Testosterone, Free (Dialysis), Total (MS), and Sex Hormone Binding Globulin - Testosterone is mostly bound to transport proteins in circulation, with less than 1% remaining free. Measuring Free Testosterone can be helpful when there are suspected disturbances in Sex Hormone Binding Globulin (SHBG), such as in cases of obesity or high estrogen levels. Testosterone measurements are used to evaluate conditions like erectile dysfunction, infertility, gynecomastia, and osteoporosis, as well as to monitor hormone replacement therapy. Preferred Specimen(s) : 3 mL serum Minimum : Volume 1 mL   Methodology : Chromatography/Mass Spectrometry • Equilibrium Dialysis • Calculation (CALC) • Immunoassay (IA)

Dihydrotestosterone (DHT) is a powerful androgen produced from testosterone through the action of 5-alpha-reductase. A deficiency in 5-alpha-reductase leads to males with incomplete virilization, appearing phenotypically female. This condition is indicated by a high testosterone to DHT ratio. Preferred Specimen(s) : 1 mL of serum collected in a red-top tube (without gel) Minimum Volume : 0.6 mL Methodology : Chromatography/Mass Spectrometry

Dihydrotestosterone, Free, Serum - This test assesses the serum levels of dihydrotestosterone (DHT) and can aid in diagnosing 5 alpha-reductase deficiency, an autosomal recessive disorder affecting male sexual development. Evaluating DHT levels is also utilized to assess treatments that influence androgen levels. DHT is the most powerful androgen and is crucial for the development of male external genitalia, prostate enlargement, and skin metabolism. DHT mainly originates from testosterone through 5-alpha-reductase activity in peripheral tissues, but it can also be produced via a "backdoor pathway" without testosterone as a precursor. Individuals with 5-alpha-reductase deficiency experience reduced conversion of testosterone to DHT, so a high testosterone-to-DHT ratio may indicate this condition. A definitive diagnosis of 5-alpha-reductase deficiency requires DNA analysis of SRD5A2. Most circulating DHT is bound to sex hormone-binding globulin (SHBG). Since SHBG levels can be affected by medications, diseases, and sex steroids, measuring free DHT provides a more accurate reflection of bioactive DHT levels than total serum DHT measurement. Although DHT has been circumstantially linked to prostate tumorigenesis, current evidence does not support a connection between high serum DHT levels and an increased risk of benign prostatic hyperplasia and prostate cancer. Test Details Include : Dihydrotestosterone and DHT, Free Preferred Specimen(s) : 1.5 mL serum collected in a red-top tube (no gel) Minimum Volume : 1 mL Methodology: Equilibrium Dialysis • Chromatography/Mass Spectrometry

The Estrogens, Fractionated, LC/MS panel quantifies the levels of estrone, estradiol, and estriol, which are the three primary types of estrogens found in serum. This panel can be useful for evaluating estrogen levels. Estrogens are naturally occurring steroids that play a role in the growth and maintenance of reproductive organs and secondary sexual characteristics in women. They also impact other bodily systems, such as bone density maintenance, liver protein production, arterial vasodilation, lowering intraocular pressure, and influencing mood. Estrogen measurements can assist in assessing sexual maturity, menstrual and fertility disorders, fetal-placental health during pregnancy, estrogen-secreting tumors, and feminization syndromes in men. Elevated estrogen levels may occur in normal pregnancy, early puberty, hyperthyroidism, liver cirrhosis, and ovarian, testicular, and adrenal tumors. Reduced estrogen levels may be seen in cases of failing pregnancy, Turner syndrome, hypopituitarism, primary and secondary hypogonadism, menopause, Stein-Leventhal syndrome, and anorexia nervosa. Estriol becomes the dominant estrogen only in late pregnancy. Estriol measurements are often used to help screen for certain fetal abnormalities, such as Down syndrome. In non-pregnant women and men, estriol measurements have limited clinical significance. Test Details Include : Estrone Estradiol, Ultrasensitive, LC/MS Estriol, Serum Preferred Specimen(s) : 2.5 mL serum collected in a red-top tube (no gel) Minimum Volume : 1.5 mL Methodology : Chromatography/Mass Spectrometry

Estradiol, Free - A significant portion of Estradiol is attached to proteins. The fraction that is unbound, along with Estradiol bound to proteins with low affinity, indicates the free concentration. Free Estradiol might have a stronger correlation with medical conditions compared to Total Estradiol concentrations. Test Details Includes : Estradiol, Free Estradiol, Ultrasensitive, LC/MS Preferred Specimen(s) : 1 mL serum collected in a red-top tube (no gel) Minimum Volume : 0.5 mL Methodology : Chromatography/Mass Spectrometry • Equilibrium Dialysis

Androsterone, Serum - Androsterone is one of the androgens, produced by both the adrenal glands and gonads. It can be reversibly converted into 3 alpha androstenediol. Elevated androsterone levels can lower cholesterol. High androsterone levels are often observed in female patients with acne, with or without hirsutism. Hirsute females without acne typically have normal androsterone levels. Elevated androsterone levels are also found in hyperthyroid patients, while lower levels are seen in patients with myxedema and cancer. Test Details : Patient Preparation: A fasting specimen is preferred. Patients should avoid hormonal medications for 2 days. Preferred Specimen(s) : 3 mL frozen serum Alternative Specimen(s) : Plasma collected in an EDTA (lavender-top) tube Minimum : Volume: 1 mL Methodology : Enzyme Linked Immunosorbent Assay (ELISA) • Extraction

Androstenedione - This compound can be helpful in assessing patients with androgen excess and in the management of those with congenital adrenal hyperplasia (CAH). Preferred Specimen(s) : 1 mL serum collected in a red-top tube (no gel) Alternative Specimen(s) : Plasma collected in: EDTA (lavender-top) tube, EDTA (royal blue-top) tube, sodium heparin (green-top) tube, or lithium heparin (green-top) tube Minimum Volume : 0.25 mL Methodology : Chromatography/Mass Spectrometry

Free Progesterone - Progesterone is a progestin primarily produced through the enzymatic metabolism of pregnenolone. It is enzymatically converted into 17-hydroxy progesterone and 11-deoxycorticosterone. It is secreted by both the gonads and adrenal glands. Most of it binds to cortisol-binding globulin and albumin, with a small portion existing in the "free" form, which is the bioactive component. This "free" progesterone is excreted in the urine mainly as "free" unconjugated progesterone and as pregnanediol. Progesterone causes cellular changes in the cervix, vagina, and uterus. Levels are lowest during the follicular phase and rise quickly after the luteal surge. Increased progesterone inhibits ovulation. "Free" progesterone significantly increases during pregnancy, reaching about 20% of the total progesterone concentration at delivery. Measuring progesterone can be useful for monitoring fertility, corpus luteum function, endometrial development, and assisting patients undergoing in-vitro fertilization. Test Details Include : Free Progesterone % Free Progesterone (% of Total Progesterone) Progesterone, Total Preferred Specimen(s) : 3 mL serum Alternative Specimen(s) : Plasma collected in: EDTA (lavender-top) tube Minimum Volume 2 mL Methodology : Radioimmunoassay following ultrafiltration of specimens

Progesterone, LC/MS - This test (1) determines the presence of a functioning corpus luteum or luteal cell activity, (2) verifies basal body temperature readings related to ovulation, (3) provides an estimate of the ovulation day, and (4) evaluates placental function during pregnancy. Preferred Specimen(s): 1 mL serum collected in a red-top tube (without gel) Alternative Specimen(s): Plasma collected in: EDTA (royal blue-top) tube, sodium heparin (green-top) tube, lithium heparin (green-top) tube, or EDTA (lavender-top) tube Minimum Volume: 0.25 mL Methodology: Chromatography/Mass Spectrometry

Steroid Panel, Comprehensive - This panel detects all primary adrenal steroid hormones and aids in diagnosing the four enzymatic defects linked to congenital adrenal hyperplasia (CAH). All steroids are measured in a single assay using LCMSMS, requiring only 0.1 mL of serum. Test Details Include : 11-Deoxycortisol 17-Hydroxyprogesterone 17-Hydroxypregnenolone 18-Hydroxycorticosterone and Cortisone Androstenedione Corticosterone Cortisol DHEA, Unconjugated Deoxycorticosterone Pregnenolone Progesterone Total Testosterone, LC/MS/MS Preferred Specimen(s) : 0.5 mL frozen serum collected in a red-top tube (no gel) Minimum Volume : 0.25 mL Methodology: Chromatography/Mass Spectrometry

Total Estrogen and Precision Medicine: Treating Patients vs. Treating the Population: Throughout the history of medicine, healthcare trends have evolved toward disease prevention instead of treating disease. Yet, the flood of lifestyle and dietary changes designed to avoid certain diseases seems to be more of a marketing strategy for food distributors, health clubs and supplement companies than realistic and actionable recommendations in practice.  In recent years, a number of studies have discussed the association between genetic mutations (SNPs), estrogen sensitive cancers (breast, uterine and prostate), and estrogen hormone replacement therapies. In these studies, there seemed to be a clear association between orally administered synthetic estrogen and the development of estrogen-sensitive cancer. However, as more and more medical practitioners switched from oral administration of synthetic estrogen to bio-identical creams, patches, gels and pellets, the association between estrogen and breast cancers appeared to decrease. Those prescribing and/or marketing bioidentical hormones made the assumption that breast cancer risk was related to the synthetic make-up in oral administration of estrogen, but although the association to synthetic oral estrogen to cancer is statistically significant, this assumption may be misleading. This article will specifically discuss the pathways of estrogen metabolism, what these pathways represent in clinical practice and how to identify and, subsequently, mitigate the risks associated with developing estrogen-sensitive breast, uterine and prostate cancers in disease prevention. Estrogen is essential in both men and women for bone health, brain health, cardiovascular health, reproductive health and has even shown positive effects in treating certain cancers. It is clear that maintaining optimal levels of estrogen is very important, because having too much or too little estrogen can present with a myriad of symptoms. Measuring Estrogen before addressing suspected hormone imbalances and monitoring estrogen levels during hormone replacement therapy are crucial in maintaining hormone balance, but the clinical science behind the risks and benefits of Estrogen relating to cancer remained a mystery until estrogen metabolism became more understood. In order to customize therapies for patients while mitigating risks associated with estrogen-sensitive cancers, all primary estrogens (Estrone, Estradiol and Estriol), as well as the downstream estrogen metabolites, must be evaluated. Most practitioners are accustomed to evaluating serum estradiol by itself when determining if a patient is a candidate for estrogen hormone therapy, but measuring a single estrogen significantly limits practitioners in developing therapies that are precise to each individual patient. As men and women age, aromatase activity takes place at the testosterone precursor, Androstenedione, at a higher rate than the aromatase activity at Testosterone observed in younger populations. The result is a migration toward estrone production over estradiol; so simply measuring estradiol can produce an inaccurate assessment of estrogen effect at the estrogen receptors (ERs). In addition to assessing Estrone, it is essential to look at the various stages of Estradiol, Estrone and Estrone metabolism collectively. Special attention should be paid to the amount of free hormone that is available to tissues based on the binding affinities of estrone (E1), estradiol (E2), estriol (E3) and 16-alpha-hydroxyestrone at the estrogen receptors. The Total Estrogen Effect (TEE) in urinary hormone and hormone metabolite testing is calculated based on the presence of estrone, estradiol, estriol, and their relative binding affinities at the estrogen receptors. Estradiol is considered to be the most estrogenic estrogen and is the prevalent primary estrogen produced by menstruating women. Estradiol has a strong and long-binding affinity at the estrogen receptors and is responsible for most cell proliferation at estrogen-sensitive tissues, such as breast and uterine tissues, in menstruating women.1-6 Based on receptor availability at the tissues, estradiol is converted to estrone and further converted to estriol through the 16-alpha-hydroxyestrone metabolism pathway during Phase I metabolism. Unless a female is pregnant or menopausal, most estrone and estriol is a result of conversion from estradiol. Based on this knowledge, we are able to assume the estrogen effect at the estrogen receptors. The calculation for the TEE assumes certain standard binding affinities at the estrogen receptors. Estriol is the weakest binding primary estrogen at the estrogen receptors and can compete with stronger-binding estrogens. Due to its competitive nature and its weaker binding affinity, estriol is considered to be a protective estrogen. Following estriol in binding affinity is estrone (4x more estrogenic than estriol), 16a-hydroxyestrone (9x more estrogenic than estriol), and estradiol (10x more estrogenic than estriol).These relative values are added together to establish the Total Estrogen Effect at the ERs. Table 1 : Fictitious Example (If a patient has an estriol of 2, an estrone of 2, a 16a-OHE1 of 2, and an estradiol of 2, then the Total Estrogen Effect (TEE) would be 48 based on their relative binding affinities) Estrogen Fictitious Result Binding-Affinity Multiplier Total Estriol 2 1 2 Estrone 2 4 8 16a-OHE1** 2 9 18 Estradiol 2 10 20 Total Estrogen Load 48 **Although 16a-OHE1 is a Phase I metabolite, it is included in the calculation due to its ability to contribute to estrogen dominance. Assessing the amount of estrogen effect at the receptor is the best way to decide if a patient is a candidate for estrogen hormone replacement, but this assessment is only the beginning; assessing Phase I and Phase II metabolism of estrogen is where inflammatory responses and cancer risk are assessed and customized therapies are derived. As shown in Figure 2, there are three distinct pathways of Phase I metabolism of Estrone, resulting in 2-hydroxyestrone (the most favorable pathway of metabolism), 16-alpha-hydroxyestrone (a result of inflammation) and 4-hydroxyestrone (a carcinogenic pathway). These Phase I pathways are directly impacted by lifestyle and dietary choices. 4-OHE1 is catalyzed predominantly through CYP1B1 2-OHE1 is catalyzed predominantly through CYP1A1 16-a-OHE1 is catalyzed predominantly through CYP3a4 Improvements in lifestyle result in a preference of metabolism down the most favorable 2-hydroxyestrone pathway. 2-hydroxyestrone does not bind to the estrogen receptors. However, COMT activity causes the methylation of 2-hydroxyestrone in Phase II metabolism. That results in stable DNA adducts and can slow estrogen-sensitive cell growth and even reverse DNA damage caused by the 4-OHE1 pathway of Phase I metabolism. 2-methoxyestrone has also been shown to reverse inflammatory responses to estrogen dominance and slow or reverse breast, uterine and prostate cancer growth. When estrogen dominance or certain mutations in the CYP1B1 gene are present, Phase I metabolism increases down the 4-hydroxyestrone pathway. 4-Hydroxyestrones are highly reactive and form 3,4 Quinones that can form unstable DNA adducts, resulting in the creation of carcinogenic mutations. The 4-hydroxyestrone pathway is additionally influenced by environmental toxins, so patients who have CYP1B1 SNPs are especially susceptible to estrogen-sensitive cancers. Because 4-OHE1 is a biomarker of CYP1B1 SNPs, patients with increased 4-OHE1 levels should avoid chemical toxins and improve methylation to drive Phase II detoxification of 4-OHE1. When 4-OHE1 is methylated in Phase II metabolism, the carcinogenic effects of 4-OHE1 are completely neutralized.  Finally, the 16-alpha-hydroxyestrone pathway increases in the presence of inflammation. While most of this inflammation stems from the gut, estrogen dominance and the presence of estrogen-sensitive cancers can increase 16-a-OHE1 during Phase I metabolism as well. The best way to assess 16-a-OHE1 levels is the relative rate of metabolism of 2-OHE1 to 16-a-OHE1 via the 2:16 ratio. When the 2:16 is low, supporting Phase I metabolism and reducing gut inflammation are the primary ways to redirect Phase I metabolism down the 2-OHE1 pathway. Additionally, driving the 16-a-OHE1 pathway through 16-a-OHE1 to Estriol (via 16-hydroxylase activity) is another way to protect the tissues from estrogen dominance through weaker competitive binding of estriol at the ERs. 16-a-OHE1 covalently binds to ERs, resulting in long-standing action at the target tissues. This covalently-binding estrogen metabolite has positive effects in cell proliferation and has even been used to treat certain cancers, but it can cause an existing cancerous tumor or damaged estrogen-sensitive tissue to grow aggressively as well. This unique action of increased 16-a-OHE1 in the presence of inflammation, followed by increase cell proliferation, makes 16-a-OHE1 advantageous in the absence of free-radicals and un-repaired DNA damage. On the other hand, this action makes 16-a-OHE1detrimentally aggressive in the presence of cancer or certain genetic predispositions to increases in 4-OHE1. This is why assessing estrogen metabolism, modulating Phase I metabolism through lifestyle, and supporting Phase II metabolism through methylation and glutathione activity are essential in customizing therapies for patients and optimizing clinical outcomes. Provided there is sufficient glutathione activity, the removal of DNA-damaging free-radicals is a regular event inside of cells. In the event of DNA mutations, insufficient DNA repair and the initiation of cancerous tissue, these mechanisms of cellular defense are likely compromised significantly. Un-repaired DNA damage is a major cause of cancer initiation. Again, the 2-hydroxy ➝ 2-methyoxyestrone pathway is the most advantageous pathway for repairing DNA damage and reversing the effect of free-radicals, as well as glutathione activity. A number of nutrients, botanicals and nutrient compounds have been identified as having varying effects on the estrogen-metabolizing and detoxifying pathways. Implementation of many of these compounds through an individualized process affords great potential for those affected by the potentially deleterious effects of aberrant estrogen metabolism. DIM (diindolylmethane) – DIM is used to stimulate 2 hydroxylation (neutral estrogen pathway) via CYP1A1 and reduce expression of 16 hydroxylation (potential harmful estrogen pathway) through inhibiting CYP3a4. There is far more potential therapeutic action to DIM, however. DIM has been shown to reduce DNA hypermethylation of CpG islands (hallmark feature of cancer activity), reduce intestinal inflammation, function to mildly inhibit aromatase, and enhance DNA repair mechanisms. Flax seeds – Flax seeds are a promoter of CYP1A1 and an inhibitor of CYP1B1. Thus, flax seeds are promoters of 2 hydroxylation (neutral estrogen) and inhibitors of 4 hydroxylation (potentially undesirable). Flax also has shown to inhibit CYP3a4 and reduce the excretion of 16OHE1, another potentially-problematic estrogen. Berries – Numerous types of berries (blackberries, raspberries, grapes, blueberries) are a rich source of polyphenolic compounds, including ellagic acid. Ellagic acid is a promoter of glutathione transferase (GSTM), as well as NQO1 (quinone reductase). These 2 enzymes are important in the detoxification of 3,4 semi-quinones. Additionally, ellagic acid has been shown to increase DNA repair genes, as well as reduce DNA adducts that have been formed by carcinogens. Grapefruit & Citrus peel – Are sources of hesperidin. Hesperidin, at high doses, inhibits CYP1B1 and also CYP3a4. Grapefruit is notorious for inhibiting CYP3a4. Citrus peel contains a considerable amount of hesperidin; that is especially true of dried tangerine peel. An assortment of studies done on hesperidin have found an overall increase in blood flow and circulation, reduction in blood pressure, and reduction in symptoms of cell adhesion factors, which may disrupt cancer activities. Calcium D-glucarate – Is a form of calcium that promotes phase 2 glucuronidation. This phase 2 reaction makes molecules more water-soluble. Additionally, it is believed that calcium d-glucarate is a beta glucuronidase inhibitor, which acts to prevent the reabsorption of detoxified estrogens through 2nd pass metabolism. Glutathione promoters and/or cofactors: NAC, lipoic acid, selenium, B-2, B-6, zinci

Congenital androgen neurotoxicity refers to the neurotoxic effects resulting from excessive androgen exposure or the body's inability to utilize androgen during fetal development. This can lead to various neurological issues. In cases of androgen insensitivity, the body does not respond to androgen properly, causing a range of symptoms. On the other hand, conditions like Congenital Adrenal Hyperplasia (CAH) can result in excess androgen exposure, which may also have neurological consequences. Symptoms Neurological and Behavioral Effects: Abnormal androgen exposure, whether from CAH or AIS, can lead to various neurological and behavioral changes. These can include:  Disrupted brain development: Androgens influence the growth and organization of brain regions, including those involved in sexual differentiation and cognitive functions.   Cognitive differences: Studies suggest that prenatal androgen exposure can impact cognitive abilities, particularly in areas related to spatial abilities and some aspects of social behavior.  Behavioral differences: Early androgen exposure can influence play behavior and other behavioral characteristics.  Neurological disorders: Some research suggests a link between prenatal androgen exposure and the development of neurological disorders, although the relationship is complex and not fully understood.  Excess or deficient androgen levels can impact the development and function of the nervous system, potentially leading to conditions like spinal pressure or other neurological issues .  SBMA and Androgen Receptor Dysfunction SBMA is an inherited neuromuscular disorder caused by an expanded polyglutamine tract in the androgen receptor gene. This mutation leads to a dysfunctional androgen receptor, making the individual susceptible to neurotoxic effects of androgens, particularly testosterone.  CAIS and Androgen Insensitivity CAIS is a condition where the body cannot respond to androgen signals, leading to a range of developmental and functional differences.  Neurotoxic Effects of Androgens Research suggests that androgens can have neurotoxic effects, potentially damaging or impairing the function of neurons, especially in individuals with genetic variations in androgen receptor function. Spinal Pressure and Androgens In some cases, hormonal imbalances, including those related to androgen levels, can affect the pressure within the spinal column or surrounding tissues. For example, idiopathic intracranial hypertension (IIH), a condition characterized by increased intracranial pressure, has been linked to excess androgen in women.  While congenital androgen neurotoxicity itself doesn't directly cause anemia , the conditions it can cause can sometimes lead to anemia. Congenital androgen insensitivity syndrome (CAIS) can sometimes be associated with anemia, particularly in severe cases. Additionally, some androgen therapies used to treat certain types of anemia can have side effects that lead to other hematological issues, including anemia.  Androgen Therapy Some androgens are used to treat certain anemias, particularly acquired aplastic anemia and bone marrow failure syndromes. While effective in some cases, androgen therapy can also have side effects, including the potential for anemia or other hematological issues. Medications Typically Used Corticosteroids are used to replace cortisol and manage severe symptoms of cortisol deficiency in CAH. Anti-androgen medications block the action of androgens, helping to control excess androgen levels, particularly in CAH. Aromatase Inhibitors prevent the conversion of androgen to estrogen, which can be beneficial in certain cases of excess androgen. GnRH agonists can suppress the release of gonadotropin-releasing hormone (GnRH), which stimulates the production of androgens and estrogens. Surgical Interventions Adrenalectomy involves the surgical removal of the adrenal glands in CAH. While effective, it is considered an experimental approach according to the National Institutes of Health (.gov). Genital reconstruction surgery may be necessary in AIS to address genital abnormalities and enhance quality of life.