Gynecol 1996;88: This study looked at the effect of laparoscopic tubal ligation on salivary progesterone levels in 55 women. Daily salivary progesterone levels were measured in 3 cycles: the cycle immediately before surgery, and cycles 3 and 12 months after the procedure. Cumulative mean progesterone and peak luteal progesterone were both decreased at 3 and 12 months, indicating that tubal ligation, even by minimally invasive means, has a significant effect on ovarian function. A longer follow-up period would be necessary to determine if this effect is reversible. Swinkels LM, Ross HA, Smals AG, Benraad TJ. Concentrations of total and free dehydroepiandrosterone in plasma and dehydroepiandrosterone in saliva of normal and hirsute women under basal conditions and during administration of dexamethasone/synthetic corticotropin. Clin Chem 1990;16: Salivary and plasma DHEA levels (free and total plasma DHEA) were measured in 22 normally cycling women. Correlation coefficients for salivary vs total and free DHEA in plasma averaged r=0.9 in both luteal and follicular phases. There was near-quantitative agreement between mean plasma free DHEA and salivary DHEA. The authors also made these same comparisons in dexamethasone-suppressed women with hirsutism given CRH, and in general, found the same good correlations. They concluded, The concentration of DHEA in saliva is a reliable index of adrenal DHEA secretion, allowing a stress-free multiple sampling regimen for the study of time-related hormonal events. Thijssen JH, Gispen-de Wied CC, van Heeswijk GM, Veeman W. Determination of dexamethasone in saliva. Clin Chem 1996;42: Assay of dexamethasone in saliva after oral ingestion was reported in this paper. Serum and saliva dexamethasone concentrations correlated extremely well (r=0.99). Moreover, free dexamethasone is known to be» 18% of total serum dexamethasone, and salivary dexamethasone was» 17% of total serum dexamethasone. Therefore salivary dexamethasone appears to quantitatively represent unbound dexamethasone in plasma. 85

95 Saliva Testing Research, continued. Tschop M, Behre HM, Nieschlag E, Dressendorfer RA, Strasburger CJ. A time-resolved fluorescence immunoassay for the measurement of testosterone in saliva: monitoring of testosterone replacement therapy with testosterone buciclate. Clin Chem Lab Med 1998;36: In this paper, the treatment of male hypogonadism via testosterone replacement was monitored via salivary measurements. Parallel serum and saliva samples were taken at increasing intervals after a single injection of testosterone buciclate, for a total of 16 weeks. The concentration versus time profiles for serum androgens (total testosterone + total DHT) and saliva testosterone were very similar. A weak, but significant correlation between total serum androgens and salivary testosterone (r=0.22) was seen, but no details on the timing of acquisition of saliva and blood samples were provided. Samples acquired at different times of the day would not be expected to correlate for a hormone such as testosterone for which significant circadian variation is seen. The authors conclude, The time resolved fluorescence immunoassay for salivary testosterone provides a useful tool for monitoring androgen status in men and women, and is well suited for the follow up of testosterone replacement therapy on an outpatient basis. Tulppala M, Bjorses UM, Stenman UH, Wahlstrom T, Ylikorkala O. Luteal phase defect in habitual abortion: progesterone in saliva. Fertil Steril 1991;56: Daily salivary progesterone levels were measured in consecutive menstrual cycles in women with a history of multiple miscarriages, and controls. Serum progesterone levels were also measured along with endometrial biopsies and LH measurements to establish accurate timing within the cycles. The saliva progesterone levels exhibited an ovulatory pattern in all cases, but there was no difference in salivary progesterone between the study groups, and the same was true for serum progesterone. Salivary progesterone measurements are not helpful in the study of women with frequent miscarriages. Tunn S, Mollmann H, Barth J, Derendorf H, Krieg M. Simultaneous measurement of cortisol in serum and saliva after different forms of cortisol administration. Clin Chem 1992;38: Cortisone was administered to 8 dexamethasone-suppressed males by intravenous, oral and rectal routes, and the resulting serum and saliva cortisol levels were measured over the ensuing 8 hours. (In serum, free and bound cortisol levels were measured.) These levels followed a nearly identical time course over the whole 8 hours for both IV and oral administration. A saturation effect was seen in which saliva levels increased rapidly once a threshold serum concentration was exceeded. This supports the contention that salivary cortisol reflects the non-protein-bound cortisol in serum. (Once all available cortisol binding sites on cortisol binding globulin and albumin are occupied, the concentration of free cortisol in serum will increase rapidly.) Also, the close agreement in time between behaviour between serum and saliva indicates that delivery of hormone to saliva is bloodbased, rather than lymphatic-based. Vedhara K, Hyde J, Gilchrist ID, Tytherleigh M, Plummer S. Acute stress, memory, attention and cortisol. Psychoneuroendocrinol 2000;25: Salivary cortisol levels were measured in university students in two 48 hour periods before, and during examinations. Cortisol levels were lower during the presumably more stressful exam period. The authors commented confounding effects of venipuncture stress were avoided by the use of saliva as a measurement vehicle. 86

96 Saliva Testing Research, continued. Vining RF, McGinley RA, Symons RG. Hormones in saliva: mode of entry and consequent implications for clinical interpretation. Clin Chem 1983;29: This is a landmark paper for salivary hormone analysis in which measurements of non-protein bound, unconjugated estriol and DHEA in serum are compared to salivary estriol and salivary DHEA. Direct numerical agreement between the two matrices indicates that salivary hormone levels represent the non-protein bound, unconjugated, or bioavailable hormone. Consideration of the anatomy of the salivary gland, along with solubility considerations, and comparison of the concentration of conjugated hormone in saliva and serum allow the following conclusions to be drawn: Unconjugated steroids make their way into saliva by partitioning between the membranes of the acinar cells and saliva. Unconjugated steroids are largely excluded from saliva since they primarily gain entry by passage through acinar cell tight junctions. Because the concentration of conjugated estriol in serum is roughly ten times that of unconjugated estriol however, the amounts of conjugated and unconjugated estriol in saliva are roughly equal despite their different mechanisms of transport into saliva. Vining RF, McGinley RA. The measurement of hormones in saliva: possibilities and pitfalls. J Steroid Biochem. 1987;27(1-3): This paper is an excellent review of the fundamental principles of salivary hormone analysis (saliva formation, transport of hormones into saliva, effect of saliva flow rate) and well summarizes the body of knowledge circa 1986 regarding the clinical applicability of saliva hormone assays. Various tentative conclusions (such as the utility of salivary testosterone and estradiol measurements) have been confirmed. The authors also enumerate a number of practical and scientific advantages of saliva analysis: minimal stress on the HPA axis, good representation of free plasma hormone levels, avoidance of ethical problems in studies of children, convenience and low cost, ease of acquisition of multiple samples. Vittek J, L Hommedieu D, Gordon G, Rappaport S, Southren L. Direct radioimmunoassay (RIA) of salivary testosterone: correlation with free and total serum testosterone. Life Sciences 1985;37: This is an earlier paper on salivary hormone testing which compares salivary testosterone levels with free and total serum testosterone in men and women. Testosterone concentration in submandibular and mixed (submandibular and parotid) saliva were quantitatively equal (r=0.99). Free serum testosterone and salivary testosterone were also quantitatively equal (r=0.97). Salivary testosterone and total testosterone were well correlated (r=0.8) and salivary testosterone was» 2% of total serum testosterone. The authors conclude that, determination of salivary testosterone is a reliable method to detect changes in the concentration of available biologically active hormone. Voss HF. Saliva as a fluid for measurement of estradiol levels. Am J Obstet Gynecol 1999;180:S This paper is a good general overview of the underlying physiology of saliva as it relates to steroid hormone analysis. It describes the characteristics of a commercially available salivary estriol assay and discusses the utility of salivary estriol for pregnancy monitoring. 87

97 Saliva Testing Research, continued. Vuorento T, Lahti A, Hovatta O, Huhtaniemi I. Daily measurements of salivary progesterone reveal a high rate of anovulation in healthy students. Scand J Clin Lab Invest 1989;49: Daily salivary progesterone levels were measured on 17 healthy, regularly menstruating university students, and compared to profiles obtained from 15 other women for whom the occurrence of ovulation was verified by ultrasound, LH surge and endometrial biopsy. On two occasions (one in the follicular phase and one in the luteal phase) parallel serum progesterone measurements were made. Serum and saliva measurements correlated well (r=0.93) with saliva progesterone roughly 1% of serum progesterone. Salivary progesterone profiles demonstrated the expected luteal phase peak. Interestingly, 47% (6/15) of the students did not ovulate as judged by the absence of a luteal phase progesterone rise. The authors conclude, Normal (indicating ovulation) and pathological (luteal dysfunction or no ovulation) progesterone profiles can be reliably discriminated. Sample collection is easy and makes ling-term monitoring of corpus luteum function a feasible means of diagnosis and follow up of treatment in infertility patients. Walker RF, Read GF, Wilson DW, Riad-Fahmy D, Griffiths K. Chronobiology in laboratory medicine: principles and clinical applications illustrated from measurements of neutral steroids in saliva. Chronobiology: its role in clinical medicine, general biology, and agriculture, Part A, pages The authors present salivary cortisol and testosterone data from subjects ranging in age from newborn to adolescence. These data illustrate the expected circadian variation demonstrated in serum and plasma studies, as well as the variation of salivary testosterone with stage of genital maturity, and confirm that salivary hormone measurements correlate well to physiology. Wang DY, Fantl VE, Habibollahi F, Clark GM, Fentiman IS, Hayward JL, Bulbrook RD. Salivary oestradiol and progesterone levels in premenopausal women with breast cancer. Eur J Cancer Clin Oncol Apr;22(4): Daily saliva specimens were submitted for estradiol and progesterone analysis by 24 women for one complete menstrual cycle. Twelve of the women had a past history of breast cancer, but were disease free at the time of study, and twelve were controls. Parallel saliva and serum specimens were also obtained on 19 women with no history of cancer. In these 19 women, salivary estradiol levels correlated well to free serum estradiol levels (r=0.78). Serum free estradiol was determined by centrifugal ultrafiltration dialysis. Salivary levels were on average,» 30% of free plasma values. Salivary estradiol also correlated well (r=0.79) to total serum estradiol. When profiles of estradiol and progesterone between breast cancer survivors and unaffected controls were compared, no differences were seen. Wang DY, Knyba RE. Salivary progesterone: relation to total and non-protein-bound blood levels. J Steroid Biochem 1985;23: Daily saliva samples were taken from 9 women throughout an entire menstrual cycle, and the expected luteal phase increase was observed. Paired random blood and saliva samples were also analyzed and a correlation coefficient of r=0.78 was derived between salivary progesterone and total serum progesterone. The authors also measured the percent binding of progesterone in serum to be roughly 3%. From the total serum progesterone values, they computed free plasma progesterone values and note that these computed values are approximate ly 5 times greater than the salivary levels. They conclude that salivary progesterone does not represent unbound progesterone in blood. However, their saliva levels are significantly lower than other reported values, including those reported by contemporary assay labs including ZRT. 88

98 Saliva Testing Research, continued. Wellen JJ, Smals AG, Rijken JC, Kloppenborg PW, Benraad TJ. Testosterone and androstenedione in the saliva of patients with Klinefelter s Syndrome. Clin Endocrinol 1983;18: This study compared saliva and plasma (total) levels of testosterone and androstenedione in controls and patients with Klinefelter s Syndrome. Both saliva and plasma assays yielded the same result: testosterone levels, but not androstenedione levels were lower in Klinefelter s patients. Correlation coefficients between saliva and plasma for testosterone and androstenedione were 0.58 and 0.7 respectively. The authors conclude, The data illustrate that measurement of salivary steroids by reflecting the free hormone fraction in blood may be useful in evaluating endocrine function in both health and disease. Wong YF, Mao K, Panesar NS, Loong EP, Chang AM, Mi ZJ. Salivary estradiol and progesterone during the normal ovulatory menstrual cycle in Chines women. Eur J Obstet Gynecol Reprod Biol 1990;34: Daily consecutive estradiol and progesterone measurements were made throughout one cycle in 10 women. The profiles generated agree with the findings of other researchers. Parallel serum samples were also acquired in 5 of the 10 women, and a correlation coefficients of r=0.93 was obtained for both estradiol and progesterone. The authors conclude that, salivary steroids may serve as an assessment of follicular dynamics especially in relation to ovulation induction, artificial insemination or in vitro fertilization. Worthman CM, Stallings JF, Hofman LF. Sensitive salivary estradiol assay for monitoring ovarian function. Clin Chem 1990;36: A method for salivary estradiol assay is discussed in this paper. Samples were obtained from 15 normallycycling women at various times in their cycles, and the expected pre-ovulatory peak was readily detected. Matched serum and saliva specimens were assayed in another group of women undergoing ovulation induction, and a correlation coefficient r=0.76 was generated. The authors conclude that, monitoring of estradiol concentrations in saliva may provide a useful diagnostic tool that allows better resolution of some clinical problems than do values for serum alone. Wren B, McFarland K, Edwards L, O Shea P, Sufi S et al. Effect of sequential transdermal progesterone cream on endometrium, bleeding pattern, and plasma progesterone and salivary progesterone levels in postmenopausal women. Cliamcteric 2000;3: Twenty-eight postmenopausal women received cyclic transdermal progesterone and continuous transdermal estradiol for 3 consecutive 28 day cycles. Saliva progesterone was measured serially through days and compared to serum. Serial endometrial biopsies were taken. There were several aberrant findings in this study. Firstly, the authors observed that the saliva progesterone results fluctuated by up to 2 orders of magnitude within 24 hours. They also detected less than 1 pg/ml of progesterone in serum before progesterone cream usage, and very little change after. Their baseline serum progesterone levels are far below accepted postmenopausal levels for commercial progesterone assays (e.g. DPC Immulite). No details on verification of compliance with cream usage were given, or the timing of saliva sampling relative to cream application. Both of these factors may exert a significant effect on variability of measured levels. The authors conclude that salivary progesterone levels were too variable to offer any assistance in the management of women receiving HRT, and that transdermal progesterone failed to induce a discernable response in the endometrium. 89

99 Saliva Testing Research, continued. Androstenedione Lac G, Lac N, Robert A. Steroid assays in saliva: a method to detect plasmatic contaminations. Arch Int Physiol Biochim Biophys 1993;101: The authors measure concurrent saliva and plasma (total) steroid hormone levels (DHEAS, Testosterone, DHEA, cortisol and androstenedione) and report correlation coefficients of 0.51, 0.77, 0.73, 0.74 and 0.92 respectively. They calculate that between microliters of plasma per ml of whole saliva must be present to perturb salivary hormone results by 20%. (Note: This is a substantial volume of exudate and would not be seen under normal circumstances such as avoidance of toothbrushing before sample acquisition, and absence of periodontal disease.) Lac G, Marquet P, Chassain P, Galen F. Dexamethasone in resting and exercising men. II. Effects on adrenocortical hormones. Matched serum and saliva specimens were obtained from men who performed a cycle ergometer workout after 4 days of dexamethasone suppression or placebo treatment. DHEA, DHEAS, cortisol and androstenedione were measured in both sample types, and testosterone, ACTH and aldosterone were measured in plasma. Dexamethasone prior to exercise suppressed the increase of all steroids except testosterone. In all cases, salivary gave the same information as plasma, and the authors conclude, the consistent results obtained from the 2 matrices allow us to consider salivary assays as a useful tool for steroid abuse detection. Lipson SF, Ellison PT. Development of protocols for the application of salivary steroid analyses to field conditions. Am J Human Biol 1989;1: Practical parameters of interest for the performance of salivary hormone assays (testosterone, progesterone, androstenedione and cortisol) were examined in this study. Samples were centrifuged and extracted prior to analysis. Samples collected in polystyrene tubes gave the same results as samples collected in glass. Storage at room temperature for 1-6 months did not affect results. Freezing introduced a constant negative offset. Stimulants to saliva flow including lemon juice and sugared gum skewed results, as did coffee or milk, although these effects varied widely between individuals. Wellen JJ, Smals AG, Rijken JC, Kloppenborg PW, Benraad TJ. Testosterone and androstenedione in the saliva of patients with Klinefelter s Syndrome. Clin Endocrinol 1983;18: This study compared saliva and plasma (total) levels of testosterone and androstenedione in controls and patients with Klinefelter s Syndrome. Both saliva and plasma assays yielded the same result: testosterone levels, but not androstenedione levels were lower in Klinefelter s patients. Correlation coefficients between saliva and plasma for testosterone and androstenedione were 0.58 and 0.7 respectively. The authors conclude, The data illustrate that measurement of salivary steroids by reflecting the free hormone fraction in blood may be useful in evaluating endocrine function in both health and disease. 90

100 Saliva Testing Research, continued. Young MC, Walker RF, Riad-Fahmy D, Hughes IA. Androstenedione rhythms in saliva in congenital adrenal hyperplasia. Arch Dis Child 1988;63: Salivary androstenedione levels were measured in 17 patients with Congenital Adrenal Hyperplasia, along with plasma levels of other steroids. The authors demonstrate that in plasma, androstenedione, testosterone and 17- hydroxyprogesterone are all well correlated, and that salivary androstenedione correlates well to plasma testosterone (and by inference, the other plasma markers). They generated saliva androstenedione nomograms which could be used to guide cortisol dosing in pubertal patients. Acknowledgements Women In Balance Permission for use of references provided by the authors of For additional information, please visit 91

101 jgfjfdjkgbvm,v,khg,fbggxgfccdbdzsgd

102