Endocrinology Assessments

2013 Edition

PRODUCT LINE GUIDE THE GENOVA DIAGNOSTICS Advantage Our comprehensive line of assessments for personalized treatment & prevention of chronic disease:

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ACCREDITATION

Genova Diagnostics is fully licensed federally under Clinical Laboratory Improvement Amendments (CLIA) and certified by Medicare (all states), and by New York State. TABLE OF CONTENTS

Endocrinology Product Line Guide for Genova Diagnostics

2-5 Steroidogenic Pathways Chart ______18 Essence Hormone Tests ______First Morning Void vs. 24-Hour Collection ______19 Specimen Selection Salivary Assessments A Guide to Choosing Sample Types ______2 Menopause Plus ______20 Complete Testing Line Rhythm ______22 Individual Components Breakdown Chart ______4 Male Hormones Plus ______24 One Day Hormone Check ______26 Therapeutic Ranges for Menopause Profile ______28 Profiles ______6-38 Serum Assessments Urinary Assessments Hormonal Health ______30 Complete Hormones ______6 Hormonal Health Support Guide ______32 Complete Male Hormones ______8 Essential Estrogens ______10 Complete Hormones Treatment & Testing Guide ______12 Miscellaneous Comprehensive Urinary Hormones ______16 Steroidogenic Pathways References ______35

Recognized as a pioneer and leader in laboratory functional testing, Genova Diagnostics proudly offers Urinary Hormone testing as part of its extensive endocrine line. Hormone testing is a critical diagnostic tool for safe and effective prevention and treatment of hormone-related symptoms and conditions. With this diagnostic focus in mind, Genova Diagnostics has developed an unequaled endocrine line, establishing itself as the only laboratory to offer comprehensive hormone testing in all three matrices: blood, saliva, and now, urine. Employing state-of-the-art laboratory equipment and world-class medical education, Genova Diagnostics strives to lead the field of endocrine diagnostics. Genova Diagnostics offers expertise in blood, saliva and urine hormone testing, and related genomic evaluations, to provide the most accurate and comprehensive diagnostics for the prevention and treatment of hormone-related symptoms and conditions.

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Specimen Selection

Hormone Testing A Guide to Choosing Sample Types

URINE SALIVA BLOOD Are reported hormones Urine reflects unbound Saliva reflects unbound Serum generally reflects total bound or unbound? (bioavailable) fraction of (bioavailable) fraction of hormones (bound and unbound), hormones. hormones. although “free testosterone”, free T3 and T4 are available. Sex hormone-binding globulin (SHBG) infers the amount of unbound testosterone, calculated as ‘Free Androgen Index’.

Can estrogen Yes. Only urine provides all No, estrogen metabolites are not Yes, although serum testing is metabolites be estrogen metabolites (2-OHE1, measureable in the saliva. limited to estrogen metabolites measured? 16 α-OHE1, 4-OHE1, 2-MeOE1, 2-OHE1 and 16 α-OHE1. 4-Me-OE1, ratios)

What is the advantage • Provides most comprehensive • Best way to evaluate diurnal • Reference ranges are well- of each specimen type? array of hormones and their patterns of cortisol and established and in agreement metabolites. melatonin. between labs. • Provides best reflection of • Provides evaluation of menstrual • Well-represented in the litera - tissue hormone utilization and cycle in premenopausal women, ture. metabolism. unless current or recent use • Provides average of hormone (within past year) of fluctuations over several hours. progesterone cream, which • Easy home collection raises salivary levels above (24 hr or FMV). normal reference range. • Easy home collection. • Reflects circulating levels of hormones.

What are some aspects • Urine assessment should • Transdermal hormone creams • Single ‘snapshot in time’ does of each specimen type not be used with diuretics or tend to result in salivary levels not account for hormone to be aware of? abnormal renal function. above the normal reference fluctuations. • In urine, steroid hormones are range, which do not reflect what • Timing with luteal peak of partly represented as their is occurring physiologically. menstrual cycle may be hard to downstream metabolites, due to • Bleeding from the gums can gauge when cycling is irregular. extensive metabolism (e.g., the falsely elevate androgens such • Stress of blood draw may need for DHEA and testosterone as testosterone and DHEA. influence hormone levels. are inferred by ‘Total • Should not be used in • Transdermal hormone creams 17-ketosteroids’; progesterone conjunction with sublingual tend to be under-represented in is not measurable and is hormone treatments. serum samples. represented by downstream metabolites.) URINE SALIVA BLOOD Can I use this for a • Yes, urine provides a good base - • Yes, saliva provides a good base - • Yes, blood provides a good base - baseline assessment of line assessment of hormone line assessment of hormones, line assessment of hormone hormones (i.e., before levels. unless patient has used a trans - levels. starting HRT)? Note: Avoid under- or over- dermal cream hormone within the hydration during urine collection. past year (elevated levels in the Aim for fluid consumption of ~2 saliva may persist after stopping qts/day for an average adult, the HRT for at least 3-12 months). spread out evenly over the day. • For past use of transdermal gel Note: (For FMV collections): If you HRT, wait at least 1 month before need to urinate during the night using saliva for baseline assess - within 6 hrs of your rising time, ment. collect this urine and refrigerate; • For past use of sublingual drops or add to sample in the morning. troches, wait ~1 week.

What about Urine can be used to monitor any Transdermal creams produce Blood provides reliable monitoring monitoring HRT? form of bioidentical form of HRT. abnormally high salivary levels. of most forms of bio-identical We recommend 24 hr collections to IF the decision is made to still hormones, with possible exception average HRT peaks and troughs. monitor with saliva, note that: of transdermal cream hormones, Due to metabolism, HRT dosing • Current reference ranges are not which are somewhat under- should be based upon parent based on transdermal HRT; represented in serum. hormones, and their metabolites: • Testing may be employed for • For Testosterone & DHEA, refer to initial monitoring of transderma l Total 17-ketosteroids; cream HRT, or when dose is • For Progesterone, refer to increased; Pregnanediol; • Salivary testing is not reliable • Normal or high levels of Total when dose is decreased, due to 17- hydroxycorticosteroids persisting elevations in the saliva do not rule out adrenal for an indefinite period of time. insufficiency.

Will all types of HRT be • All bioidentical forms of HRT are • All bioidentical forms of HRT are • All bioidentical forms of HRT are reflected on the report? reflected on report. reflected on report. reflected on report. • Conjugated equine estrogens • Conjugated equine estrogens • Conjugated equine estrogens (e.g., Premarin™) are only (e.g., Premarin™) are only (e.g., Premarin™) are only partially represented as estrone; partially represented as estrone; partially represented as estrone other estrogens are NOT repre - other estrogens are NOT repre - and estrone- sulfate; other estro - sented. sented. gens are NOT represented. Medroxyprogesterone acetate Medroxyprogesterone acetate Medroxyprogesterone acetate (e.g., Provera™) is synthetic and (e.g., Provera™) is synthetic and (e.g., Provera™) is synthetic and NOT represented. NOT represented. NOT represented.

When should specimen 24 hour urine collections should be Collect saliva 8-12 hours after last Collect blood 8-10 hours after last be collected relative to used with HRT, in order to average dose of HRT (all forms except dose of HRT (all forms except HRT dosing? hormone peaks and troughs with patch). patch). once or twice a day dosing. Once/week patch collect ~3 Once/week patch collect ~3 Once/week patch collect ~3 days after applying. days after applying. days after applying. Twice/week patch collect ~19 Twice/week patch collect ~19 Twice/week patch collect 1 day hours after applying. hours after applying. after applying.

Can the test be run on No. The hormones in the pill will not No. The hormones in the pill will not No. The hormones in the pill will not a patient using oral be represented and the patient’s be represented and the patient’s be represented and the patient’s contraceptives? own hormone production will be own hormone production will be own hormone production will be suppressed for those supplemented suppressed for those supplemented suppressed for those supplemented hormones. hormones. hormones.

This information is for the sole use of a licensed health care practitioner and is for educational purposes only. It is not meant for use as diagnostic information. All claims submitted to Medicare/Medicaid for Genova Diagnostics laboratory services must be for tests that are medically necessary. “Medically necessary” is defined as a test or procedure that is reasonable and necessary for the diagnosis or treatment of illness or injury or to improve the functioning of a malformed body member. Consequently, tests performed for screening purposes will not be reimbursed by the Medicare program. 3 4 SERUM m u r e t S n

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BL OOD G H C M E D ONL Y CP T I Estrone Sulfate - 83519 Estrone 82679 82671

S Estradiol 82670 82671 Estriol 82677 82671 Sex Hormone-Binding Globulin - 84270

T 2-Hydroxyestrone 82679 82542 4-Hydroxyestrone - 82542 2-Methoxyestrone - 82542

N 4-Methoxyestrone - 82542 16 α -Hydroxyestrone 82679 82542 2 Hydroxy: 16α Hydroxyestrone Ratio - - 2-Methoxyestrone: 2-Hydroxyestrone Ratio - - E Progesterone 84144 Pregnanediol 84135 Cortisol 82530 N DHEA-S 82627 Androstanediol 82542 Testosterone 8440 3

O Allo-Tetra-hydrocortisol (a-THF) 83491 Pregnanetriol 84138 Tetra-hydrocortisol (THF) 83491 P Tetra-hydrocortisone (THE) 83491 Tetra-hydrodeoxycortisol (THS) 82634 17-Hydroxysteroids (Total) - DHEA 82626 M Androsterone 82160 Etiocholanolone 82696 11-Hydroxy-androsterone 83593

O 11-Hydroxy-etiocholanolone 83593 11-Keto-androsterone 83593 11-Keto-etiocholanolone 83593 17-Ketosteroids (Total) C - Anabolic/Catabolic Balance - E/A: 5-ß /5-α Ratio - THF/a-THF Ratio - 11-ß-HSD Index - L Aldosterone (24 hr only) 82088 Human Growth Hormone (hGH) 83003

A Melatonin 83519 T3 84480 Hypersensitive TSH 84443

U Free T3 84481 Free T4 84439 Reverse T3 84482 Anti-TG Antibodies, IgG 86800 D Anti-TPO Antibodies, IgG 86376

I Free Testosterone 8440 2 Dihydrotestosterone (DHT ) 82651 Prostate Specific Antigen (PSA ) 84153 8430 5 V Insulin-Like Growth Factor 1, IGF-1 Insulin-Like Growth Factor Binding Protein-3, IGFBP-3 82397 Add-On -

I IGF-1/IGFBP-3 Ratio Deoxypyridinoline, Urine 82523 Pyridinium Crosslinks, Urine 82523

D Andr ostenedione 82157 Add-On Add-On Follicle Stimulating Hormone 83001 Add-On Add-On Lut einizing Hormone 83002 Add-On Add-On Pr olactin 84 146 Add-On Add-On N Vitamin D 82306 Add-On Add-On

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*Ratioed to Creatinine CPT= 82679 6

Urinary Assessments

Complete Hormones is the most comprehensive urinary hormone metabolism evaluation designed to assist in the prevention and treatment of hormone-related symptoms and conditions. This provides insights about menstrual irregularities, infertility, menopause, fatigue, breast cancer risk, and osteoporosis.

Complete Hormones is ideal for: • The prevention and treatment of hormone-related symptoms and conditions • Prescribing and monitoring Hormone Replacement Therapy (HRT) - 24-hour sample recommended • Assessing the risk of hormone-related disease

Complete Hormones evaluates • Analytes: • Estrogens and metabolites Estrone • Progesterone metabolites Estradiol • Adrenal hormones and metabolites Estriol • Androgens and metabolites 2-Hydroxyestrone • Anabolic/Catabolic Balance 4-Hydroxyestrone • Methylation capacity 2-Methoxyestrone 4-Methoxyestrone Complete Hormones is ideal for the prevention and treatment of the 16 a-Hydroxyestrone following symptoms and conditions: 2:16 Ratio 2-Methoxyestrone/ Cognitive and Mood Ovarian Dysfunction 2-Hydroxyestrone Ratio Pregnanediol Anxiety and Depression Menopausal symptoms Androstanediol Fatigue Premenstrual syndrome Testosterone Poor memory Polycystic ovarian syndrome allo-Tetrahydrocortisol (a-THF) Loss of libido Osteoporosis Pregnanetriol Uterine fibroids Tetrahydrocortisol (THF) Tetrahydrocortisone (THE) Other symptoms and conditions Tetrahydrodeoxycortisol (THS) Muscle weakness and atrophy Headaches/Migraines 17-Hydroxy-corticosteroids (Total) Hypoglycemia and diabetes Proinflammatory immune dysregulation DHEA Androsterone Cardiovascular disease Etiocholanolone 11-Hydroxy-androsterone Treatment 11-Hydroxy-etiocholanolone Therapy is directed at the source of the abnormality, which may include: 11-Keto-androsterone • HRT – estrogen, progesterone, androgens, thyroid, cortisol, etc. 11-Keto-etiocholanolone • Methylation support - S-adenosyl-methionine (SAMe), B vitamins 17-Ketosteroids (Total) • Estrogen Metabolism support - Indole-3-carbinol (I3C) and/or Anabolic/Catabolic Balance Diindolemethane (DIM) E/A: 5- b/5- aRatio 11- b-HSD Index

Add-On: Cortisol, Free Triiodothyronine, T3 Aldosterone (available on 24-hour only) Further treatments may be indicated by specific genomic testing (EstroGenomics™), which identifies modifiable genetic influences that can impair • Specimen Requirements: healthy hormone metabolism. 120ml urine (24-hour collection or first-morning void) Patient: SAMPLE PATIENT ID: Page 3

Anabolic/Catabolic Balance (FMV) 1.42 1.00-3.86

885 143-1,897 nmol/dl (SG) 1,260 576-3,142 nmol/dL (SG)

* Total values equal the sum of all measurable parts

2 - 1 O E H O 16D-OHE1 2-OHE1 E e NR 1 -M Higher Risk of Lower Risk of 2 Breast/Prostate RR = 1.7 - 2.8 Breast/Prostate Cancer Cancer

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This sample pie-chart represents the optimal More 5D-Reductase 1.00 Less 5D-Reductase balance of estrogen RR = 0.34 - 1.76 metabolites. The metabolites in green are considered protective, whereas metabolites in red are associated with increased risk of auto-immune disease, Less Cortisol 1.99 More Cortisol breast & prostate cancer. RR = 0.63 - 1.37 The dark line separates Phase 1 and Phase 2 detoxification pathways.

Urinary Assessments

Complete Male Hormones analyzes key urinary markers, providing insights into a wide range of disorders, from reduced libido and muscle mass to cardiovascular disease.

Complete Male Hormones is ideal for: • The prevention and treatment of hormone-related symptoms and conditions • Prescribing and monitoring Hormone Replacement Therapy (HRT) - 24-hour sample recommended • Assessing the risk of hormone-related disease, including prostate cancer

Complete Male Hormones evaluates: • Androgens and metabolites • Adrenal hormones and metabolites • Anabolic/Catabolic Balance • Estrogens • Progesterone metabolites

Interpretation Complete Male Hormones is ideal for the prevention and treatment of the following symptoms and conditions:

Diminished Cognition and Mood: Loss of Well Being: • Poor memory • Muscle weakness and atrophy • Analytes: • Poor attention/focus • Poor healing Estrone • Fatigue • Loss of libido Estradiol • Anxiety and Depression Estriol 2-Hydroxyestrone Other Conditions: 4-Hydroxyestrone • Diabetes and hypoglycemia 2-Methoxyestrone • Cardiovascular disease 4-Methoxyestrone • Infammatory conditions 16 a-Hydroxyestrone 2:16 Ratio • Male pattern balding 2-Methoxyestrone/ 2-Hydroxyestrone Ratio Treatment Pregnanediol Therapy is directed at the source of abnormality, which may include: Androstanediol • HRT - testosterone, DHEA, hGH, thyroid, cortisol, etc. Testosterone • Nutritional support and hormones (e.g. chrysin increases testosterone by 17-Hydroxy-corticosteroids (Total) inhibiting aromatase) DHEA 17-Ketosteroids (Total) Anabolic/Catabolic Balance E/A: 5- b/5- aRatio 11- b-HSD Index

Add-On: Cortisol, Free Triiodothyronine, T3 Aldosterone (available on 24-hour only) Further treatments may be indicated by specific genomic testing (EstroGenomics™), which identifies modifiable genetic influences that can impair • Specimen Requirements: a healthy hormone metabolism. 120ml urine (24-hour collection or first-morning void) #OMPLETE 3DJH 3DWLHQW6$03/(3$7,(17 ;OZS6]`[]\Sa ,'

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Urinary Assessments

Essential Estrogens is designed to assist in the prevention and treatment of estrogen-related symptoms and conditions. This robust urine analysis is ideal for prescribing and monitoring Estrogen Replacement Therapy (ERT) and for assessing the risk of estrogen-related diseases, including breast cancer risk, prostate disease, osteoporosis, and auto-immune disease.

This profile measures the following urinary estrogens and estrogen metabolites, and includes an assessment of estrogen methylation, which is a critical step in the protective mechanism of estrogen metabolism.

Estrogens “2:16 ratio” • Estrone (E1) • 2-Hydroxy-estrone/16 a-Hydroxy-estrone ratio • Estradiol (E2) • Estriol (E3) Estrogen Metabolites Estrogen Methylation • 2-Hydroxy-estrone • 2-Methoxy-estrone/2-Hydroxy-estrone ratio • 2-Methoxy-estrone • 4-Hydroxy-estrone • 4-Methoxy-estrone • 16 a-Hydroxy-estrone Interpretation Estrogens, particularly estrone and estradiol, are well described in the literature regarding the health and disease of estrogen sensitive tissues. Estrogen deficiency Estrogen excess • Osteoporosis • Menorrhagia • Amenorrhea • Uterine Fibroids • Menopausal symptoms • Anxiety/Irritability • Breast Cancer Estrogen metabolites are proven to convey protection (if balanced) or increased risk (if imbalanced) for disease: Estrogen metabolite imbalance results in increased risk of: • Analytes: Estrone • Breast Cancer • Osteoporosis • Prostate Cancer Estradiol Estrogen methylation is a critical step in the protective mechanism of Estriol estrogen metabolism. Methylation is required for the protective effects of 2-Hydroxyestrone 4-Hydroxyestrone 2-hydroxy-estrone, and for the safe detoxification of 4-hydroxy-estrone. 2 Methoxyestrone 4 Methoxyestrone Treatment 16 a-Hydroxestrone Therapy is directed at the source of the abnormality, which may include 2:16 Ratio treatments such as ERT in estrogen deficiency, S-adenosyl-methionine (SAMe) 2 Methoxyestrone: 2 Hydroxyestrone Ratio to improve estrogen methylation, and Indole-3-carbinol (I3C) and/or diindolylmethane (DIM) for balancing estrogen metabolites. Add-On: Cortisol Triiodothyronine, T3 Aldosterone (available on 24-hour only)

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T REATMENT & T ESTING G UIDELINES

Progesterone

Pregnanediol

Estrone 1.98 2.0-39.0 mcg/L

Estradiol 0.90 1.0-13.0 mcg/L

Estriol 2.90 3.0-48.0 mcg/L If Estrone and/or Estradiol is low

Possible Medications : Decreased conversion Low body mass index Chronic stress Ketoconazole, from androgens Hypothalamic or Causes or strenuous exercise (shunting of cimetidine, (if high Total pituitary insufficiency (esp. in women) precursors to cortisol) oral contraceptives, 17-ketosteroids) megestrol

Treatment Consider: Options Rule out: -Glandulars of hypothalamus, Stress management -Aromatase inhbitors pituitary, and/or ovary (Refer to section for -Smoking -Increase BMI, -(Females): Estrogenic High Total -Dioxin toxicity if relevant herbs, e.g., red clover, 17-hydroxysteroids) -Ketoconazole or metformin fennel, sage, hops, black cohosh, panax ginseng, anise

If Estrone and/or Estradiol is high

Treatment Reduce aromatase Identify contributing Lifestyle adjustments Nutritional support activity (check for low Options medications Total 17-ketosteroids)

-Reduce excess fat -Nutritional/Herbal : -Broad-spectrum Chrysin, stinging -Estrogen -Reduce excess fat nutrient support for nettles & other -Correct underlying metabolism supplementation flavonoids, soy -Clomiphene hypothyroidism or -Consider soy isoflavones, flaxseed, hyperinsulinemia isoflavones, flaxseed, -Tamoxifen procyanidins, EGCG, -Digoxin fiber to increase SHBG epilobium, vitamin C -Pharmaceutical : Anastrazole (Arimidex 1 mg/d), letrozole (Femara 25 mg/d), exemestane (Aromasin 2.5 mg/d)

Estriol Quotient

E3 Estriol Quotient /(E1+E2) 5.12 > 1.0 If Estriol Quotient is low Implication Possibly higher risk of breast & endometrial cancer, benign breast disease

Treatment Reduce E1 or E2 if elevated (refer to section Consider E3 administration Options for high E1 or E2) Estrogen Metabolism

Phase 1 Phase 2 Phase 1 Hydroxylation Estrone 2-Hydroxyestrone/16α-Hydroxyestrone Ratio α 2-OHE1 16 -OHE1 <0.38 Higher Risk of Lower Risk of 2-OHE1 4-OHE1 16 α-OHE1 Breast Cancer 2.1 15.8 Breast Cancer 1 eOE 2-OH 2-M Phase 2 Methylation (Methylation) E1

2-Methoxyestrone/2-Hydroxyestrone Ratio 4 1 -M 1 E eO Less Methylation More Methylation E H E >2.60 H 1 2-MeOE1 4-MeOE1 O O - - 4

>=0.2 α 6 1 If high 4-OHE1 and/or low 2-OHE1/16 α-OHE1 Ratio

Implication Higher risk of breast or prostate cancer

Treatment Rule out hypothyroidism and toxicity Consider I3C (150 mg 2-3 X day), DIM Increase cruciferous vegetables (e.g., pesticides, polycyclic aromatic Options (120 1-2 X day), or sulforaphane (30 mg/day) hydrocarbons, PCBs)

If low 2-MeOE1/2-OHE1 Ratio

Implication Impaired methylation, higher risk of breast or prostate cancer

Treatment Ensure adequate methionine and Ensure adequate cofactors: Mg, vitamins B2, Consider betaine (trimethylglycine, or TMG) Options protein assimilation B6, B12, folic acid, serine 500 mg 2-3 X day

Androgens

17-Ketosteroids, Total* 4.82 15.70-44.60 micromol/24 hr

* Sum of all values

Testosterone 1.65 <= 0.53 micromol/24 hr

Androstanediol 1.85 0.18-3.30 micromol/24 hr

If low level of 17-Ketosteroids or other androgens Treatment Reduce aromatase activity Lifestyle adjustments Consider supplementation Options (check for higher estrogen levels)

-Reduce excess fat -Reduce stress -Nutritional/Herbal: -Reduce excess adipose Chrysin, stinging nettles, & other flavonoids, -Improve insulin sensitivity and glucose soy isoflavones, flaxseed, procyanidins, -DHEA (15-100 mg/day) regulation, if relevant EGCG, epilobium, vitamin C -Other androgens -Increase dietary protein -Pharmaceutical : -Zinc, if deficient -Quit smoking Anastrazole (Arimidex 1 mg/d), -Minimize alcohol letrozole (Femara 25 mg/d), consumption exemestane (Aromasin 2.5 mg/d)

If high level of 17-Ketosteroids or other androgens Treatment Identify and treat causes of Identify causes of reduced Rule out Options low sex hormone-binding globulin aromatase activity

-Aromatase inhibitors -Hypothyroidism (see section for low androgens) -PCOS (in women) -Hyperinsulinemia -Smoking -Congenital adrenal hyperplasia -Obesity -Dioxin toxicity -Tumors of adrenals, ovaries, or testes -Androgen supplementation -Ketoconazole or metformin

13 14

5α-Reductase Activity Etiocholanolone/Androsterone (E/A) Ratio

Androstenedione Testosterone More 5α-Reductase 2.0 Less 5α-Reductase (5 α) (5 β) (5 α) Androsterone Etiocholanolone DHT 0.4 1.8 If low E/A Ratio

Implication Greater production of dihydrotestosterone (DHT) from testosterone

Treatment Options Consider 5 α-reductase inhibitors Additional modifiers

-Flaxseed (5-10 gms/day) Herbal : Saw palmetto (160 mg 2 X d), -Soy isoflavones (20-80 mg/day) pygeum (50-150 mg/d), -Green tea and/or green tea extract stinging nettles (300 mg 1-2 X d) (500 mg 1-3 X day) Pharmaceutical : Finasteride -Quercetin (200-400 mg 1-3 X day) (Proscar ® 5 mg/d; Propecia ® 1mg/d); -Progesterone dutasteride (Avodart ® 0.5 mg/d) -Reduce carbohydrate intake

Adrenals

Anabolic/Catabolic Balance 17 Ketosteroids/17 Hydroxysteroids Ratio

Anabolic/Catabolic Balance (FMV urine) 0.30 1.00-3.86

Catabolic (Wear & Tear) Anabolic (Growth & Healing) 1.0

Catabolic Anabolic 17-Hydroxysteroids, Total 2,067 168-1,751 nmol/dL (SG) 17-Ketosteroids, Total 612 303-2,184 nmol/dL (SG)

If low A/C Balance If high A/C Balance

Possible Chronic stress, excessive wear and tear, Possible Androgen therapy, PCOS, Causes poor recovery from illness or injury Causes congenital adrenal hyperplasia, adrenal tumor

Treatment Refer to Treatment Options for Treatment Refer to Treatment Options for high Total 17-hydroxysteroids high Total 17-ketosteroids Options or low Total 17-ketosteroids Options or low Total 17-hydroxysteroids

11 b-HSD Index (a-THF + THF)/THE

Less Cortisol1.91 More Cortisol

0.7 1.3 Cortisone (11 β-HSD, type 1 ) Cortisol (Inactive) (Active) (11 β-HSD, type 2 ) If low 11 β-HSD Index If high 11 β-HSD Index Implication Less cortisol activation Implication More cortisol activation

To increase Rule out hyperthyroidism; consider licorice, To reduce Improve insulin sensitivity, reduce abdominal fat, vitamin D, or grapefruit (naringenin); correct hypothyroidism or growth hormone active cortisol rule out contributing meds active cortisol insufficiency, control inflammation, (e.g., rosiglitazone, ketoconazole) rule out hypoxia, reduce sodium intake 17-Hydroxysteroids

17-Hydroxysteroids, Total * 2,067 168-1,751 micromol/24 hr

* Sum of all values

Cortisol, Free 4.8 1.4-14.3 mcg/dL 24 hr

If high Total 17-hydroxysteroids, with or w/out high cortisol

Possible High cortisol production, e.g., stress, strenuous exercise, Normal or low cortisol production in conjunction with Causes anorexia nervosa, Cushing’s disease increased clearance of cortisol

Further Confirm adrenal function with salivary AdrenoCortex Stress Profile or serum a.m. cortisol Testing

Treatment Identify sources of Adrenal nutrient Consider additional Stress management Dietary adjustments Options for cortisol activation support support Adrenal Excess (confirmed Inflammation, Vitamin C, zinc, Eleutherococcus, infections, B vitamins, consider ashwagandha, Reduce stimulants by saliva hypoglycemia, Adequate sleep, extra B6 (100 -200 astragalus, schizandra, such as caffeine, dysbiosis/leaky gut, meditation, yoga, or mg/d) and B5 rhodiola, passion or serum) ephedra, yerba mate; allergies, abdominal other stress-reducing (500-1500 mg/d); flower, St. John’s wort, minimize high fat/insulin resistance, activities consider valerian, skullcap, glycemic-load foods hypothyroid, licorice phosphatidyl-serine, hops, magnolia bark, ingestion GABA, L-theanine DHEA

If low Total 17-hydroxysteroids, with or w/out high cortisol

Possible Suppression of endogenous cortisol Low cortisol production, e.g., adrenal Causes production from glucocorticoids, insufficiency or congenital adrenal Pituitary insufficiency e.g., Prednisone hyperplasia (enzyme deficiency)

Treatment Address physiologic Adrenal nutrient Consider additional Stress management Dietary adjustments Options for sources of stress support support Adrenal Insufficiency Eleutherococcus, Inflammation, Vitamin C, zinc, Panax ginseng, Adequate sleep, Reduce intake infections, B vitamins, consider ashwagandha, meditation, yoga, or of sweets and other hypoglycemia, extra B6 (100 -200 astragalus, other stress-reducing high-glycemic dysbiosis/leaky gut, mg/d) and B5 schizandra, activities load foods allergies (500-1500 mg/d) rhodiola, licorice

T REATMENT & T ESTING G UIDELINES

15 16

Urinary Assessments

Comprehensive Urinary Hormone Assessments

First-Morning-Void (8 hour) OR 24-hour urine collection These simple, at-home collection options offer the advantage of a time average, effectively integrating spikes that may occur in these hormones over time.

Measures metabolites of important steroid hormones These metabolites contribute to provide a robust analysis of hormonal excess or deficiency, and help to assess the adequacy and safety of hormone replacement therapy (HRT).

Measures the free, bio-available fraction of: • Progesterone metabolites (Pregnanediol & Pregnanetriol) • Androgens and their metabolites • Adrenal hormones and their metabolites • Estrogens and their metabolites

Anabolic/Catabolic Balance (ACB) The Anabolic/Catabolic balance evaluates the ability for growth and healing (anabolic) versus the state of wear and tear (catabolic); this ratio decreases with unhealthy aging and chronic stress, and is associated with increased risk of cardiovascular disease, impaired immune function, diabetes, depression, and cognitive disturbances.

Provides reference ranges for: • Premenopausal Women • Menopausal Women • Men

S UPPORT G UIDES Urinary Androgens Urinary Estrogens c i

• Testosterone l Urinary Hormones • Estrone • 4-Methoxy-estrone • Dehydroepiandrosterone (DHEA) • Estradiol • 16 a-Hydroxy-estrone • Total 17-Ketosteroids o • Estriol • 2-Hydroxy-estrone:16 a-Hydroxy-estrone ratio Androsterone • 2-Hydroxy-estrone • 2-Methoxy-estrone:2-Hydroxy-estrone ratio Etiocholanolone • 2-Methoxy-estrone • 4-Methoxy-estrone:4-Hydroxy-estrone ratio 11-Hydroxy-androsterone b • 4-Hydroxy-estrone 11-Hydroxy-eticholanolone 11-Keto-androsterone a 11-Keto-etiocholanolone Estrogens and their metabolites are important in the health n DHEA of both men and women. Urinary estrogens and estrogen metabolites provide a robust analysis Androgens are important hormones in the health of both men andA women. Testosterone and DHEA are metabolized into what is for evaluating hormone replacement therapy and the risk of collectively known as the 17-ketosteroids (DHEA is formally included estrogen-related disease, including breast cancer and prostate cancer. as a 17-ketosteroid). Additionally, the results assist the clinician in choosing therapies that modulate estrogen metabolism, if necessary. Together, these markers provide a comprehensive assessment of androgen sufficiency, as well as evaluating the need for, and monitoring of, androgen hormone therapy. Estrogen Metabolite imbalance Estrogen deficiency Estrogen excess Increases risk of Total 17-ketosteroids (Anabolic) are used to calculate the Anabolic/Catabolic Balance (ACB). ❑ Osteoporosis ❑ Menorrhagia ❑ Breast Cancer ❑ Amenorrhea ❑ Uterine Fibroids ❑ Osteoporosis Androgen deficiency Androgen excess Anabolic/Catabolic Balance (ACB❑) Menopausal symptoms ❑ Anxiety/Irritability ❑ Prostate Cancer

Anabolic/Catabolic Balance (ACB) is a simple yet powerful tool that assists in the prevention of disease and the promotion of healthy aging by ❑ Fatigue ❑ Metabolic Syndrome (“Syndrome X”) balancing the processes that direct growth and healing (anabolic process) versus wear and tear (catabolic process) ❑ Muscle Weakness and Atrophy ❑ Acne . ❑ Anxiety ❑ Polycystic Ovarian Syndrome ❑ Depression ❑ Prostatism (nocturia, dysuria) Anabolic/Catabolic Balance and Healthy Aging ❑ Loss of Libido ❑ Male Pattern Balding Excessive catabolic or anabolic activity has been linked with disease process. More commonly seen, catabolic dominance, or “catabolic shift,” increases Urinary Adrenal Steroids with age and is associated with the whole-body effects of aging. c i

• Cortisol l Total 17-Hydroxy-corticosteroids Catabolic shift is associated with: Catabolic shift causes: •

Allo-Tetra-hydrocortisol (a-THF) o Aging ❑ Poor Healing Tetra-hydrocortisol (THF) ❑ Insomnia ❑ Cognitive Decline Tetra-hydrocortisone (THE) b ❑ Hypoxia ❑ Muscle and Tissue Degeneration Tetra-hydrodeoxycortisol (THS) Pregnanetriol ❑ Chronic Stress ❑ Cardiovascular Disease a ❑ ❑ ❑ Chronic Illness Proinflammatory Immune Dysregulation t ❑ Hyperandrenalism ❑ Anxiety and Depression These hormones and metabolites provide critical information ❑ Hypoandrogenism a Urinary Progesterones regarding the health of the adrenal gland, and the activity of ❑ Hyperglycemia and Diabetes its hormones, in both men and women.

• Pregnanediol C • Pregnanetriol 17-Hydroxy-corticosteroids (cortisol-related metabolites) assist in ACB is the ratio of the total androgen metabolites providing a robust analysis of cortisol status, and assessing cortisol’s availability at target tissues (low levels = poor stress tolerance; high levels = wear Progesterone is an active hormone and a prohormone in both men (Total 17-Ketosteroids) and the total catabolic metabolites (Total 17-Hydroxy-corticosteroids). and tear). Urinary Cortisol reflects the adrenal gland’s Total Output of this and women. It is completely metabolized into two primary molecules: hormone, averaging fluctuations that occur during its circadian rhythm. Pregnanedioalnd Pregnanetriol . Together, these markers provide a robust analysis These metabolites are easily measured in the urine and together provide a for evaluating adrenal therapies. robust analysis of progesterone status, which may be used to evaluate the need for, and monitoring of, progesterone hormone therapy. Total 17-Hydroxy-corticosteroids (Catabolic) are used to calculate the Anabolic/Catabolic Balance (ACB).

Progesterone deficiency Progesterone excess ❑ PMS ❑ Sedation ❑ Headaches/Migraines ❑ Depression Cortisol deficiency Cortisol excess ❑ Anxiety ❑ Amenorrhea ❑ Fatigue ❑ Anxiety ❑ Insomnia ❑ Hypoglycemia ❑ Depression ❑ Breast, uterine and ovarian cancer risk ❑ Poor stress tolerance ❑ Wear and Tear (tissue degeneration) ❑ Polycystic ovarian syndrome ❑ Allergies ❑ Obesity ❑ Benign prostatic hypertrophy ❑ Inflammation (arthritis, pain, etc.) ❑ Metabolic Syndrome ❑ Male pattern balding Progesterone is rarely able to be measured directly in urine.

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Urinary Assessments

First Morning Void vs. 24-Hour Collection Options for Urine Hormone Collection

Urine hormone testing is well established as a valid and accurate form of hormone testing. This sample type also provides a “time-average”, which integrates multiple spikes that may occur in some hormones throughout the day.

There are two options for urine specimen collection: First Morning Void (FMV) 24-Hour Collection The FMV is a single urine collection upon waking. The 24-Hour Collection captures all urine over a It provides a time-average for hormone spikes that 24-hour time period. It provides a time-average for may occur during the hours of sleep (approxi - hormone spikes that may occur throughout a mately 8-hours), and includes the early morning complete circadian rhythm (24 hours). As a result, peak excretion expected in a number of hormones. the 24-Hour provides the highest level of sensitivi - As a result, the time-average of the overnight col - ty to assess very low levels of hormones by provid - lection paired with capturing the peak excretion ing a direct measurement of the Total Output of provides an estimate of both the Total Output and each hormone. Peak Output of measured hormones. Specimen collection comparison: First Morning Void (FMV) 24-Hour Collection • Single collection convenience • Multiple collections • Includes early morning hormone peaks • Includes entire circadian rhythm • Excellent measure of Peak Output • Direct measure of Total Output • Convenient estimate of Total Output • Best measure for low total hormone levels

Both specimen collection options provide the following advantages: • Time-average of hormone spikes that may occur • Measures parent hormones and metabolites • Measures the free, bio-available fraction of each hormone • Simple, at-home specimen collection • Both collection options can be used effectively for prescribing and monitoring hormone replacement therapy (HRT).

Salivary Assessments

Menopause Plus is a comprehensive, noninvasive salivary assay that examines seven specimens for levels of b-estradiol, estrone, estriol, progesterone, and testosterone as well as cortisol, DHEA, and melatonin. Results can be used to identify imbalances contributing to menopause symptoms and various systemic disorders, and to determine the need for hormone replacement therapy as well as the effect of sex hormones on stress hormones.

Menopause Plus is ideal for: • The prevention and treatment of hormone-related symptoms and conditions • Prescribing Hormone Replacement Therapy (HRT) • Identifying irregular hormone patterns over time • Analytes: Menopause Plus evaluates • Estrogens • Adrenal hormones • Progesterone • Melatonin • Androgens

Interpretation estradiol estrone Menopause Plus is ideal for the prevention and treatment of the following estriol symptoms and conditions: progesterone testosterone Cognitive Ovarian Other symptoms cortisol and Mood Dysfunction and conditions DHEA Anxiety and Depression Menopausal Muscle weakness and atrophy melatonin Fatigue symptoms Hypoglycemia and diabetes Poor memory Osteoporosis Cardiovascular disease Loss of libido Headaches/Migraines estradiol estrone The Benefits of Salivary Hormone Testing: estriol Multiple, non-invasive, at-home collections progesterone testosterone These simple, at-home collections offer the advantage of measuring hormones at different times of the day, providing insight into irregular patterns of hormone secretion over time. • Specimen Requirement: Measures the free, bio-available fraction of hormones Salivary testing measures the portion of hormones that are active in the body.

Treatment Therapy is directed at the source of the abnormality, which may include: • HRT – estrogen, progesterone, testosterone, cortisol, and DHEA. 7 (3ml) saliva samples collected over a 6-day period • Nutritional support – vitamins and minerals are helpful for supporting glandular health (e.g. B-vitamins for adrenal health)

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Salivary Assessments

Rhythm is a comprehensive salivary assessment of estradiol, progesterone and testosterone spanning a full 28 day period. Imbalances revealed in this profile can help illuminate root causes of disorders such as premenstrual syndrome (PMS), infertility, and menstrual irregularities.

Balance is the key. Research has long shown that fluctuating levels of estradiol, progesterone and testosterone play a major role in a woman’s overall health, effecting: • Menstrual Cycle • Appetite Level • Mood Swings • Sex Drive • Sleep Patterns Chronic imbalances of these hormones are implicated in disorders such as: • PMS • Breast Cancer • Anovulation • Endometriosis • Infertility • Polycystic Ovary Disease • Amenorrhyea • Osteoporosis

Interpretation:

Saliva testing provides a thorough analysis of estradiol and progesterone over a full 28 day cycle. Testosterone is measured once from the 28th day specimen. By utilizing 11 saliva samples for analysis, the relationship and balance of these • Analytes: essential hormones are analyzed more precisely through time. The levels of estradiol and progesterone, as well as the ratio between the two, are clearly graphed for easy reference and patient education. estradiol progesterone Unlike serum measurements that typically reflect both bound and unbound testosterone fractions of hormones, salivary samples represent only the free (unbound) bioavailable fraction of hormone. Factors that effect binding globulin such as obesity and thyroid function do not influence test results with false high or low test levels. estradiol Indications : progesterone testosterone Rhythm is indicated for both premenopausal and perimenopauseal women not cortisol currently supplementing with hormones. An analysis of estrogen, DHEA progestesterone and testosterone can reveal ovulatory function and trends in melatonin hormone production. Imbalances such as unopposed estrogen, high follicular •Specimen Requirement: progesterone, anovulation and luteal phase defects are easily identified. This test is especially useful in treatment of patients with chronic gynecologic disorders.

The comprehensive version, Rhythm Plus , includes a circadian analysis of 11 (3ml) saliva samples collected cortisol, melatonin, and an assay of DHEA. over a 28-day cycle;

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Salivary Assessments

Male Hormones Plus is a noninvasive salivary assessment of the daily circadian activity of bioavailable testosterone. This profile provides insights into a wide range of disorders, from reduced libido and muscle mass to cardiovascular disease and osteoporosis.

Male Hormones Plus is ideal for: • The prevention and treatment of hormone-related symptoms and conditions • Prescribing and monitoring Hormone Replacement Therapy (HRT) • Identifying irregular hormone patterns over time

Male Hormones Plus evaluates: • Testosterone x 4 • DHEA • Cortisol x 4 • Melatonin x 3

Interpretation Male Hormones Plus is ideal for the prevention and treatment of the following symptoms and conditions:

Cognitive and Mood Other symptoms and conditions

Loss of libido Muscle weakness and atrophy • Analytes: Fatigue Hypoglycemia and diabetes Poor memory Poor endurance Anxiety and Depression Sleep disturbance Prostate abnormalities testosterone The Benefits of Salivary Hormone Testing: cortisol Multiple, non-invasive, at-home collections DHEA melatonin These simple, non-invasive, at-home collections offer the advantage of measuring hormones at different times of the day, providing insight into irregular patterns of hormone secretion over time.

Measures the free, bio-available fraction of hormones testosterone Salivary testing measures the portion of hormones that are active in the body. • Specimen Requirements: Treatment Therapy is directed at the source of the abnormality, which may include: • HRT – Testosterone, DHEA, cortisol, melatonin • Nutritional support – many vitamins and minerals are helpful for supporting glandular health (e.g. B-vitamins for adrenal health) 4 saliva samples (3ml) collected over a 24-hour period

Further treatments may be indicated by specific genomic testing (DetoxiGenomics™), which identifies modifiable genetic influences that can 4 saliva samples (3ml) collected impair healthy hormone metabolism. over a 24-hour period NOTE: Not an actual report. This is a representation of the data produced on this particular report.

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Salivary Assessments

The One Day Hormone Check is a convenient, noninvasive salivary assay that combines the technology of three of our most comprehensive salivary hormone tests and examines one specimen for levels of adrenal hormones and melatonin, as well as estradiol, estrone, estriol, progesterone, and testosterone for women who are menopausal.

Adrenal Hormones The adrenal hormones cortisol and DHEA function to influence: • Metabolism • Thyroid function • Anti-inflammatory response • Resistance to stress Changing the amounts of cortisol and DHEA can profoundly affect: • Energy levels • Resistance to disease • Emotional states • General sense of well-being

Melatonin Melatonin is synthesized within the pineal gland from tryptophan during the dark phase of the day. With its unique ability to pass through all blood barriers in the body, melatonin acts as the central hub of physiological function. Its role is to: • Orchestrate the complex interactions between the mind, body and environment • Influence most of the autonomic, hormonal, and behavioral functions of the human organism • Advance sleep time and duration • Modulate annual and circadian biorhythms (thereby reducing symptoms of jet- lag) • Regulate body temperature • Regulate cardiovascular function • Regulate immune function, with a possible role in fighting cancer • Act as an antioxidant • Regulate female reproductive hormones

Menopausal Hormones Testing for estradiol, estrone, estriol, progesterone and testosterone in menopausal women is ideal for the prevention and treatment of the following symptoms and conditions: Cognitive Ovarian Other symptoms and Mood Dysfunction and conditions Anxiety and Depression Menopausal Muscle weakness and atrophy Fatigue symptoms Hypoglycemia and diabetes Osteoporosis Poor memory Cardiovascular disease • Analytes: Loss of libido Headaches/Migraines estradiol Sexual Dysfunction estrone estriol progesterone The Benefits of Salivary Hormone Testing: testosterone Non-invasive, at-home collection DHEA cortisol This simple, non-invasive, at-home collection offers the convenience of measuring melatonin many hormones from a single salivary sample. Measures the free, bio-available fraction of hormones • Specimen Requirement: Salivary testing measures the portion of hormones that are active in the body. 1 (5ml) saliva sample 2QH'D\+RUPRQH&KHFN

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Therapeutic Ranges For Genova Diagnostics’ Menopause Profile

Therapeutic Range Determination: This therapeutic range has been determined by transdermal BiEst (E2/E3) cream, Testosterone, statistical analysis of women receiving personalized and Progesterone. Follow-up clinical analysis has dosages of transdermal bio-identical HRT for demonstrated that these patients are doing well. menopausal symptoms. It is known that transdermal We will continue to expand the number of healthy delivery of HRT will lead to higher levels of salivary menopausal women in this cohort in our effort to hormones. In this analysis women have significantly provide the best salivary reference ranges for improved their menopausal symptoms by taking transdermal, bio-identical hormone therapies.

Hormone Reference Range Testosterone 33.5 - 183.2 pmol/L Estrone 5.5 - 26.1 pmol/L Estriol 16.5 - 162.4 pmol/L Estradiol 2.90 - 13.65 pmol/L Progesterone 174 - 1417 pmol/L P/E2 Ratio 29 - 192

For Example: Reference Range Reference Range Hormone Reference Range Hormone Reference Range

68.5 33.5-183.2 pmol/L 27.2 2.90-13.65 pmol/L

9.0 5.5-26.1 pmol/L 1243 174-1417 pmol/L

133.3 16.5- 162.4 pmol/L 182 29-192 Ratio Notes

29 30

Serum Assessments

Using a single serum sample, Hormonal Health provides a focused overview of sex steroid hormones and their inter-relationships in both pre- and post-menopausal women, helping to better assess the impact of hormonal imbalances on each woman’s health.

Associated Reproductive Conditions Systemic Conditions

Amenorrhea Galactorrhea Osteoporosis Menstrual irregularities Infertility / Miscarriage Cardiovascular Disease Ovarian cysts Symptoms of Peri- or Breast Cancer Uterine fibroids Post-Menopause Autoimmune Disease Sexual dysfunction Hypo- or Hyperthyroidism Polycystic Ovarian Syndrome • 3 versions of the profile: Vaginal Dryness/Dyspareunia Fibrocystic Breast Disease Insulin Resistance Pre-menopause Luteal Incontinence Chronic Stress Post-menopause Menopause & HRT (This version uses pre-menopausal follicular Estrogens (Estrone, Estrone Sulfate, Estradiol, Estriol) : reference ranges. Non-bio-identical hormones will - Secreted from the ovaries pre-menopausally; aromatized from adrenal androgens post- not be reflected in the report) menopausally; Deficient estrogens augment bone resorption, collagen breakdown, dyslipidemia, & menopausal Sx; Excessive estrogen affects PMS, ovarian cysts, fibroids, • Analytes: menstrual irregularities, and breast cancer. Estrone Progesterone: Estrone Sulfate - Counters effects of estrogen on the endometrium and serves as precursor to other Estradiol steroid hormones; Low levels pre-menopausally associated with anovulation, infertility, or Estriol ‘estrogen-excess’ conditions; post-menopausally may indicate adrenal fatigue and risk of Progesterone endometrial hyperplasia. DHEA-S Sex-Hormone Binding Globulin, Binding Proteins (Sex-hormone binding globulin) : Testosterone - SHBG binds steroid hormones, regulating the amount of bioavailable (unbound) Free Androgen Index hormone in the body; SHBG is up- and downregulated by various factors, including 2-Hydroxyestrone testosterone and estrogen. 16alpha-Hydroxyestrone Androgens (DHEA-Sulfate, Testosterone, Free Androgen Index) : 2:16alpha-Hydroxyestrone Ratio - DHEA-S serves as precursor to other androgens that may also be converted to • Optional Add-On Analytes: estrogens; Maintains immune function, libido, lean body mass, insulin sensitivity, and the stress response; Testosterone protects against osteoporosis and helps maintains libido FSH (Follicle Stimulating Hormone) and lean muscle mass; Free Androgen Index (calculated using SHBG) represents the LH (Luteinizing Hormone) Androstenedione amount of bioavailable testosterone. Prolactin Estrogen Metabolism (2-Hydroxyestrone, 16a-Hydroxyestrone, 2:16a-Hydroxyestrone Ratio) : - 2-OHE1 is a weak metabolite of estrone; 16a-OHE1 is a potent estrogen metabolized to • Specimen Requirement: estriol; The 2:16a-OHE1 Ratio serves as a gauge of net estrogen activity in the body; A 8 ml serum in SST (shipped frozen) low 2:16 ratio correlates with breast cancer risk, a high ratio with risk of osteoporosis. • Pre-menopause Luteal: Specimen Optional Add-On Markers (FSH, LH, Androstenedione, Prolactin) : collected Mon-Thurs, Days 19-25 of menstrual cycle - Low levels of FSH and LH are associated with hypogonadism; in peri-menopause, • Post-menopause: Specimen elevated FSH and LH serve as a gauge of declining ovarian function; Androstenedione collected on any day serves as precursor for testosterone and estrone; Prolactin has been implicated in • Menopause & HRT: Specimen hyperandrogenism, visceral fat accumulation, breast cancer risk, and autoimmunity, collected 8-10 hrs after last dose of including celiac disease. oral or transdermal HRT, and 13 hrs after application of a patch. Collection during estrogen phase if cyclic HRT regimen; otherwise anytime 3DWLHQW 6$03/( 2UGHU1XPEHU 3$7,(17 &RPSOHWHG)HEUXDU\ $JH 5HFHLYHG)HEUXDU\ 6H[) &ROOHFWHG)HEUXDU\ 051

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Women’s Hormonal Health Understanding Estrogen Metabolism

A growing body of research reveals that it is not sim - promote proper balance, interpreted in light of each ply the amount of total estrogen circulating in a woman’s unique health risks. woman’s body that is critical to her health. How estro - For example, a woman may have "normal" total levels gen is broken down, or metabolized, in the body may of estrogen, but if her 2/16 OHE1 ratio is low, indicating also play an important role in the pathogenesis of a dominance of the active metabolite, she may feel wide variety of estrogen-dependent conditions — symptoms of, or be at increased risk for, conditions including osteoporosis, autoimmune disorders, and linked to estrogen excess. 6, 8, 19 Alternately, if her 2/16 certain cancers. OHE1 ratio is high, pointing to a surplus of the inactive Two competing pathways represent a critical "fork in metabolite, her body may lack the stimulatory estro - the road" in estrogen metabolism (see Figure 1). In the genic "fuel" it requires to adequately maintain bone dominant pathway, estrogen is metabolized into 2- tissue 14,15 — thus increasing her risk of osteoporosis. 1, 6, 17 hydroxyestrone, an inactive, possibly anti-estrogenic The 2/16 OHE1 ratio is very useful for tracking the metabolite. 2-hydroxyestrone (or 2OHE1, for short) is clinical effects of therapies designed to optimize sometimes called the "good" estrogen.1 This is estrogen metabolism. Studies indicate that the bal - because 2OHE1 is not likely to stimulate cell division in ance between these two unique metabolites can be target tissues; thus, it is not likely to promote the pro - modulated through a variety of lifestyle, dietary, and liferation of cells in the breast or endometrium, a supplement interventions. 18 Substances such as plant process linked to DNA damage and tumor growth. 1 lignans (flaxseed, grains, legumes) 19,20 , indole-3- Moreover, by latching onto available estrogen cell carbinol (cruciferous vegetables) 21 , omega-3 fatty receptors, 2OHE1 may exert a blocking action that pre - acids (coldwater fish) 22 , and isoflavones (soy) 23 have vents more potent estrogen metabolites from gaining been shown to increase the 2/16 OHE1 ratio very sig - a foothold into the cell. nificantly, reducing the risk of estrogen-dependent Along a competing pathway, estrogen is metabolized health disorders by shifting estrogen metabolism into 16 α-hydroxyestrone (16alpha-OHE1). This metabo - toward the inactive 2OHE1. Follow-up testing is able lite is much more active and powerful, with a potent to assess the clinical impact of these interventions stimulatory effect. 16 α-OHE1 binds strongly to special after just a few weeks. receptors inside cells that can accelerate the rate of Hydroxyestrone levels do not exhibit significant circa - DNA synthesis and cell multiplication .2-5 In this way, higher levels of 16alpha-OHE1 may increase the risk of dian variation, so they can be measured in a single estrogen-dependent conditions, such as lupus and serum or spot urine specimen. Studies on estrogen metabolism have historically focused on urine testing. breast cancer. 6-13 Genova Diagnostics provides serum testing, as this The levels of 2OHE1* and 16 α-OHE1, as well as the bal - assessment reflects circulating levels of the hydroxye - ance between these metabolites (measured in a 2/16 strones to which estrogen-sensitive tissues are directly OHE1 ratio), provide important clinical information exposed. Because each hydroxyestrone metabolite is about estrogen metabolism. The ultimate goal is to cleared by the kidneys into urine at different rates, ref -

*NOTE: 2-OHE1 is the primary 2-hydroxyestrogen metabolite measured in both serum and urine. Urinary assays, however, also detect small amounts of other 2-hydroxyestro - gen(2OHE) metabolites. Here we follow the convention of the published literature which, for the sake of simplicity, commonly refers to urinary 2-OHE as 2-OHE1. erence range values for these markers (and their ratio) assessment provides physicians with a comprehensive differ in serum and urine. 12,13 tool to assess levels of hormones and metabolites that play a vital role in a woman’s health. The hydroxyestrone Similar to other estrogens, hydroxyestrone levels vary dur - subpanel, called the Estrogen Metabolism Assessment, is ing the course of the menstrual cycle. Peak values ordinari - a convenient, easy-to-use tool for monitoring response to ly occur just after ovulation, and minimal values are found therapy designed to normalize estrogen metabolism. 12,13 during menstruation. Serum levels of the hydroxyestrones are most constant and remain relatively high in the early These profiles allow physicians to assess hormone bal - to midluteal phases, that is, 4 to 10 days after ovulation. ance and estrogen metabolism in both premenopausal Specimen samples collected during this post-ovulatory and menopausal women and to evaluate the impact of period are reproducible and representative of estrogen hormone replacement therapy. All assays meet FDA metabolism. requirements for clinical use as in vitro diagnostic tests.

The first ultrasensitive assay to measure 2 and 16 α-OHE1 metabolites directly in serum is included as a subpanel in the Hormone Health assessment. The Hormone Health *NOTE: References on Page 32.

Figure 1

33 34

Serum Assessments

Women’s Hormonal Health Understanding Estrogen Metabolism

References

1 Bradlow HL, Telang NT, Sepkovic DW, Osborne MP. 2-hydroxye - 9 Lahita RG, Bradlow HL, Fishman J, Kunkel HG. Abnormal estrogen 17 Leelawattana R, Ziambaras K, Roodman-Weiss J, Lyss C, Wagner strone: the 'good' estrogen. J Endocrinol 1996 Sep;150 and androgen metabolism in the human with systemic lupus ery - D, Klug T, Armamento-Villareal R, Civitelli R. The oxidative metab - Suppl:S259-65. thematosus. Am J Kidney Dis 1982 Jul;2(1 Suppl 1):206-11. olism of estradiol conditions postmenopausal bone density and 2 Bradlow HL, Hershcopf R, Martucci C, Fishman J. 16 alpha-hydrox - 10 Lahita RG, Bucala R, Bradlow HL, Fishman J. Determination of 16 bone loss. J Bone Miner Res 2000 Dec;15(12):2513-20. ylation of estradiol: a possible risk marker for alpha-hydroxyestrone by radioimmunoassay in systemic lupus 18 Fowke JH, Longcope C, Hebert JR. Macronutrient intake and breast cancer. Ann N Y Acad Sci 1986;464:138-51. erythematosus. Arthritis Rheum 1985 Oct;28(10):1122-7. estrogen metabolism in healthy postmenopausal women. Breast Cancer Res Treat 2001 Jan;65(1):1-10. 3 Swaneck GE, Fishman J. Covalent binding of the endogenous 11 Yoo HJ, Sepkovic DW, Bradlow HL, Yu GP, Sirilian HV, Schantz SP. breast cancer cells: characterization and intranuclear localization. Estrogen metabolism as a risk factor for head and neck cancer. 19 Haggans CJ, Travelli EJ, Thomas W, Martini MC, Slavin JL.The Proc Natl Acad Sci USA 1988;85:7831-5. Otolaryngol Head Neck Surg. 2001 Mar;124(3):241-7. effect of flaxseed and wheat bran consumption on urinary estro - gen metabolites in premenopausal women. Cancer Epidemiol 4 Newfield L, Goldsmith A, Bradlow HL, et al. Estrogen metabolism 12 Dupont E, Klug T, Salud C, Nygun K, McCann C, Reintgen D, Biomarkers Prev 2000 Jul;9(7):719-25. and human papillomavirus-induced tumors of the larynx: chemo- Cantor A, Cox C. Prognostic value of altered estrogen metabolism prophylaxis with indole-3-carbinol. Anticancer Res 1993;13:337- in breast cancer patients on premarin. Poster session abstract 20 Haggans CJ, Hutchins AM, Olson BA, Thomas W, Martini MC, 42. presented May 13, 2001 at the 37th American Society of Clinical Slavin JL. Effect of flaxseed consumption on urinary estrogen Oncology (ASCO) Meeting 2001 May 11-15; San Francisco, CA. metabolites in postmenopausal women. Nutr Cancer 5 Newfield L, Bradlow HL, Sepkovic DW, Auborn K. Estrogen metab - 1999;33(2):188-95. olism and the malignant potential of human papillomavirus 13 Dupont E, Klug T, McCann C, Vincent D, Shons A, Teng S, immortalized keratinocytes.Proc Soc Exp Biol Med 1998 Reintgen D, Cantor A, Cox C. The prognostic value of altered 21 Fowke JH, Longcope C, Hebert JR. Brassica vegetable consump - Mar;217(3):322-6. estrogen metabolism in breast cancer. Ann Surg Oncol tion shifts estrogen metabolism in healthy postmenopausal 2000;7(1):supplement. [Abstract originally presented at women. Cancer Epidemiol Biomarkers Prev 2000 Aug;9(8):773-9. 6 Muti P, Bradlow HL, Micheli A, Krogh V, Freudenheim JL, Schunemann HJ, Stanulla M, Yang J, Sepkovic DW, Trevisan M, 53rd Annual Cancer Symposium Society of Surgical Oncology 22 Bradlow HL, Davis DL, Lin G, Sepkovic D, Tiwari R. Effects of pes - Berrino F.Estrogen metabolism and risk of breast cancer: a Meeting 2000 Mar 16-19;New Orleans, LA.] ticides on the ratio of 16 alpha/2-hydroxyestrone: a biologic prospective study of the 2:16alphahydroxyestrone ratio in pre - 14 Westerlind KC, Gibson KJ, Malone P, Evans GL, Turner RT. marker of breast cancer risk. Environ Health Perspect 1995 menopausal and postmenopausal women. Epidemiology 2000 Differential effects of estrogen metabolites on bone and repro - Oct;103 Suppl 7:147-50. Nov;11(6):635-40. ductive tissues of ovariectomized rats. J Bone Miner Res. 1998 23 Lu LJ, Cree M, Josyula S, Nagamani M, Grady JJ, Anderson KE. 7 Fishman J, Schneider J, Herschcope RJ, Bradlow HL. Increased Jun;13(6):1023-31. Increased urinary excretion of 2-hydroxyestrone but not 16alpha- estrogen-16 alpha-hydroxylase activity in women with breast and 15 Robinson JA, Waters KM, Turner RT, Spelsberg TC. Direct action hydroxyestrone in premenopausal women during a soya diet con - endometrial cancer. J Steroid Biochem 1984;20(4B):1077-81. of naturally occurring estrogen metabolites on human osteoblas - taining isoflavones. Cancer Res 2000 Mar 1;60(5):1299-305. 8 Meilahn EN, De Stavola B, Allen DS, Fentiman I, Bradlow HL, tic cells. J Bone Miner Res 2000 Mar;15(3):499-506. Sepkovic DW, Kuller LH Do urinary oestrogen metabolites predict 16 Lim SK, Won YJ, Lee JH, Kwon SH, Lee EJ, Kim KR, Lee HC, Huh breast cancer? Guernsey III cohort follow-up. Br J Cancer 1998 KB, Chung BC. Altered hydroxylation of estrogen in patients with Nov;78(9):1250-5. postmenopausal osteopenia. J Clin Endocrinol Metab. 1997 Apr;82(4):1001-6.

For more information on hormone health, visit www.GDX.net STEROIDOGENIC PATHWAYS REFERENCES FROM PAGE 18

3bHSD (3beta-hydroxysteroid dehydrogenase) 17 b-HSD (17beta-hydroxysteroid dehydrogenase) Lower activity: Lower activity of 17 ‚HSD type 1 (e.g., resulting in less E2 or testosterone) Alcohol: Chiao YB, et al. Alcohol Clin Exp Res. 1981 Spring;5(2):2306. Phytoestrogens: Krazeisen A, et al. Adv Exp Med Biol. 2002;505:15161. PCBs: Andric SA, et al. Environ Health Perspect. 2000 Oct;108(10):9559 Licorice: Armanini D, et al. N Engl J Med. 1999 Oct 7;341(15):1158. Progestins: Lee TC, et al. J Clin Endocrinol Metab. 1999 Jun;84(6):210410. Pasqualini JR, Ebert C. Gynecol Endocrinol. 1999 Jun;13 Suppl 4:119. Isoflavonoids: Deluca D, et al. J Steroid Biochem Mol Biol. 2005 Feb;93(25):28592. Tamoxifen: Speirs V, et al. J Steroid Biochem Mol Biol. 1993 Nov;46(5):60511. Higher activity: Higher activity of 17 bHSD type 1 (e.g., resulting in more E2 or testosterone) Hyperadrenalism: Simonian MH. J Steroid Biochem. 1986 Dec;25(6):10016. Alcohol: Sarkola T, et al. Alcohol Alcohol. 2000 Jan;35(1):8490. Hyperinsulinemia: DeClue TJ, et al. J Clin Endocrinol Metab. 1991 Jun;72(6):130811. Abdominal obesity: Corbould AM, et al. Int J Obes Relat Metab Disord. 2002 Feb;26(2):16575. PCOS: Strauss JF 3rd. Ann N Y Acad Sci. 2003 Nov;997:428. DHEA: Bonney RC, et al. J Steroid Biochem. 1983 Jan;18(1):5964. Forskolin: Bird IM, et al. J Endocrinol. 1996 Sep;150 Suppl:S16573. CYP19 (Aromatase) 5a (5alpha-reductase) Lower activity: Lower activity: -Dioxins: Drenth HJ, et al. Toxicol Appl Pharmacol. 1998 Jan;148(1):505. Flaxseed, isoflavones: Evans BA, et al. J Endocrinol. 1995 Nov;147(2):295302. Phytoestrogens: Krazeisen A, et al. Mol Cell Endocrinol. 2001 Jan 22;171(12):15162. EGCG: Hiipakka RA, et al. Biochem Pharmacol. 2002 Mar 15;63(6):116576. EGCG, green tea catechins: Kapiszewska M, et al. Br J Nutr. 2006 May;95(5):98995. Progesterone: Cassidenti DL, et al. Obstet Gynecol. 1991 Jul;78(1):1037. Chrysin: Edmunds KM, et al. Reprod Nutr Dev. 2005 NovDec;45(6):70920. Saw palmetto: Drsata J, et al. Cas Lek Cesk. 2002 Oct 11;141(20):6305. Higher activity: Higher activity: Inflammation: Cutolo M, et al. Ann N Y Acad Sci. 2006 Nov;1089:53847. Insulin resistance, obesity: Westerbacka J, et al. J Clin Endocrinol Metab. 2003 Licorice: Takeuchi T, et al. Am J Chin Med. 1991;19(1):738. Oct;88(10):492431. Vitamin D3: Lou YR, et al. J Steroid Biochem Mol Biol. 2005 Feb;94(13):1517. Epub 2005 Essential HT: Soro A, et al. Hypertension. 1995 Jan;25(1):6770. Feb 17. DHEA: Stomati M, et al. Gynecol Endocrinol. 2000 Oct;14(5):34263. CYP21 (21alpha-hydroxylase) 5b (5beta-reductase) Lower activity: Lower activity: Lateonset adrenal hyperplasia: Carmina E, et al. J Endocrinol Invest. 1984 Apr;7(2):8992. Licorice: Tamura Y, et al. Arzneimittelforschung. 1979;29(4):6479. Primary adrenal insufficiency: Nikfarjam L, et al. Eur J Endocrinol. 2005 Jan;152(1):95101. Higher activity: Resveratrol: Supornsilchai V, et al. Horm Res. 2005;64(6):2806. Epub 2005 Nov 1. Insulin resistance, fatty liver: Westerbacka J, et al. J Clin Endocrinol Metab. 2003 DHEA: Stomati M, et al. Gynecol Endocrinol. 2000 Oct;14(5):34263. Oct;88(10):492431. Higher activity: Sodium depletion: Tremblay A, et al. J Biol Chem. 1991 Feb 5;266(4):224551. CYP11 b1 (11beta-hydroxylase) High prolactin: Kau MM, et al. J Cell Biochem. 1999 Feb 1;72(2):28693. Lower activity: Azole antifungals: Ayub M, et al. J Steroid Biochem. 1989 Apr;32(4):51524. CYP1A1 DHEA: Stomati M, et al. Gynecol Endocrinol. 2000 Oct;14(5):34263. Lower activity: Excess sugar: Peters LP, Teel RW. Anticancer Res. 2003 JanFeb;23(1A):399403. 11 ‚HSD (11beta-hydroxysteroid dehydrogenase) Excess omega 6 fats: Lord RS, et al. Altern Med Rev. 2002 Apr;7(2):11229. More cortisol: Oral contraceptives: Jernstrom H, et al. Carcinogenesis. 2003 May;24(5):9911005. Metabolic syndrome: Seckle JR, et al. Recent Prog Horm Res. 2004;59:35993. Cimetidine: Galbraith RA, Michnovicz JJ. N Engl J Med. 1989 Aug 3;321(5):26974. Inflammation: Cai TQ, et al. J Steroid Biochem Mol Biol. 2001 May;77(23):11722. Higher activity: Hypothyroid: Hoshiro M, et al. Clin Endocrinol (Oxf). 2006 Jan;64(1):3745. Strawberries, blackberries, raspberries: Sowers MR, et al. J Nutr. 2006 Jun;136(6):158895. Licorice: Ferrari P, et al. Hypertension. 2001 Dec 1;38(6):13306. Soy isoflavones: Lu LJ. Cancer Res. 2000 Mar 1;60(5):1299305. Less cortisol: Caffeine: Sowers MR, et al. J Nutr. 2006 Jun;136(6):158895. Hyperthyroidism: Taniyama M, et al. Thyroid. 1993 Fall;3(3):22933. Thyroxine: Michnovicz JJ, Galbraith RA. Steroids. 1990 Jan;55(1):226. Coffee: Atanasov AG, et al. FEBS Lett. 2006 Jul 24;580(17):40815. Epub 2006 Jun 27 Ketoconazole: Stiefel P, et al. Endocrine. 2002 Aug;18(3):27984. CYP1B1 Rosiglitazone: Berthiaume M, et al. Am J Physiol Regul Integr Comp Physiol. 2004 Lower activity: Nov;287(5):R111623. Hops: Henderson MC, et al. Xenobiotica. 2000 Mar;30(3):23551. Bioflavonoids: Doostdar H, et al. Toxicology. 2000 Apr 3;144(13):318. 17 ·-hydroxylase Grapefruit: Girennavar B, et al. Bioorg Med Chem. 2006 Apr 15;14(8):260612. Lower activity: Higher activity: Smoking: Yeh J, et al. J Steroid Biochem. 1989 Oct;33(4A):62730. PAHs, PCBs: Carpenter DO, et al. Environ Health Perspect. 1998 Dec;106 Suppl 6:126370. Antifungals: Weber MM, et al. Clin Investig. 1993 Nov;71(11):9338. Spironolactone: Kossor DC, et al. Mol Pharmacol. 1991 Aug;40(2):3215. CYP3A4 Higher activity: Lower activity: Hyperglycemia: Ueshiba H, et al. Eur J Endocrinol. 2002 Mar;146(3):37580. Grapefruit: Fukuda K, et al. Pharmacogenetics. 1997 Oct;7(5):3916. Hyperinsulinemia: Nestler JE, et al. N Engl J Med. 1996 Aug 29;335(9):61723. Rosemary: Zhu BT, et al. Carcinogenesis. 1998 Oct;19(10):18217. Stress: Sirianni R, et al. J Clin Endocrinol Metab. 2005 Jan;90(1):27985. Epub 2004 Oct 19. Wild yam: Wu WH, et al. J Am Coll Nutr. 2005 Aug;24(4):23543. Alcohol: Chiao YB, et al. Alcohol Clin Exp Res. 1981 Spring;5(2):2306. Peppermint oil: Dresser GK, et al. Clin Pharmacol Ther. 2002 Sep;72(3):24755. Higher activity: 17,20 lyase Hypothyroidism: Liddle C, et al. J Clin Endocrinol Metab. 1998 Jul;83(7):24116. Lower activity: Pesticides: Bradlow HL, et al. Environ Health Perspect. 1995 Oct;103 Suppl 7:14750. -Hyperglycemia: Ueshiba H, et al. Eur J Endocrinol. 2002 Mar;146(3):37580. Smoking or caffeine: Sowers MR, et al. J Nutr. 2006 Jun;136(6):158895. Azole antifungals: Ayub M, Levell MJ. J Steroid Biochem. 1989 Apr;32(4):51524. Dioxins: Moran FM, et al. Biol Reprod. 2003 Jan;68(1):24451. COMT—Methylation support Licorice: Deluca D, et al. J Steroid Biochem Mol Biol. 2005 Feb;93(25):28592. Rule out Hg toxicity: Houston MC. Altern Ther Health Med. 2007 MarApr;13(2):S12833. Higher activity: Rule out oxidative stress: James SJ, et al. Am J Clin Nutr. 2004 Dec;80(6):16117. PCBs: Andric SA, et al. Environ Health Perspect. 2000 Oct;108(10):9559. Reduce stress: Dubey RK, et al. J Clin Endocrinol Metab. 2004 Aug;89(8):392231. DHEA: Stomati M, et al. Gynecol Endocrinol. 2000 Oct;14(5):34263. Antiepileptics (valproate): NelsonDeGrave VL, et al. Endocrinology. 2004 Feb;145(2):799 808. Epub 2003 Oct 23.

35 Notes

36 Notes

37 About Genova Diagnostics

Headquartered in Asheville, N.C., Genova Diagnostics is a global specialty clinical laboratory, pioneering a systems approach that supports healthcare providers in the personalized treatment and prevention of chronic disease. Unlike traditional labs that focus on disease pathology, Genova specializes in comprehensive panels that combine standard and innovative biomarkers to provide a more complete understanding of specific biological systems. Chronic diseases are often complex and Genova’s system-based testing helps physicians develop targeted, personalized treatments for their patients. Easy-to-read color graphic reports synthesize test results into actionable information and facilitate physician-patient communication. The internationally renowned lab is committed to the highest standards and has a team of medical experts who provide consultation to healthcare professionals, as well as a robust array of educational resources.