Glucocorticoid Therapy and Adrenal Suppression

ABSTRACT

Glucocorticoids are steroid hormones produced by the adrenal cortex. They have pleiotropic effects and contribute substantially to the maintenance of resting and stress-related homeostasis. Although the molecular mechanisms of their actions are not fully understood, most of glucocorticoid effects are mediated by a ubiquitously expressed transcription factor, the glucocorticoid receptor. The latter influences the transcription rate of several glucocorticoid-target genes or interact physically with other transcription factors regulating their transcriptional activity in a positive or negative fashion. We present the molecular mechanisms of glucocorticoid action, and we discuss glucocorticoid treatment in endocrine and non-endocrine disorders, the side effects of glucocorticoids, their concomitant use and interactions with other drugs, and the risk factors for adrenal suppression. We suggest regimens for weaning patients from long-term glucocorticoid therapy, describe the glucocorticoid withdrawal syndrome, and provide some future perspectives on glucocorticoid treatment. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION

Glucocorticoids are steroid hormones produced by the zona fasciculata of the adrenal cortex. These molecules are secreted into the peripheral blood under the control of the hypothalamic-pituitary-adrenal (HPA) axis in an ultradian, circadian and stress-related fashion (1). Glucocorticoids influence a myriad of physiologic functions contributing substantially to the maintenance of resting and stress-related homeostasis. At the cellular level, glucocorticoids regulate proliferation, differentiation and programmed cell death (apoptosis) of various cell types and may change the methylation status of cytosine-guanine dinucleotides (CpG) located in the regulatory regions of many genes, leading to important epigenetic alterations (1, 2).

Although glucocorticoids have been introduced in the treatment of rheumatoid arthritis since 1949, their molecular mechanisms of actions remain an evolving field of molecular and cellular endocrinology. Their anti-inflammatory and immunosuppressive effects are mediated mostly by their cognate receptor, the glucocorticoid receptor (GR), a transcription factor that belongs to the steroid receptor subfamily of the nuclear receptor superfamily (3). The therapeutic applications of synthetic glucocorticoids have been greatly broadened to encompass a large number of non-endocrine and endocrine diseases. Indeed, the prevalence of long-term glucocorticoid use worldwide is estimated at between 1% and 3% of adults (4).

When glucocorticoids are used at supraphysiologic doses, glucocorticoid-induced Hypothalamic-pituitary-adrenal (HPA) axis suppression renders the adrenal glands unable to generate sufficient cortisol if glucocorticoid treatment is abruptly stopped. In addition to adrenal suppression, a growing list of glucocorticoid adverse effects have been documented.

Glucocorticoid resistance has become another limitation in the therapeutic use of glucocorticoids. Our ever-increasing and deeper understanding of the molecular mechanisms of glucocorticoid actions might provide the basis for designing selective GR agonists that will optimize the therapeutic outcome, while minimizing undesired side effects.

MOLECULAR MECHANISMS OF GLUCOCORTICOID ACTION

Within the glucocorticoid target cell, the human (h) GR interacts with heat shock proteins (HSP90, HSP70) and immunophillins (FKBP51 and FKBP52), forming a multiprotein complex. Upon glucocorticoid-binding, the hGR dissociates from its protein partners, translocates into the nucleus, and forms homo- or hetero-dimers that bind to specific DNA sequences, termed “glucocorticoid response elements” (“GREs”), influencing the transcription of several glucocorticoid target genes in a positive or negative fashion (3, 5). For additional information please see the

Endotext chapter on Glucocorticoid receptors (6). Many anti-inflammatory genes are trans-activated by glucocorticoids, while pro-inflammatory genes are trans-repressed by these hormones. Aside from the genomic actions, accumulating evidence suggests that glucocorticoids may exert some effects in a very short time frame, independently of gene transcription and/or translation (7). These nongenomic glucocorticoid effects are believed to be mediated by membrane-bound hGRs that trigger specific kinase signaling pathways (8).

In addition to the above-mentioned actions, glucocorticoids can influence gene expression independently of hGR binding to DNA. These actions are mediated by physical interaction between the monomeric hGR with other transcription factors, such as the nuclear factor κB (NF-κB), the activator protein 1 (AP-1), and the signal transducers and activators of transcription (STATs), influencing the transcription rate of target genes of the latter (3, 5, 6).

SYNTHETIC GLUCOCORTICOIDS

Since the introduction of glucocorticoids (GCs) in the treatment of rheumatoid arthritis in 1949, intense efforts have been made by science and industry to maximize the beneficial effects and minimize the side effects of glucocorticoids. Thus, many synthetic compounds with glucocorticoid activity were manufactured and tested (9). The pharmacologic differences among these chemicals result from structural alterations of their basic steroid nucleus and its side groups. These changes may affect the bioavailability of these compounds - including their gastrointestinal or parenteral absorption, plasma half-life, and metabolism in the liver, fat, or target tissues - and their abilities to interact with the glucocorticoid receptor and to modulate the transcription of glucocorticoid - responsive genes (10, 11). In addition, structural modifications diminish the natural cross-reactivity of glucocorticoids with the mineralocorticoid receptor, eliminating their undesirable salt-retaining activity. Other modifications increase glucocorticoids' water solubility for parenteral administration or decrease their water solubility to enhance topical potency (11).

Synthetic GCs' clinical efficacy depends on their pharmacokinetics and their pharmacodynamics. Pharmacokinetic parameters such as the elimination half-life and pharmacodynamic parameters such as the concentration producing the half-maximal effect determine the duration and intensity of GC effects (12). It is known that the presence of an 11β-hydroxyl group is essential for the anti-inflammatory and immunosuppressive effects of GCs and for the sodium retaining effects of the mineralocorticoids (MCs). The most important pharmacokinetic systems for GCs and MCs are the 11β- hydroxysteroid dehydrogenases (11β-HSDs) because they regulate the target cell adjustment between the active hydroxy- and the inactive oxo- form of a steroid (13, 14). 11β- hydroxysteroid dehydrogenase type 2 (11β-HSD2, oxidizing enzyme) catalyzes the conversion of cortisol to cortisone, the inactive metabolite, whereas 11β- hydroxysteroid dehydrogenase type 1 (11β-HSD1, reductase) converts cortisone to cortisol. Thus, 11β-HSD1, which is expressed in a wide range of tissues, mainly in the liver, facilitates GC hormone action whereas the major role of 11β-HSD2 is to prevent cortisol from gaining access to high-affinity MC receptors and, therefore, the enzyme is predominantly expressed in the MC responsive cells of the kidney and other MC target tissues (colon, salivary glands) and the placenta (13).

The main structural features determining GC potency are the size and the polarity of the substituent in position 6 or 16. A hydrophobic residue increases GC activity (statistically significant enhancement with 6-α methyl and 16-methylene substitution). The more polar 16-hydroxy substitution decreases GC potency. The 6α and 9α-fluorination (such as in 6α and 9α fluorocortisol respectively) leads to increased GC and MC activity and double fluorination in the same positions augments this shift. Moreover, the Δ1-dehydro-configuration (in prednisolone) enhances GC activity but opposite to that effect it attenuates MC potency. The same effect is observed with the 16-methylene, 16α-methyl (dexamethasone) and 16β-methyl (betamethasone) groups. Thus, the more selective GC transactivation activity of GCs with a 16α-methyl or 16β-methyl group and a Δ1-dehydro-configuration, results from a significantly decreased activity via the mineralocorticoid receptor (MR) and an enhanced activity via the glucocorticoid receptor (GR) (15). Moreover, whereas GC selectivity can be improved by hydrophobic substituents in position 16 and the Δ1-dehydro-configuration, maximal GC activity needs additional fluorination in position 9α (such as in dexamethasone) (16). Figure 1 presents the chemical structures of cortisol and the most commonly used synthetic GCs.

Figure 1: Chemical Structures of the Most Commonly Used Synthetic GCs.

Protein binding is another pharmacokinetic property that influences GCs biological activity because only the unbound GC fraction is biologically active (14). In humans, endogenous cortisol binding to cortisol binding globulin (CBG) ranges between 67% and 87%, whereas a further 7-19% of total cortisol is bound to albumin, leading to about 95% of cortisol being protein-bound in the plasma. Except for prednisolone, synthetic GCs bind predominantly to albumin and only marginally to CBG. Plasma binding e.g. of dexamethasone and betamethasone is 75% and 60% respectively, and this is quite constant across a wide concentration rate (17). Thus, CBG binding is not a major determinant of plasma and biological half-lives of synthetic GCs.

However, especially for hydrocortisone and prednisone, pharmacokinetics are non-linear due to protein binding. As a result, higher doses result in more rapid clearance rates. It has to be mentioned that prednisone itself is biologically inactive and its 11-keto group must be reduced by hepatic 11βHSD1 to form the active drug, prednisolone. Moreover, clearance rate depends on age and is more rapid in children than adults (18) and also depends upon individual variability. Finally, certain diseases may influence synthetic GCs' pharmacokinetics. Thus, clearance is reduced particularly in renal and hepatic diseases and hypothyroidism and increased in hyperthyroidism. The concomitant use of other drugs influences synthetic GCs' half-lives and, thus, their final effect in target tissues (18, 19). Classic bioassays measure synthetic GC potency by testing the ability to suppress eosinophils and inhibit inflammation and the ability to stimulate hepatic glycogen deposition. The biologic effective half-life of glucocorticoids divides them into short-, intermediate-, or long-acting, based on the duration of corticotropin suppression after a single dose of the compound. The main corticosteroids used in clinical practice together with their relative biologic potencies and their plasma and biological half-lives are listed in Table 1.

Table 1: Glucocorticoid Equivalencies (11, 20, 21)
Glucocorticoids	Equivalent dose (mg)	Gluco-corticoid potency	HPA Suppression	Mineralo-corticoid potency	Plasma half-life (min)	Biologic half-life (h)
*Short-acting*
Cortisol	20.0	1.0	1.0	1.0	90	8-12
Cortisone	25.0	0.8		0.8	80-118	8-12
*Intermediate-acting*
Prednisone	5.0	4.0	4.0	0.3	60	18-36
Prednisolone	5.0	5.0		0.3	115-200	18-36
Triamcinolone	4.0	5.0	4.0	0	30	18-36
Methylprednisolone	4.0	5.0	4.0	0	180	18-36
*Long-acting*
Dexamethasone	0.75	30	17	0	200	36-54
Betamethasone	0.6	25-40		0	300	36-54
*Mineralocorticoids*
Fludrocortisone	2.0	10	12.0	250	200	18-36
Desoxycorticosterone acetate		0		20	70

SYSTEMIC GLUCOCORTICOID ADMINISTRATION

Therapeutic Indications

GCs are used in both endocrine and non-endocrine disorders (11, 22). First of all, they are administered as replacement therapy in patients with primary or secondary adrenal insufficiency, and as adrenal suppression therapy in congenital adrenal hyperplasia and glucocorticoid resistance (11). They are also used in patients with Grave's opthalmopathy and for some diagnostic purposes such as in establishing Cushing's syndrome (11). Moreover, due to their immunosuppressive and anti-inflammatory properties they are used in a broad range of non-endocrine disorders affecting many different systems (22, 23). Thus, they are given to treat skin disorders such as dermatitis and pemphigus, rheumatologic diseases such as systemic lupus erythematosus, polyarteritis and rheumatoid arthritis, and also polymyalgia rheumatica and myasthenia gravis. In hematology, they are used, along with chemotherapy, for the treatment of lymphomas and leukemias (24) and in hemolytic anemias and idiopathic thrombocytopenic purpura. In addition, they are administered in gastrointestinal diseases such as inflammatory bowel disease, in liver diseases (chronic active hepatitis) and in respiratory diseases (angioedema, anaphylaxis, asthma, sarcoidosis, tuberculosis, obstructive airway disease). Moreover, GCs are used in nephrotic syndrome and vasculitis and also in the suppression of the host-versus-graft and graft-versus-host reaction in cases of organ transplantation. In nervous disorders such as cerebral edema and raised intracranial pressure the use of GCs is also beneficial (25, 26).

Acute administration of pharmacologic doses of glucocorticoids is advocated in a small number of nonendocrine diseases, such as for patients suffering from acute traumatic spinal cord injury, although two recent meta-analyses support that the use of methylprednisolone should be limited (27, 28). Moreover, steroid administration should be considered as a post-operative additional therapy for cases with severe neurological deficits even after surgery (29). Glucocorticoids are also used for postoperative pain relief after severe bone operations (30). In addition, as it is known that premature birth is associated with an increased risk of neonatal mortality and morbidity, including respiratory distress syndrome (RDS), and because 7-10% of all pregnancies in North America are under such risk, in 1994 the National Institutes of Health (NIH) Consensus Developmental Conference on the Effects of Corticosteroids for Fetal Maturation on Perinatal Outcomes concluded that all fetuses between 24 and 34 week gestation at risk of preterm delivery should be considered as candidates for antenatal treatment with GCs. Recommended treatment consisted of 2 doses of 12mg betamethasone given IM 24 hours apart or 4 doses of 6mg dexamethasone given 12 hours apart. In 2001 the NIH Consensus Developmental Panel recommended that repeat courses should not be used routinely until insightful findings are available. However, the Australian Collaborative Trial (ACTORDS), that has been completed, reported that repeat course synthetic GCs improved short-term neonatal outcome compared to single course therapy (31).

Acute administration of pharmacologic doses of glucocorticoids is also necessary in some types of acute illness. For years it is known that any type of acute illness or trauma results in loss of the diurnal variation in cortisol secretion. In the early phase of critical illness cortisol levels frequently rise and levels of CBG and albumin are substantially depleted. In the chronic phase of critical illness, however, high ACTH and cortisol levels are generally sustained and CBG levels gradually increase. Both very high and very low cortisol levels have been associated with increased mortality from critical illness. High cortisol levels reflect severe stress, whereas low levels reflect an inability to sufficiently respond to stress (32). The term "critical illness-related cortisol insufficiency" (CIRCI) defines a state of both the inadequate production of GCs as well as a corticosteroid tissue resistance. It has been estimated that the overall incidence of adrenal insufficiency in critically ill patients is approximately 20%, with an incidence as high as 60% in patients with severe sepsis and septic shock. It is possible that CIRCI is an epiphenomenon and a marker of illness severity (33).

According to the current recommendations, CIRCI should be suspected in hypotensive patients who respond poorly to fluids and vasopressor agents, particularly in the setting of sepsis. To diagnose CIRCI, the clinician may use a delta serum cortisol <9 μg/dl after cosyntropin (250μg) administration or a random plasma cortisol <10μg/dl. The authors suggest that clinicians should not use plasma-free cortisol or salivary cortisol level over plasma total cortisol. For patients with septic shock that is not responsive to fluid and moderate- to high-dose vasopressor therapy, the authors suggest IV hydrocortisone < 400 mg/day for ≥ 3 days at full dose. They, however, do not suggest using corticosteroids in adult patients with sepsis without any evidence of shock. The dose regimen in patients with early moderate to severe ARDS is methylprednisolone 1mg/kg/day for at least 14 days. Finally, glucocorticoids are not suggested for cases of major trauma (34). In a second part of the guidelines, the authors formulated statements for or against the use of synthetic corticosteroids for other common pathologic conditions, including community-acquired pneumonia, influenza, meningitis, and non-septic systemic inflammatory response syndrome (SIRS) that may be associated with shock, namely burns, cardiac arrest and cardiopulmonary bypass surgery (35).

Benefits of GCs replacement has been demonstrated in a number of other patient populations including low cardiac output syndrome after cardiac surgery (36), acute exacerbation of chronic obstructive pulmonary disease (37), and cirrhosis (38).

Adverse Effects (AEs)

Although synthetic GCs remain an important component of therapy for many conditions, in recent years there are arguments against their use based mainly on the concern of toxicity. Nowadays, GCs toxicity is one of the commonest causes of iatrogenic illness associated with chronic inflammatory disorders. Despite the fact that the adverse effects of GCs have been known for decades, the actual risk-benefit ratio is incomplete and/or inconsistent. This happens because it is in general difficult to separate the effects of GCs from the outcome of the underlying disease, other comorbidities, or the use of other medications. Moreover, toxicity reports usually concern patients using high doses of GCs, different types of GCs with different relative drug potencies, for a heterogeneous group of related diseases, and for different periods of time (39, 40).

Only recently there has been intense effort by scientists and clinicians to explore and quantify the incidence and severity of the AEs of GC therapy. Generally, it is known that GCs' toxicity is related to both the average and cumulative dose during their use (41). The question that arises is whether or not patterns relating the frequency of AEs to GC dosage and/or length of GC treatment exist (39).

Historically, GCs at a prednisone equivalent of 5-10mg/day are considered low dose. However, a review of "the 4 extensively reviewed trials on low dose GCs in rheumatoid arthritis" led to the conclusion that definitive association of low dose GCs with many AEs such as osteoporosis, myopathy, cardiovascular disease, glaucoma, increased incidence of any kind of infection, and behavior disturbances remains elusive, and that the fear of GCs toxicity is probably overestimated based on extrapolation from observations with higher dose treatment. However, according to the same analysis, the use of 5-10mg/day of prednisolone (or equivalent) for over 2 years is associated with an increase of mean body weight in the range of 4-8% (40).

The prevalence of GC associated AEs was identified in a large survey of 2167 long term (≥60 days) users of GCs with mean prednisone equivalent dose of 16±14mg/day. The AE with the greatest prevalence was weight gain, experienced by 70% of the individuals, followed by skin bruising/thinning, and sleep disturbances. Cataracts (15%) and fractures (12%) were among the most serious AEs. All AEs demonstrated a strong dose-dependent association with cumulative GC use. Acne, skin bruising, weight gain and cataracts were significantly associated with longer duration (>90 days) of low-dose GCs (≤7.5mg/day of prednisolone), while fractures and sleep disturbances were more strongly associated with small increments in daily dosage (within the 0-7.5mg/d range). In conclusion, this survey adds further evidence that more GC associated AEs are dependent on both the average dose and the duration of therapy and that even low dose GC therapy could lead to serious AEs (42).

As in more severe cases of chronic inflammatory diseases long-term (≥ 1month) dosage of GCs is medium to low (≤30mg/d prednisolone or equivalent), a systematic review of 28 studies (2382 patients) concerning patients with rheumatoid arthritis (RA), polymyalgia rheumatica, and inflammatory bowel disease was the first to present a pooled analysis of the commonest reported AEs associated with this pattern of administration. The AE rate depends both on the quality of the study and primarily- on the disease in the study population. The overall mean rate of AEs was 150 per 100 patient-years, varying from 43/100patient years in rheumatoid arthritis and 80/100patient years in polymyalgia rheumatica to 555/100 patient years in inflammatory bowel disease. Psychological and behavioral disturbances (e.g. minor mood disturbances) were most frequently reported, followed by gastrointestinal events (e.g. dyspepsia, dysphagia) (43).

A recent retrospective population-based cohort study and self-controlled case series aimed to assess the risk of sepsis, venous thromboembolism, and fracture in 327,452 adults aged 18 to 64 years who received at least one prescription for less than 30 days over a three-year period (44). The authors found increased rates of sepsis (incidence rate ratio 5.30, 95% confidence interval 3.80 to 7.41), venous thromboembolism (3.33, 2.78 to 3.99), and fracture (1.87, 1.69 to 2.07), which decreased within the next 90 days. The increased risk for these adverse effects was observed at prednisolone doses lower than 20mg per day (44).

An important observational study aimed to identify patterns of self-reported health problems relating to dose and duration of GCs in 1066 unselected patients with RA (39). The study identified 2 distinct dose-related patterns of AEs. A continuous, approximately linear rising with increasing dose was found for cushingoid phenotype, ecchymosis, leg edema, mycosis, parchment-like skin, shortness of breath, and sleep disturbance. The most clearly attributable adverse drug reaction to GCs, Cushing syndrome, becomes evident after at least one month of treatment and was observed in 2.7, 4.3, 15.8, 24.6% of patients with no GCs in the past 12 months, and <5, 5-7,5 and >7,5mg/d of prednisolone or equivalent for >6 months, respectively. The second pattern identified describes an elevation in the frequency of health problems beyond a certain threshold value and is defined as a "threshold pattern". The threshold for the increase in glaucoma, depression, and an increase in blood pressure was observed at dosages of greater than 7.5mg/d. Dosages of 5mg/d or more were associated with epistaxis and weight gain. A very low threshold was observed for eye cataract (<5mg/d). All the associations found are in agreement with biological mechanisms and clinical observations (39). However, more extensive research on the risk-benefit ratio of long-term GCs is needed and could help to create new targets for drug development.

An overview of the most common and most serious AEs associated with GC therapy is discussed below.

ADRENAL INSUFFICIENCY (AI)

Iatrogenic, tertiary adrenal insufficiency induced by chronic administration of high doses of GCs is the most common cause of adrenal insufficiency (45). Physiologically, the hypothalamus secretes CRH which stimulates the release of ACTH from the anterior pituitary. ACTH leads to the release of cortisol from the zona fasciculata of the adrenal gland, which in turn exerts negative feedback on CRH and ACTH release. Administration of exogenous GCs even in small doses for only few days leads to a measurable suppression of the HPA axis by decreasing CRH synthesis and secretion and by blocking the trophic and ACTH-releasing actions of CRH on the anterior pituitary. This leads to suppressed synthesis of POMC, ACTH and other POMC derived peptides and later, to the atrophy of the corticotrophin cells of the anterior pituitary. As a result, in the absence of ACTH, the adrenal cortex loses the ability to produce cortisol. Nevertheless, the adrenal cortex retains the ability to secrete enough cortisol for some period of time and also mineralocorticoids, as this latter function depends mainly on the renin-angiotensin system rather than on ACTH.

The association between AI and treatment with oral GCs has been recognized for decades, although the magnitude of the risk has not been determined until recently. It has also been reported that the inhibition of the HPA axis function induced by exogenous GCs may persist for 6 to 12 months after treatment is withdrawn (46). Based on the literature the absolute risk of adrenal crisis after cessation of oral and inhaled GCs might be considered rare, but it is likely to be substantially underreported in clinical practice (10, 47).

The first study that quantified the increased risk of AI in people prescribed oral and inhaled GCs in the general population was published in 2006 (48). This case-control study, that used data from a cohort of 2.4 million people, found a strong dose-response relationship between oral GCs exposure and the risk of AI with an OR of 3.4 (95% CI, 1.6-2.5) per course of treatment per year. Furthermore, the study indicated, that administration of inhaled GCs within 90 days of diagnosis is associated with an increased risk of AI (OR 3.4, 95% CI 1.9-5.9) and this effect was dose related. However, after adjustment for oral GCs exposure, this association was reduced (OR 1.6, 95% CI 0.8-3.2) although the dose relation remains. The largest increase in risk occurred in association with a recent prescription for fluticasone proprionate (48). These findings were confirmed by more recent studies that aimed to investigate the prevalence of AI in patients treated either with inhaled (49) or with oral GCs (47, 50). Interestingly, in a recently published systematic review the authors found that the percentage of patients with glucocorticoid-induced AI had a median (IQR) of 37.4%, ranging from 13%-63% (51). Three years after glucocorticoid withdrawal, AI persisted in 15% of retested patients. AI occurred in patients receiving <5mg prednisolone equivalent dose/day, for less than 4 weeks, and with a cumulative dose <0.5g (51).

CARDIOVASCULAR DISEASE

A population-based study that compared the risk for CVD in 68,781 patients using GCs versus 82,202 nonusers identified that the relative risk for a cardiovascular event in patients receiving high-dose GCs (≥7,5mg/d prednisolone) was 2.56 (CI 2.18-2.99) after adjustment for known covariates (52). Similar associations were noted in another observational study that included 50,656 patients and an equal number of matched controls. According to this study, current use of GCs was associated with an increased risk of heart failure (OR 2.66, 95% CI 2.46-287) and a smaller risk of ischemic heart disease (OR 1.20, 95% CI 1.11-1.29) (53). However, the previous results are not confirmed by other studies (54, 55). Additionally, an association of GCs use and the risk for atrial fibrillation and flutter has been established by several studies (56-58).

GLUCOSE HOMEOSTASIS

The alterations in glucose homeostasis induced by GCs are multifactorial and could be explained by several potential mechanisms including the induction of enzymes involved in hepatic gluconeogenesis, the decrease in glucose uptake in peripheral tissues, the stimulation of lipolysis, the prevention of insulin production, and the induction of ceramide biosynthesis leading to insulin resistance (59). An interesting review of the existing literature published between 1950-2009 shows that GC-induced hyperglycemia is common among patients with and without diabetes mellitus. The OR for new onset diabetes mellitus ranges from 1.5 to 2.5 and the induction of the disease is strongly predicted by GC accumulative dose and duration of therapy (60).

INFECTIOUS EVENTS

GC therapy is associated with an increased risk of infectious complications, as GCs are known to have suppressive effects upon both innate and acquired immunity. This is confirmed by several studies. According to a large observational study of 16,788 patients with RA, prednisone use, even at doses of 5mg/kg, increased the risk of hospitalization for pneumonia. Furthermore, there was a dose related relationship between prednisone use and pneumonia risk in RA (61). Another study of 15,597 patients with RA found that GC use doubled the rate of serious bacterial infections compared with methotrexate with a dose response relationship for doses greater than 5mg/d (62). The latter results were confirmed by additional studies that have identified GCs as an independent risk factor for infections (63, 64). Moreover, a more recent study demonstrated that patients receiving 5mg prednisolone continuously for the last 3 months, 6 months or 3 years, had a 30%, 46% or 100% increased risk of serious infection, respectively (65). Also, caution about GC use in patients with active or dormant TB is well accepted as these individuals are susceptible to contract or to sustain activation of the disease (66). An epidemiological study of patients with TB showed that they were nearly 5 times more likely to have been using GCs at the time of their diagnosis (67).

OSTEOPOROSIS

The effects of GCs on bone homeostasis are both systemic and local. Systemic effects include a reduction in calcium absorption from the intestine and a reduction in calcium reabsorption in the kidney, both enhancing PTH secretion and thus bone loss. Furthermore, the attenuation of sex steroids and growth hormone by GCs enhances bone loss. The direct effects of GCs on bone cells include induction of osteoblast and osteocyte apoptosis through activation of pro-apoptotic molecules, impairment of Wnt signaling, and induction of RANKL, a potent stimulator of osteoclastogenesis produced by osteoblasts (68). As a result, GCs induced osteoporosis is the most common type of iatrogenic osteoporosis. This has been confirmed by several studies. One of them showed that therapy with high-dose oral GCs caused significant decrease in BMD even in the first 2 months of therapy (69). As a result, there is an increased risk of osteoporotic fractures (70) and it has been estimated that fractures may occur in up to 30-50% of patients on GC therapy (71) but fortunately there is a rapid decrease of the risk on cessation of therapy (70, 72). Similar findings were observed by a more recent study showing that low daily dose prednisone (≤7,5mg/d) with high cumulative doses increases the risk for fractures. Intermittent high-dose regimens with cumulative doses less than 1gr, however, did not show an increased risk. Risk declines rapidly, the decrease beginning 3 months after cessation of therapy (73). For additional details please see the Endotext chapter on Glucocorticoid-induced osteoporosis (74).

NEUROPSYCHIATRIC EVENTS

Despite a slight increase in their overall sense of well-being independent of improvement in disease activity, it has been established that synthetic GC treatment may induce behavioral, psychic, and cognitive disturbances (75). These disturbances can be detected by structural, functional, and spectroscopic imaging. Behavioral changes in feeding and sleeping are commonly observed. Among psychic AEs, hypomania and mania are the most common during acute GC therapy and depression during long-term treatment. Suicides have also been reported (76). These AEs are usually mild/moderate but are severe in 5-10% of cases. Cognitive changes affect mostly declarative and working memory. All these AEs are generally dose and time dependent (infrequent at prednisone equivalent doses <20mg/d) and usually reversible. There has to be greater concern for pediatric patients. Several medications such as lithium, phenytoin, lamotrigine, memantine and other anti-seizure, anti-psychotics, and anti-depressants could be useful for treating such disorders (77, 78).

PEDIATRIC EVENTS

Prolonged GC treatment of children with chronic illnesses impairs their longitudinal growth (79). GCs exert multiple growth suppressing effects, such as inhibition of GH secretion and IGF-1 expression, reduction of bone and collagen formation, bone mineralization, and vascularization. These effects are more pronounced with daily oral GCs than alternate day oral GC therapy (80). According to a study of 224 children with cystic fibrosis who have received alternate day treatment with prednisone, boys but not girls, had persistent growth impairment (mean final height 4cm less than children who were treated with placebo) after discontinuation of treatment (81).

Apart from growth retardation, children may also be more susceptible to other AEs associated with GCs such as osteoporosis, glaucoma and cataracts. Moreover, fracture risk seems to be higher in GC-treated children (82).

Intrauterine exposure to GCs is able to affect fetal HPA axis development causing reduction in fetal and, in some cases newborn and infant HPA axis activity under basal conditions and more consistently after pain-related stress. Although baseline HPA axis function seems to recover within the first 2 weeks postpartum, there is initial evidence that blunted HPA axis reactivity to pain-related stress persists throughout the first 4 months of life. These effects are dose dependent and vary with the time between GC exposure and HPA assessment. It seems that programming of the HPA axis involves interaction with other endocrine systems such as the Hypothalamus-Pituitary-Gonadal axis (HPG). Moreover, exposure to GCs during pregnancy has been linked to impaired fetal growth and modulated fetal immune functions, indicators of compromised cognitive, neurological and psychological functions, and increased blood pressure into adolescence. Furthermore, there is some evidence that reduced HPA axis activity early in life will switch to a hyperactive state later in life due to over-adjustment and because of that, affected infants may be vulnerable to stress related disorders associated with hypercortisolemia such as depression and cardiovascular disease. Finally, it seems that changes in HPA axis function following antenatal exposure to GCs are trans-generational and likely involve epigenetic mechanisms (17).

In addition, according to a recent meta-analysis, early postnatal GC treatment (≤7 days), particularly with dexamethasone, causes short term AEs including gastrointestinal bleeding, intestinal perforation, hyperglycemia, hypertension, hypertrophic cardiomyopathy and growth failure (83, 84). Long term follow-up studies report an increased risk of abnormal neurological examination and cerebral palsy (85).

PHEOCHROMOCYTOMA CRISIS

Severe isolated cases of pheochromocytoma crisis have been reported after administration of exogenous GCs (86, 87). Thus, GCs should be avoided or administered only if absolutely necessary in patients with known or suspected pheochromocytomas.

The most common AEs of GC therapy are summarized in Table 2.

Table 2: Common AEs of Glucocorticoid Therapy (88)

Onset early in therapy, essentially unavoidable

Emotional lability

Enhanced appetite, weight gain, or both

Insomnia

Enhanced in patients with underlying risk factors or concomitant use of other drugs

Glucocorticoid-related acne

Diabetes mellitus

Hypertension

Peptic ulcer disease

When supraphysiologic treatment is sustained

Cushingoid appearance

Hypothalamic-pituitary-adrenal suppression

Impaired wound healing

Myopathy

Osteonecrosis

Increased susceptibility to infections

Delayed and insidious, probably dependent on cumulative dose

Atherosclerosis

Cataracts

Fatty liver

Growth retardation

Osteoporosis

Skin atrophy

Rare and unpredictable

Glaucoma

Pancreatitis

Pseudotumor cerebri

Psychosis

COMPARTMENTAL GLUCOCORTICOID ADMINISTRATION

Topical Glucocorticoids

Glucocorticoids are the first line of treatment for various skin disorders such as atopic dermatitis, vitiligo, psoriasis, etc. (10, 89-94). They are quite effective when applied topically and nontoxic to the skin in the short term. The factors that determine local penetration are the structure of the compound employed, the vehicle, the basic additives, occlusion versus open use, normal skin versus diseased skin, and small areas versus large areas of application. Fluorinated steroids (e.g. dexamethasone, triamcinolone acetonide, betamethasone, and beclomethasone) penetrate the skin better than nonfluorinated steroids, such as hydrocortisone. However, fluorinated steroids also produce more local complications and may be associated with systemic absorption and side effects.

The most frequent AEs are local and include atrophy, striae, rosacea, perioral dermatitis, acne, and purpura. Less frequently, hypertrichosis, pigmentation alterations, delayed wound healing, and exacerbation of skin infections occur. Furthermore, the rate of contact sensitization against GCs is greater than previously believed. Systemic reactions such as hyperglycemia, glaucoma and adrenal insufficiency are less frequent (95). Some cases of Cushing's syndrome following overuse of topical GCs have also been described (96). The frequency of systemic effects by topical corticosteroids is increased in newborns and small children compared to adolescents and adults, because GCs penetrate the skin of newborns and small children more easily and in larger proportional amounts. Infants, especially, have a greater risk for Cushing's syndrome or adrenal insufficiency and also hepatosteatosis. An infant's death due to generalized CMV infection following administration of topical GCs has been reported (97). Based on the Body Surface Area, a simple guideline for how much topical GC to prescribe for a child has been proposed. Roughly, infants require one fifth of adults' doses, children two fifths and adolescents two thirds of adults' doses (98). Finally, the use of skin lightning cosmetics used in most African countries includes corticosteroids and may have many serious and sometimes fatal complications, including adrenal insufficiency (99).

Opthalmic Glucocorticoids

In the past 10 years intravitreal GCs injections have been increasingly used for patients with a variety of posterior segment diseases, including diabetic macular edema, branch and central retinal vein occlusion, pseudophakic cystoid macular edema, and uveitic macular edema (100). Currently, novel agents including preservative-free and sustained-release intravitreal implants are being studied in clinical trials. Potential complications of intravitreal steroid treatment are divided into steroid-related and injection-related side effects. Steroid-related side effects include cataract formation and glaucoma (101). Injection related side effects include retinal detachment, vitreous hemorrhage, and bacterial and sterile endopthalmitis.

Inhaled Glucocorticoids

GC inhalation therapy is widely used in patients with asthma and chronic obstructive pulmonary disease. Their relative topical to systemic effect ratio or therapeutic index depends upon the pharmacokinetic differences for inhaled GCs. Factors that enhance the therapeutic index are: decreased oral absorption retention in the lung and rapid systemic clearance once the drug is absorbed into the systemic circulation. More recently, it has been posited that the therapeutic index is also enhanced by high plasma protein binding. Inhaled GCs have important pharmacokinetic differences (102).

In general, inhaled GCs have fewer and less severe AEs than oral and systemic GCs. However, systemic AEs may be observed and this risk is influenced by the dose, the period of treatment, the delivery system used, the site of delivery (i.e. gastrointestinal tract, lung), the concomitant use of other medications, and the altered steroid metabolism due to individual's differences in the patient's response to GCs.

As far as growth deceleration in children is concerned, the results are somewhat contradictory. Although inhaled GCs seem to cause a dose-dependent reduction in height velocity (103), these changes are not significantly associated with final adult height (104). However, a study of 1041 asthmatic children treated with budesonide, nedocromil, and placebo for 4.3 years, a decrease in growth velocity was observed in the budesonide group which was most evident in the first year of treatment (105). When 90% of these children were followed-up for an additional 4.8 years, a lower mean height was found in the budesonide group and this was more pronounced in girls than in boys (106).

Moreover, as GCs effect on bone metabolism is of great concern, some studies have shown that inhaled GC therapy is associated with increased fracture risk (107, 108) but this has not been confirmed by a meta-analysis (109). Nevertheless, several studies confirm a negative relation between total accumulative dose of inhaled GCs and bone mineral density (110). The loss in BMD is of concern, especially in early postmenopausal women and boys during puberty (111, 112). Again, these results have been argued (113).

It has been shown that adrenal insufficiency is also associated with inhaled GCs, although with lower prevalence (114). The newest inhaled GC, Ciclesonide, appears to have different pharmacokinetics enhancing its therapeutic index. It is administered as a pro-drug converted to the active metabolite des-Ciclesonide in the lung. Thus, it has low oral bioavailability and also rapid clearance and high protein binding, factors that reduce pharmacologically relevant systemic exposure (115). Furthermore, Ciclesonide appears to have less suppressive effects on HPA axis function (116, 117).

Nasal Glucocorticoids

Intranasal GCs are effectively used for the treatment of allergic rhinitis, rhinosinusitis, rhinoconjunctivitis, and nasal polyposis (118, 119). Topical steroid drops are used for the treatment of sinus ostia stenosis in the postoperative period (120). Interestingly, molecules designed specifically to achieve potent localized activity with minimum risk of systemic exposure such as mometasone furoate, fluticasone proprionate, and fluticasone furoate may be preferable. Studies in children have not found any adverse effects including HPA axis suppression or growth retardation (118). Yet, some studies suggest a relationship between intranasal steroids and increased intraocular pressure (119). Generally, frequent and chronic use should be avoided to prevent local and systemic complications (121).

Intraarticular Glucocorticoids

The main beneficial effect of intraarticular GC injection is pain relief. Most favorable results are seen in juvenile idiopathic arthritis patients. Local AEs are either rare or insignificant and include joint infection, intraarticular and periarticular calcifications, cutaneous atrophy, cutaneous depigmentation, avascular necrosis, rapid destruction of the femoral head, acute synovitis, Charcot's arthropathy, tendinopathy, Nicolau's syndrome, and joint dislocation (122). Moreover, some systemic AEs have also been reported. These include a transient HPA axis suppression, a transient increase in blood glucose in diabetic patients, and other metabolic, hematologic, vascular, allergic, visual and psychological AEs (123). The most used intraarticular glucocorticoids are triamcinolone hexacetonide, triamcinolone acetonide, and methylprednisolone acetate (124).

MONITORING OF PATIENTS ON GLUCOCORTICOID TREATMENT

As osteoporosis, with resultant fractures, constitutes one of the most serious morbid complications of GC use, worsening patients quality of life, recently, the American College of Rheumatology (ACR) updated the 2010 recommendations for patients receiving oral GC therapy (125). In this systematic review, the authors addressed the initial assessment in patients that began or continue glucocorticoid treatment for longer than 3 months, and discussed the advantages and disadvantages of lifestyle modification, as well as for calcium, vitamin D and pharmaceutical treatment, including bisphosphonates, raloxifene, teriparatide, and denosumab. According to the ACR guidelines, there are three categories of fracture risk. High risk criteria include patients aged over 40 years, previous osteoporotic fracture, hip or spine BMD T-score ≤ −2.5, or 10-year fracture probability of ≥20% (major osteoporotic fracture) or ≥3% (hip fracture) (125, 126). Moderate and low risk criteria are based only on FRAX-derived fracture probability (10–19% and >1 to ≤3% respectively for moderate

risk, and <10% and ≤1% respectively for low risk) (125, 126). In patients aged less than 40 years, a previous osteoporotic fracture is considered as a high-risk criterion, whereas the criteria of moderate and low risk are based on the BMD. Patients at low fracture risk are recommended to receive only calcium and vitamin D, whereas adults at moderate-to-high fracture risk should be treated with calcium and vitamin D plus an oral bisphosphonate. However, adults in whom oral bisphosphonates are not appropriate, are recommended to continue calcium plus vitamin D but switch from an oral bisphosphonate to another anti-fracture medication (125). Finally, adults who complete a planned regimen with oral bisphosphonates and continue glucocorticoid treatment, are recommended to continue oral bisphosphonate treatment or switch to another anti-fracture medication (125). Recommendations were also suggested for children, women of childbearing potential, and people with organ transplants, as well as patients receiving very high doses of glucocorticoids (125). For additional details please see the Endotext chapter on Glucocorticoid-induced osteoporosis (74).

CONCOMITANT USE OF GLUCOCORTICOIDS WITH OTHER DRUGS

Special attention is required in the concomitant use of glucocorticoids with other drugs because of potential interactions, and because some drugs may affect the metabolism of the steroids, which may lead to a decreased or increased glucocorticoid effect on their target tissues (20). Such interactions and effects are shown in Tables 3, 4 and 5.

Table 3: Interactions of Glucocorticoids with Other Drugs (20)
Drug	Side effect	Comments
Amphotericin B	Hypokalemia	Monitor potassium levels frequently
Digitalis glycosides	Digitalis toxicity Hypokalemia	Monitor potassium levels frequently
Growth hormone	Ineffective	-
Potassium-depleting diuretics	Hypokalemia	Monitor potassium levels frequently
Vaccines from live attenuated viruses	Severe generalized infections	-

Table 4: Effects of Glucocorticoids on Blood Levels of Other Drugs (20)
Drug	Drug blood levels	Comments
Aspirin	Decreased	Increased metabolism or clearance. Monitor salicylate level
Coumarin anticoagulants	Decreased	Frequent control of prothrombin levels
Cyclophosphamide	Increased	Inhibition of hepatic metabolism. Adjust the dosage
Cyclosporine	Increased	Inhibition of hepatic metabolism
Insulin	Decreased	Adjust the dosage of the drug
Isoniazid	Decreased	Increased metabolism and clearance
Oral hypoglycemic agents	Decreased	Adjust the dosage of the drug

Table 5: Effect of Drugs on Plasma Glucocorticoid Concentrations (20, 127, 128)
Drug	Drug blood levels	Comments
Antacids	Decreased	Possible physical absorption to antacid
Carbamazepine	Decreased	Increased cytochrome P450 activity
Cholestyramine	Decreased	Decreased gastrointestinal absorption of glucocorticoids
Colestipol	Decreased	Decreased gastrointestinal absorption of glucocorticoids
Cyclosporine	Increased	Inhibition of hepatic metabolism
Ephedrine	Decreased	Probably increased metabolism
Erythromycin	Increased	Impaired elimination
Itraconazole	Increased	Decreased cytochrome P-450 activity
Mitotane	Decreased, with elevated transcortin	Total plasma cortisol unreliable. Adjust glucocorticoid levels
Oral contraceptives	Increased	Impaired elimination, increased protein binding
Phenobarbital	Decreased	Increased cytochrome P-450 activity. Adjust glucocorticoid dosage
Phenytoin	Decreased	Increased cytochrome P-450 activity. Adjust glucocorticoid dosage
Rifampin	Decreased	Increased cytochrome P-450 activity (?) Adjust glucocorticoid dosage
Ritonavir	Increased	Decreased cytochrome P-450 activity
Troleandomycin	Increased	Partially resulting from impaired elimination

PREDICTING GLUCOCORTICOID-INDUCED HPA AXIS SUPPRESSION

Several predictors of glucocorticoid-induced HPA axis suppression have been discussed, the major of which are the following:

Kind of Steroid Used and GC Potency

As shown in Table 1 long acting preparations have a longer tissue life which induces a chronic state of tissue hypercortisolism, making HPA axis suppression more likely. Thus, hydrocortisone and cortisone acetate are the least potent and, therefore, least suppressive agents. Prednisone, prednisolone, methylprednisolone and triamcinolone are moderately suppressive, and dexamethasone suppresses ACTH the longest.

Systemic Versus Compartmental Therapy

Systemic GC therapy, particularly parenterally, is more likely to suppress the HPA axis. However, other routes of administration such as inhalation, topical, intra-ocular cause HPA axis suppression as well as other systemic AEs and this depends on the systemic bioavailability of the drug (19, 48, 49, 95, 114, 123).

Daily Therapy

There is evidence that patients are at lower risk for adrenal insufficiency if they can take glucocorticoids on alternate days from the outset or if they can convert to alternate-day therapy before the HPA axis is suppressed (21, 129).

Split Doses and Night Doses

Administering GCs in several different doses during the day imposes a greater risk for HPA axis suppression. In the same way, evening doses of glucocorticoids tend to suppress the normal early morning surge of ACTH secretion, resulting in greater adrenal suppression. Whenever possible, it is better to treat patients with a single morning dose. Once-a-day dosing is usually feasible for intermediate or long acting GCs e.g. prednisone, triamcinolone and dexamethasone. The short-acting hydrocortisone and cortisone acetate are usually given twice a day, at waking and around 5 PM. To mimic normal diurnal cortisol rhythms, the morning dose is two thirds, and the afternoon dose one third of the total daily dose (19, 130, 131).

Duration and Cumulative Dose of Glucocorticoid Treatment

Although traditionally the duration of glucocorticoid therapy and the cumulative dose of glucocorticoid received have been considered as predictive of the likelihood of HPA axis suppression, several studies suggest that they only roughly predict HPA axis suppression (132-134). Adrenal insufficiency is extremely rare in patients treated for 1 week or less (135, 136). Nevertheless, with a so called "short-term" 14 day course of systemic GCs, generally considered safe, in patients with acute exacerbation of chronic obstructive pulmonary disease, suppression of the HPA axis has been defined (47).

Cushingoid Features

Patients with Cushing's syndrome symptoms due to GC therapy are more likely to have a suppressed HPA axis and adrenal atrophy (19).

It has been suggested that the best predictor of HPA axis suppression is the patient's current glucocorticoid dosage. A strong correlation has been found between prednisone maintenance doses above 5 mg/d and a subnormal ACTH-stimulation test result (137). Finally, it can be assumed that patients who are more likely to develop HPA axis suppression are those who receive high doses (>20-30mg prednisolone or equivalent) of systemic GCs for long periods (>3weeks) and those who appear to have Cushingoid features. As the HPA axis function in patients treated with synthetic GCs cannot be reliably estimated from the above parameters several tests are commonly used in order to assess the axis' recovery.

WEANING PATIENTS FROM GLUCOCORTICOID THERAPY

Besides their multiple therapeutic uses, GC withdrawal is indicated when their use is no longer recommended as the maximum therapeutic benefit has been obtained or when significant side effects appear and become uncontrollable, such as GC induced psychosis, diabetes mellitus, severe hypertension, and incapacitating osteoporosis. The goal of a successful GC withdrawal regimen can be described as the rapid transition from a state of tissue hypercortisolism to a state of total exogenous GC deprivation without resurgence of the underlying disease and without adrenal insufficiency or any other GC dependency. Although there are no consensus documents, several tapering regimens have been published so far. In clinical practice, the majority of physicians develop their own withdrawal regimens. The common point is that GC withdrawal should never be abrupt (19).

A systematic review published in 2002 found 9 randomized, controlled clinical trials, 7 of which investigated bronchial asthma and chronic obstructive pulmonary disease, which compared different GC tapering regimens. According to this review there was no significant difference between rapid or slow tapering, regarding the diseases' exacerbation and relapse rates, suggesting that prolonged withdrawal may not be necessary for a better outcome of the underlying disease. However, the same review highlighted the uncertainty about the safety and efficacy of GC withdrawal in many chronic diseases, emphasizing the need for further research in this area (131).

In general, patients taking any steroid dose for less than 2 weeks are not likely to develop HPA axis suppression and can stop therapy suddenly without tapering. The possible exception to this is the patient who receives frequent "short" steroid courses e.g. in asthma. Where there has been chronic therapy, the objective is to rapidly reduce the therapeutic dose to a physiological level (equivalent to 7.5mg/d prednisolone) e.g. by reducing 2.5mg every 3-4 days over a few weeks, and then proceed with slower withdrawal in order to permit the HPA axis to recover (19, 21).

As far as patients with underlying disease are concerned it is recommended that all available clinical, biochemical and laboratory data on the activity status of the disease be collected in order to easily identify signs of recurrence. In such a case prescribed doses should be increased (19).

After the initial reduction to physiological levels, doses should be reduced by 1mg/d of prednisolone or equivalent every 2-4 weeks depending upon patient's general condition, until the medication is discontinued. Alternatively, after the initial reduction to 5-7.5mg of prednisolone, the clinician can switch the patient to HC 20mg/d and reduce by 2.5mg/d every week until the dose of 10mg/d is achieved. After 2-3 months on the same dose, the HPA axis function should be assessed through a Corticotropin (ACTH-Synachten) test or through an Insulin Tolerance test (ITT). A pass response to these tests indicates adequate function of the axis and GCs can be safely withdrawn. If the axis has not fully recovered, treatment should be continued and the axis function should be reassessed (21).

Other tapering regimens have been published some of them dealing with switching the patient to an alternate dosage of GC before discontinuation (138).

Irrespectively of the tapering regimen used, if GC withdrawal syndrome, adrenal insufficiency's symptomatology, or exacerbation of the underlying disease appears, the dose being given at the time should be elevated or maintained for a longer period of time. Moreover, in the absence of evidence of HPA axis full recovery in patients who have been treated with GCs for prolonged periods, supplementation equivalent to 100-150mg of HC is recommended during situations of severe stress such as major surgery, fractures, severe systemic infections, major burns, etc.

Finally, it has become obvious, that all patients treated long-term with GCs should be treated in a similar fashion to patients with chronic ACTH deficiency, thus, they should be instructed to carry some type of identification (worn around the neck or wrist or carried as a card) (19, 21).

ACUTE ADRENAL CRISIS

Full HPA axis recovery after cessation of GC therapy may take as long as 1 year or more (11, 139). Abrupt cessation of glucocorticoid treatment or quick tapering can precipitate an acute adrenal insufficiency crisis. The main symptoms range from anorexia, fatigue, nausea, vomiting, dyspnea, fever, arthralgia, myalgia, and orthostatic hypotension to dizziness, fainting, and circulatory collapse. Hypoglycemia is occasionally observed in children and very thin adult individuals. The diagnosis is a medical emergency, and treatment should be immediate administration of fluids, electrolytes, glucose, and parenteral glucocorticoids.

GLUCOCORTICOID WITHDRAWAL SYNDROME (GWS)

Chronic administration of high doses of GCs and also other hormones such as estrogens, progestins, androgens and growth hormone induce varying degrees of tolerance, resulting in a progressively decreased response to the effect of the drug, followed by dependence and rarely "addiction". Traditionally, the term "Endocrine Withdrawal Syndromes" has been used to describe symptoms and signs of specific hormone deficiency after discontinuation of hormonal therapy or removal of an endocrine gland. However, discontinuation of hormonal therapy frequently results in a mixed picture of two different syndromes: a typical hormone deficiency syndrome and a generic withdrawal syndrome. Four aspects of GCs withdrawal after cessation of pharmacological high-dose therapy are important: 1) relapse of the underlying disease for which the drug was prescribed 2) HPA axis suppression which can persist for a long time 3) psychological dependence 4) a non-specific withdrawal syndrome despite normal HPA axis function and even while patients are receiving physiological replacement doses of GCs (140, 141).

Amatruda et al. first defined the steroid withdrawal syndrome as a symptom complex resembling true adrenal insufficiency, with nonspecific symptoms like weakness, nausea, and arthralgias, occurring in patients who have finished a dosage reduction of glucocorticoid therapy and who respond normally to HPA axis testing (142). Thus, after cessation of GC therapy patients may develop anorexia, nausea, emesis, weight loss, fatigue, myalgias, arthralgias, weakness, headache, abdominal pain, lethargy, postural hypotension, fever, skin desquamation, tachycardia, emotional lability, and even delirium, and psychotic states even if the response of the HPA axis to stimuli has returned to normal (140). Children and adolescents may experience signs and symptoms of GWS even when GCs are still being administered in supraphysiological doses (19). Biochemical evidence related to the GWS includes hypercalcemia and hyperphosphatemia (140).

The GWS has been considered a withdrawal reaction due to established physical dependence on supraphysiological GC levels (140). It has also been described as a state of relative GC resistance in these patients, effectively rendering them hypoadrenal (141). The mechanisms responsible for GWS have not been fully elucidated. Nevertheless, several mediators should be considered and include CRH, vasopressin, POMC, several cytokines such as IL-1β, IL-6, TNF-α, prostaglandins such as E2, I2, phospholipase A2 and also alterations of the noradrenergic and dopaminergic systems (19, 140).

The severity of GWS depends on the genetics and developmental history of the patient, on his environment, and on the phase and degree of dependence the patient has reached (140). The syndrome is self-limited with a median duration of 10 months. Its management should include a temporary increase in the dose of GCs followed by gradual, slow tapering to a maintenance dose (141).

BIOCHEMICAL DIAGNOSIS OF ADRENAL INSUFFICIENCY

Glucocorticoid treatment may not suppress the HPA axis at all, or it may cause central suppression and adrenal gland atrophy of varying degrees. Several endocrine tests have been used to define progression of glucocorticoid-induced adrenal insufficiency. The insulin tolerance test and the metyrapone test have been employed in the diagnosis of adrenal suppression and are quite sensitive, however, the risks involved with both tests do not justify their use when a rapid ACTH stimulation test can distinguish clinically significant adrenal suppression.

To evaluate the adequacy of hypothalamic-pituitary-adrenal axis recovery, the rapid Synachten (or high-dose ACTH stimulation test) is mostly used. An intravenous bolus of 250 ug of corticotropin 1-24 is administered and cortisol is measured after 30 or 60 minutes or both. A plasma cortisol concentration > 18 - 20 μg/ dL at these times indicates adequate recovery of the hypothalamic-pituitary-adrenal axis (139).

The low-dose Synachten test (1ug or 500 ng ACTH(1-24)/1.73 m2) is also being used for the assessment of the HPA axis after prolonged use of GC medication (143-145). It is unclear if the low-dose test is superior to the high-dose test for the detection of secondary adrenal insufficiency. Some studies have shown that the low-dose Synachten test is more sensitive in detecting partial secondary adrenal insufficiency (as can occur in chronic use of GCs), which is not detected by the standard high-dose test because the latter provides a supraphysiologic stimulus able to stimulate a partially damaged adrenal (146-149). A meta-analysis of 28 studies evaluated the utility of the high and low-dose ACTH test. At a specificity of 95% the sensitivity of the high-dose test for primary adrenal insufficiency was 97%, greater than that for secondary adrenal insufficiency (57%). The sensitivities for secondary adrenal insufficiency were similar between the high-dose (57%) and the low-dose Synachten test (61%) (150). In contrast, a review of the literature published between 1965 and 2007 suggests that the low-dose test is the best test currently available for establishing the diagnosis of secondary AI (151). Further studies are needed to establish if the low-dose Synachten test is preferable for the diagnosis of secondary AI.

The Corticotropin Releasing Hormone (CRH) test can also be used in patients taking GC treatment for prolonged periods, as it can assess both the ACTH and cortisol responses and can distinguish between secondary and tertiary adrenal insufficiency (133, 152).

The Dexamethasone Suppression Test has been shown to predict the later development of an impaired adrenal function after a 14-day course of prednisone in healthy volunteers and this information may allow a more targeted approach for the patients after cessation of steroid therapy (153).

FUTURE PERSPECTIVES ABOUT GLUCOCORTICOID THERAPY

Although hydrocortisone (HC) is the most commonly used regimen for replacement in patients with primary and secondary adrenal insufficiency, it is evident that this conventional therapy cannot provide the physiological rhythm of cortisol release. Moreover, with current replacement therapy, the majority of patients with adrenal insufficiency report impaired health-related quality of life, early morning fatigue, socioeconomic health problems and, finally, increased mortality (154). Circadian infusions of HC delivered by a programmable pump can mimic the normal rhythm of cortisol secretion and improve biochemical control and quality of life in patients with adrenal insufficiency and congenital adrenal hyperplasia. Because such infusions are not a practical solution, new formulations of oral HC, which mimic cortisol physiology have been evaluated. A dual-release hydrocortisone tablet with an immediate-release outer layer covering a sustained-release core, has been used in patients with Addison’s disease showing improvements in cardiovascular risk factors, including body weight, hemoglobin A1C and blood pressure, as well as a significant improvement in fatigue (154-156). In the long-term, this once-daily formulation was well-tolerated with a small number of adverse effects (157). Another modified-release multi-particulate hydrocortisone capsule formulation has been developed recently (158). This formulation was well tolerated and very effective in controlling disease biomarkers of congenital adrenal hyperplasia, such as androstenedione and 17-hydroxyprogesterone, with a lower hydrocortisone dose equivalency (154).

Apart from their use for hormonal replacement, the clinical success of synthetic GCs as anti-inflammatory agents is largely attributed to their ability to reduce the expression of proinflammatory genes, via activation of the GR and the concomitant inhibition of the activity of proinflammatory transcription factors, including NF-κB and AP-1, through a mechanism called trans-repression. On the other hand, the appearance of their AEs mainly arise from their ability to activate, after induction of the GR, target genes involved in the metabolism of sugar, protein, fat, muscle and bone via a mechanism called trans-activation (159, 160). There is a plethora of recent work dealing with the characteristics of novel selective GR ligands with equal efficacy and improved side-effects profiles, in other words ligands that show an improved therapeutic index (159-162). These efforts have resulted in a number of different terminologies: Selective GR modulators, selective GR agonists, gene-selective compounds, dissociated compounds, etc. (161, 163), which have been developed and are still being developed mainly focusing on the trans-repression mechanism and stimulating the side-effect pathway to a lesser extent, at least in specific tissues. Nevertheless, the likelihood of finding a compound that actually separates all activated genes from all repressed genes is highly unlikely mainly because the transactivation vs trans-repression characteristics are highly cell-type and gene specific. Moreover, it is also unclear whether such a compound would be truly desirable, as upregulation of anti-inflammatory genes may also play a role in the treatment of many diseases (159-161). In addition, many non-steroidal dissociated GR modulators, some of which do not support trans-activation, have shown promising benefit to side-effect ratios (e.g. ZK216348, CpdA) (159).

Considering the complexity of pathways regulated by GR, it is clearly too naive to assume that an ideal exogenous GR modulator only eliciting the beneficial anti-inflammatory effects without any trace of side-effects will ever be found. Complementing genome-wide gene profiling studies and transcription factor/DNA binding patterns on various target tissues at once will become an adamant strategy for the future (159). However, recent reports of Selective GR modulators provide fertile ground for additional efforts and it is obvious that any progress in this area would be a major benefit for thousands of patients receiving GC therapy (161).

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Adrenocortical Carcinoma

CLINICAL RECOGNITION

Adrenocortical cancer (ACC) is a rare disease with an annual incidence of 0.7-2 cases per million per year and two distinct age distribution peaks, the first occurring in early adulthood and the second between 40-50 years with women being more often affected (55-60%). Although the great majority of ACCs are of sporadic origin, they can also develop as part of familial diseases the most common being the Beckwith-Wiedeman syndrome, the Li-Fraumeni syndrome, the Lynch syndrome, the multiple endocrine neoplasia (MEN) 1, and familial adenomatous polyposis (FAP) (Table 1). In recent years several multi-center studies have shed light on the pathogenesis of ACC but ‘multi-omic’ studies reveal that only a minority of ACC cases harbour pathogenic driver mutations.

Table 1. Clinical and Genetic Features of Familial Syndromes Associated with ACC

Genetic disease

Gene and chromosomal involvement

Organ involvement

Beckwith-Wiedemann syndrome

CDKN1C mutation

KCNQ10T1, H19 (epigenetic defects)

11p15 locus alterations

IGF-2 overexpression

Macrosomia, macroglossia, hemihypertrophy (70%), omphalocele, Wilm’s tumor, ACC (15-20% adrenocortical tumors)

Li-Fraumeni syndrome P53(17p13)

Soft tissue sarcoma, breast cancer, brain tumors, leukemia, ACC

Multiple Endocrine Neoplasia syndrome 1

Menin (11q13)

Parathyroid, pituitary, pancreatic, bronchial tumors

Adrenal cortex tumors (30%, rarely ACC)

Familial Adenomatous polyposis

APC (5q12-22)

Multiple adenomatous polyps and cancer colon and rectum

Periampullary cancer, thyroid tumors, hepatoblastoma, rarely ACC

SBLA syndrome

Sarcoma, breast and lung cancer, ACC

The clinical features of sporadic ACCs are due to hormone hypersecretion and/or tumor mass and spread to surrounding or distant tissues. An increasing number of cases (≈ 10-15%) are increasingly been diagnosed within the group of incidentally discovered adrenal masses (incidentalomas). However, the likelihood of an adrenal incidentaloma being an ACC is rather low. Approximately 50-60% of ACCs exhibit evidence of hormonal hypersecretion, usually that of combined glucocorticoid and androgen secretion (Table 2). Nearly 30-40% of patients with primary ACC present with a mass syndrome as abdominal or dorsal pain, a palpable mass, fever of unknown origin, signs of inferior vena cava (IVC) compression, and signs of left-sided portal hypertension. Rarely, complications as hemorrhage or tumor rupture may also develop. Lately the number of patients that are identified while being investigated for an adrenal incidentaloma is rapidly increasing.

Table 2. Signs and Symptoms of ACC and Recommended Testing for Confirmation of Hypersecretory Syndromes

Symptoms/Signs

Hormonal testing (ENSAT 2005)

Hypercortisolism

Centripetal fat distribution

Skin thinning – striae

Muscle wasting – myopathy

Osteoporosis

Increased blood pressure (BP)

Diabetes Mellitus

Psychiatric disturbance

Gonadal dysfunction

Overnight dexamethasone

suppression test (1mg)

24-hour free cortisol

Basal ACTH (plasma)

Basal cortisol (serum)

[for diagnosis minimum 3 out of 4 tests)

Androgen hypersecretion

Hirsutism

Menstrual irregularity – infertility

Virilization (baldness, deepening of the voice, clitoris hypertrophy)

DHEA-S

Androstendione

Testosterone

17-OH-progesterone

Mineralocorticoid hypersecretion

Mineralocorticoid excess with increased BP, hypokalemia

Potassium (serum)

Aldosterone to renin ratio

Estrogen hypersecretion

Gynecomastia (men)

Menorrhagia (post-menopausal women)

17β-estradiol

Non-hypersecretory syndrome

PATHOPHYSIOLOGY

Although studies of hereditary neoplasia syndromes have revealed various chromosomal abnormalities related to ACC development the precise genetic alterations involved are still unknown. The Wnt/β-catenin constitutive activation and insulin growth factor 2 (IGF2 overexpression) are the most important implicated genetic pathways. Germline TP53 mutations and dysregulation of the Gap 2/mitosis transition and the insulin-like growth factor 1 receptor (IGF1R) signalling have also been described. Steroidogenic factor 1 (SF1) plays an important role in adrenal development and is frequently overexpressed in ACC.

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

A palpable mass causing abdominal pain in the presence of the inferior vena cava syndrome IVC syndrome is highly suggestive of an ACC. This is substantiated further by the presence of symptoms/signs of combined hormonal secretion (cortisol and androgens), virilizing or rarely feminizing signs confirmed with the use of specific endocrine testing (Table 2). As the majority of ACCs are relatively large (size > 8cm, weight >100g) at diagnosis, specific imaging features are used to distinguish them from other adrenal lesions. If adrenal imaging indicates an indeterminate mass other parameters should be considered including tumor size > 4 cm, combined cortisol/androgen hormone excess, rapidly developing symptoms and/ rapid tumor growth and/or young age (e.g. < 40 years) that might point to an ACC.

Other adrenal lesions that need to be considered in the differential diagnosis are myelolipomas, adrenal hemorrhage, lymphoma, adrenal cysts, metastases, and mainly adrenal adenomas, the majority of which have distinctive imaging features. There is no role for biopsy in a patient who is considered suitable for surgery of the adrenal mass.

Computerized Tomography (CT) scanning of the adrenals is the major tool showing a unilateral non-homogenous mass, >5cm in diameter, with irregular margins, necrosis, and occasionally calcifications. Due to the low-fat content X-Ray density is high (>20 Hounsfield Units, HU); in a recent series of 51 ACC none had a density of less than 13 HU. The presence of enlarged aorto-caval lymph nodes, local invasion, or metastatic spread are highly suggestive of ACC. For 3-6 cm size lesions, measuring X-Ray tumoral density before and after contrast administration and estimating washout percentage can be helpful; less than 50% after 15 minutes, is associated with >90% specificity. On Magnetic Resonance Imaging (MRI), ACC appears hypo or isointense to the liver on T1-weighted images, and using gadolinium enhancement and chemical shift techniques the diagnostic accuracy obtained is 85-100%. Recently Positron Emission Tomography (PET-scan) with 18F-fluoro-2deoxy-D-glucose (18FDG) has been proposed as possibly the best second-line test to assess indeterminate masses by unenhanced CT exhibiting 95-100% sensitivity and 91-94% specificity that increases further when fused with CT imaging. Furthermore, 18FDG-PET can also be used as a staging procedure identifying metastatic adrenal disease missed by conventional imaging studies including CT of the chest. With the proper implementation of imaging studies there is no need for adrenal biopsy.

HISTOPATHOLOGICAL DIAGNOSIS

The expression of SF1 is a valid marker to document the adrenal origin (distinction of primary adrenocortical tumors and non-adrenocortical tumors) with a sensitivity of 98% and a specificity of 100%. If this marker is not available, a combination of other markers can be used which should include inhibin-alpha, melan-A, and calretinin. ENSAT has shown that KI67 is the most powerful prognostic marker in both localized and advanced ACC and that higher Ki67 levels are consistently associated with worse prognosis. Weiss system, based on a combination of 9 histological criteria that can be applied on hematoxylin and eosin-stained slides, for the distinction of benign and malignant adrenocortical tumors is the best validated score to distinguish adenomas from ACC although with high inter-observer variability.

PROGNOSIS

As survival depends on stage at presentation several different classification histopathological systems have evolved with the reported 5-year survival using the ENSAT system being 82% for stage I, 61% for stage II, 50% for stage III, and 13% for stage IV (Table 3). Tumor size remains an excellent predictor of malignancy as tumors > 6cm have a 25% chance of being malignant compared to 2% of those with a size < 4cm. As there is no single distinctive histopathological feature indicative of malignancy the Weiss score has been used with a score >3 being suggestive of malignancy and recently ki67 labelling index >10%.

Table 3. Staging system for adrenocortical carcinomas proposed by the International Union against cancer (WHO 2004) and the European Network for the study of adrenal tumors (ENSAT).
Stage	WHO 2004	ENSAT 2008
I	T1,N0,M0	T1,N0,M0
II	T2,N0,M0	T2,N0,M0
III	T1-2,N1,M0 T3,N0,M0	T1-2,N1,M0
IV	T1-4,N0-1,M1 T3,N1,M0 T4,N0-1,M0	T1-4,N0-1, M1
M0: No distant metastasis, M1: Presence of distant metastasis, N0: No positive lymph nodes, N1: Positive lymph node(s), T1: Tumor ≤ 5cm, T2: Tumor > 5 cm, T3: Tumor infiltration to surrounding tissue, T4: Tumor invasion into adjacent organs or venous tumor thrombus in vena cava or renal vein.

The median overall survival (OS) of all ACC patients is about 3-4 years. The prognosis is, however, heterogeneous. Complete surgical resection provides the only means of cure. In addition to radical surgery, disease stage, proliferative activity/tumor grade, and cortisol excess are independent prognostic parameters. Five-year survival rate is 60-80% for tumors confined to the adrenal space, 35-50% for locally advanced disease, and significantly lower in case of metastatic disease ranging from 0% to 28%. European Network for the Study of Adrenal Tumors (ENSAT) staging is considered slightly superior to the Union for International Cancer Control (UICC) staging. Additionally, the association between hypercortisolism and mortality was consistent. As Ki67 has been shown to be related with prognosis in both localized and advanced ACC threshold levels of 10% and 20% have been considered for discriminating low from high Ki67 labelling index; however, it is not clear whether any single significant threshold can be determined. Patients with stage I-III disease treated with surgical resection had significantly better median OS (63 vs. 8 months; p= 0.001). In stage IV disease, better median OS occurred in patients treated with surgery (19 vs. 6 months; p=0.001), and postsurgical radiation (29 vs 10 months; p=0.001) or chemotherapy (22 vs. 13 months; p= 0.004). Overall survival varied with increasing age, higher comorbidity index, grade, and stage of ACC at presentation. There was improved survival with surgical resection of the primary tumor, irrespective of disease stage; post-surgical chemotherapy or radiation was of benefit only in stage IV disease.

THERAPY

The management of patients with ACC requires a multidisciplinary approach with initial complete surgical resection in limited disease (stage I, II and occasionally III). Mitotane (1,1-dichloro-2(o-chlorophenyl)-2-(p-chlorophenyl) ethane [o,p’DDD]) is the only currently available adrenolytic medication achieving an overall response of approximately 30%.

Surgery

The aim of surgery is to achieve a complete margin-negative (R0) resection as patients with an R0 resection have a 5-year survival rate of 40-50% compared to the < 1year survival of those with incomplete resection. Patients with stage III tumors and positive lymph nodes can have a 10-year OS rate of up to 40% after complete resection. When a preoperative diagnosis or high level of suspicion of ACC exists, open surgical oncological resection is recommended as locoregional lymph removal might improve diagnostic accuracy and therapeutic outcome. However, the wide range of reported lymph node involvement in ACC (from 4 to 73%) implies that regional lymphadenectomy is neither formally performed by all surgeons nor accurately assessed or reported by all pathologists. Laparoscopic adrenalectomy should be considered for tumors with size up to 6 cm without any evidence of local invasion. Routine locoregional lymphadenectomy should be performed with adrenalectomy for highly suspected or proven ACC and it should include (as a minimum) the peri-adrenal and renal hilum nodes.

Preservation of the tumor capsule is essential whereas involvement of the IVC or renal vein with tumor thrombus is not a contraindication for surgery. However, even following an apparently complete surgical resection, 50-80% of patients develop locoregional or metastatic recurrence. Although such patients may be candidates for aggressive surgical resection, routine debulking is not recommended except for control of hormonal hypersecretion. Ablative therapies particularly targeting hepatic disease are used to decrease tumor load and the hypersecretory syndromes. Individualized treatment decisions are made in cases of tumors with extension into large vessels based on multidisciplinary surgical team. Such tumors should not be regarded ‘un-resectable’ until reviewed in an expert center.

Mitotane

Mitotane has traditionally been used for ACCs obtaining a partial or complete response in 33% of cases mainly by metabolic transformation within the tumor and through oxidative damage. Besides its cytotoxic adrenal action mitotane also inhibits steroidogenesis.

Adjuvant mitotane treatment is proposed in those patients without macroscopic residual tumor after surgery but who have a perceived high risk of recurrence (stage III, KI-67%>10%). However, for patients at low/moderate risk of recurrence (stage I-II, R0 resection, and Ki67 ≤ 10%) treatment with adjunct mitotane is still under investigation (results from ADIUVO trial are pending). When indicated mitotane should be initiated within six weeks and not later than 3 months. Adjuvant mitotane should be administrated for at least 2 years, but no longer than 5 years.

The tolerability of mitotane may be limited by its side effects mainly nausea, vomiting, neurological (ataxia, lethargy), hepatic and rarely hematological toxicity. Measurement of serum mitotane levels, targeting a range of 14-20 mg/l, seems to correlate with a therapeutic response while minimizing toxicity using variable dosing regimens. Mitotane causes hyperlipidemia and increased hepatic production of hormone binding globulins (cortisol, sex hormone, thyroid and vitamin D) increasing total hormone concentration while impairing free hormone bioavailability. The induction of hepatic P450-enzymes by mitotane induces the metabolism of steroid compounds requiring high dose glucocorticoid and mineralocorticoid replacement.

Hormonal excess causes significant morbidity in ACC patients. Although mitotane reduces steroidogenesis it has a slow onset of action necessitating the use of other adrenostatic medications (ketoconazole, metyrapone, aminoglutathemide, and etomidate). As adrenal insufficiency may occur close supervision is required to titrate adrenal hormonal replacement therapy.

Cytotoxic Chemotherapy

Although cisplatin containing regimens have shown some responses most studies lack power and comparisons between different regimens. The most encouraging results originate from the combinations of etoposide, doxorubicin and cisplatin with mitotane (EDP-M) achieving an overall response of 49% of 18 months duration (FIRMA-CT study). This regimen was equally effective as first line treatment or after failing of the combination of streptozotocin with mitotane and is the currently the preferred scheme. In patients who progress under mitotane monotherapy, EDP treatment is also recommended. The combination of gemcitabine with capecitabine is used for patients failing EDP- and for not responding patients targeted therapies with tyrosine kinase inhibitors (mainly sunitinib) could be used. Although initially promising treatment with IGF-1R antagonists did not prove to be efficacious suggesting that combination of therapies may be the way forward.

Radiation Therapy

Radiotherapy has a role in symptomatic metastatic disease particularly bone disease with positive responses in up to 50% - 90% of cancer patients.

Evolving Therapies

Targeting mTOR pathway alone using everolimus did not produce significant responses. An extended phase I study of the anti-IGF-1R monoclonal antibody cixutumumab with an mTOR inhibitor showed a partial but short-lived response. The use of the multikinase inhibitors sorafenib and sunitinib have also shown partial responses leading to a number of phase II studies whereas angiogenesis inhibitors have not been successful (http://www.clinicaltrials.gov). Other potential targets are antagonists of β-catenin and Wnt signaling pathway and SF-1 inverse agonists. The application of radionuclide therapy using ¹³¹I-metomidate has recently been explored. However, despite recent advances in dysregulated molecular pathways in ACCs, these findings have not yet been translated into meaningful clinical benefits. Lately immunotherapy (pembrolizumab) in phase II studies is under investigation.

FOLLOW-UP

Patients who have undergone an apparently curative resection should be followed up regularly using endocrine markers and abdominal imaging. After complete resection, radiological imaging every 3 months for 2 years and then every 3-6 months for a further 3 years is proposed. 18FDG-PET should be performed at regular intervals to detect recurrent disease at high risk patients. Patients on mitotane therapy should be regularly monitored measuring serum mitotane levels ensuring adequate replacement therapy. In case of recurrence not amenable to surgical excision patients should be enrolled in prospective clinical trials.

GUIDELINES

Fassnacht M, Dekkers O, Else T, Baudin E, Berruti A, de Krijger RR, Haak HR, Mihai R, Assie G, Terzolo M. European Society of Endocrinology Clinical Practice Guidelines on the Management of Adrenocortical Carcinoma in Adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol. 2018 Jul 24.

Berruti A, Baudin E, Gelderblom H, Haak HR, Porpiglia F, Fassnacht M & Pentheroudakis G. Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 2012 23 131-138.

REFERENCES

Petr EJ & Else T. Genetic predisposition to endocrine tumors: Diagnosis, surveillance and challenges in care. Semin Oncol 2016 43 582-590

Kassi E, Angelousi A, Zografos G, Kaltsas G, Chrousos GP. Current Issues in the Diagnosis and Management of Adrenocortical Carcinomas. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2016 Mar 6. PMID: 25905240

Chatzellis E, Kaltsas G. Adrenal Incidentalomas. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): DText.com, Inc.; 2000-2016 Feb 5. PMID: 25905250

Tacon L, Prichard R, Soon PSH, et al (2011) Current and emerging therapies for advanced adrenocortical carcinoma. The Oncologist 16:36-48

Fassnacht M, Kroiss M, Allolio B (2013). Update in adrenocortical carcinoma. J Clin Endocrinol Metab, 98:4551-4564

Tella SH, Kommalapati A, Yaturu S, Kebebew E. Predictors of survival in Adrenocortical Carcinoma: An analysis from the National Cancer Database (NCDB). J Clin Endocrinol Metab. 2018 Jun 21

Adrenal Suppression

CLINICAL RECOGNITION

Adrenal suppression, a form of secondary adrenal insufficiency (SAI), is a common clinical problem most often due to sudden cessation of chronic exposure to exogenous glucocorticoid administration or, rarely, after correction of endogenous hypercortisolism. It results from the inability of suprahypothalamic and hypothalamic centers of the hypothalamic-pituitary-adrenal (HPA) axis to recover their function and can last from days to months or years, depending on the dose and duration of the exposure to the glucocorticoid and patient’s idiosyncrasy. Exogenous glucocorticoids cause decreased secretion of corticotropin-releasing hormone (CRH) and other adrenocorticotropic hormone (ACTH) secretagogues, such as arginine-vasopressin (AVP) and alter the function of higher brain centers that regulate their secretion. Recovery of adrenal function may take as long as 1 to 2 years. In cases of endogenous hypercortisolemia adrenal suppression develops after the removal of a functional adrenal tumor secreting cortisol, or following successful removal of ACTH-secreting pituitary adenoma or other sources of ectopic ACTH secretion. Interestingly, the period to recover from adrenal suppression after the removal of ACTH-secreting pituitary adenoma caused by prolonged suppression of normal corticotrophs may be a predictor of sustained remission.

Regarding the definitions of adrenal insufficiency (AI), it is a disorder characterized by impaired adrenocortical function and decreased production mainly of glucocorticoids. Primary AI (PAI) is characterized, in addition by decreased production of mineralocorticoids (MCs) and/or adrenal androgens that occur in the setting of diseases affecting the adrenal cortex. Secondary AI (SAI) arises in diseases or conditions affecting the pituitary gland and the secretion of ACTH, while the affected hypothalamus resulting in abnormal secretion of corticotropin-releasing hormone (CRH) and other ACTH secretagogues defines the tertiary form of AI (TAI).

As adrenal suppression refers to decreases of cortisol secretion from the adrenal zona fascicularis the function of zona glomerulosa remains normal. Thus, hyponatremia is the main electrolytic disturbance observed, while circulating plasma potassium, renin, and aldosterone concentrations are within the normal range.

A broad range of severity can be seen as a result of complete or partial HPA axis suppression and concomitant adrenal gland atrophy. The true prevalence of overt adrenal insufficiency (AI) is probably rare as glucocorticoid treatment is gradually tapered before complete discontinuation leaving enough time for HPA axis recovery. However, due to the lack of specific symptoms the exact prevalence of AI following glucocorticoid tapering may be under-reported. Two recent systematic reviews reported that the percentage of patients with AI ranged from 0% to 100%, with a median (IQR) = 37.4% (13–63%), while when the studies were stratified by administration route, the percentages of patients with AI ranged from 4.2% for nasal administration (95% confidence interval [CI], 0.5–28.9) to 52.2% for intra-articular administration (95% CI, 40.5– 63.6); by disease, from 6.8% for asthma with inhalation glucocorticoids only (95% CI, 3.8 –12.0) to 60.0% for hematological malignancies (95% CI, 38.0 –78.6); by the dose from 2.4% (95% CI, 0.6 –9.3) (low dose) to 21.5% (95% CI, 12.0 –35.5) (high dose); by treatment duration from 1.4% (95% CI, 0.3–7.4) (less than 28 days) to 27.4% (95% CI, 17.7–39.8) (more than 1 year) in asthma patients.

The main symptoms of glucocorticoid insufficiency range from anorexia, fatigue, nausea, vomiting, dyspnea, fever, arthralgias, myalgias, and orthostatic hypotension to dizziness, fainting, and circulatory collapse. Hypoglycemia is occasionally observed in children and very thin adult individuals. Since 1-3% of adults worldwide are under long-term glucocorticoid therapy (Table 1), the awareness for adrenal suppression and the associated risk for glucocorticoid deficiency, as well as the appropriate treatment, are important clinical issues.

Table 1: Use of Glucocorticoid Therapy in Clinical Practice
Long-Standing Treatment
ENDOCRINE CAUSES
Replacement therapy	Primary AI Secondary AI
Adrenal suppression Therapy	Congenital adrenal hyperplasia Glucocorticoid resistance
Anti-inflammatory therapy	Grave's opthalmopathy
NON-ENDOCRINE CAUSES
Immunosuppressive/ anti-inflammatory therapy	Rheumatic diseases- (lupus erythematosus, polyarteritis, rheumatoid arthritis, polymyalgia rheumatica) Skin disorders- (dermatitis, pemphigus) Other autoimmune diseases- (multiple sclerosis, myasthenia Gravis, vasculitis) Hematological disorders- (lymphomas/ leukemias, hemolytic anemias, idiopathic thrombocytopenic purpura) Gastrointestinal diseases- (inflammatory bowel disease) Liver diseases- (chronic active hepatitis) Respiratory diseases- (angioedema, anaphylaxis, asthma, sarcoidosis, tuberculosis, obstructive airway disease). Nephrotic syndrome Suppression of host-versus-graft/graft-versus-host reaction- (bone marrow or organ transplantation) Nervous disorders- (cerebral edema, raised intracranial pressure)
Acute Treatment
ENDOCRINE CAUSES
Suppression hypothalamic-pituitary-adrenal axis	Cushing syndromediagnostic tests
NON-ENDOCRINE CAUSES
Several conditions	Acute traumatic spinal cord injury Post-operative additional therapy in severe neurological deficits even after surgery Postoperative pain relief after severe bone operations Fetuses between 24 and 34wk gestation (risk of preterm delivery)
Acute illness or trauma	"Critical illness-related cortisol insufficiency"(CIRCI): vasopressor dependent septic shock and early severe Acute Respiratory Distress Syndrome

AI: adrenal insufficiency

Many synthetic compounds with glucocorticoid activity have been developed in an attempt to maximize the beneficial and minimize the deleterious effects of glucocorticoids. The clinical efficacy of synthetic glucocorticoids depends on their pharmacokinetic, pharmacodynamic and molecular properties, which in turn determine the duration and intensity of glucocorticoid effects. According to their potency synthetic glucocorticoids are subdivided into short-, intermediate-, or long-acting. Treatment modifying factors, such as the age of the patient and the nature and severity of the underlying disease also influence synthetic glucocorticoid effects, duration, and doses administered.

The British National Formulary and the National Institute for Health and Care Excellence Clinical Knowledge Summary, both advise gradual glucocorticoid withdrawal in cases of patients that have received more than 40 mg prednisolone (or equivalent) daily for longer than one week; repeated glucocorticoid doses in the evening; glucocorticoids for more than three weeks; a short course of glucocorticoids within one year of stopping long-term glucocorticoid therapy; or have other risk factors for adrenal suppression.

PATHOPHYSIOLOGY

Supraphysiologic doses of glucocorticoids given even in small doses and/or for only a few days may result in considerable suppression of the HPA axis by decreasing CRH synthesis and secretion. The trophic and ACTH-releasing effects of CRH on pituitary corticotrophs are attenuated and the synthesis of propiomelanocortin (POMC), ACTH, and other peptides, are substantially decreased. In the absence of ACTH, the adrenal cortex temporarily loses the ability to produce cortisol, and when treatment with glucocorticoids is abruptly stopped transient glucocorticoid insufficiency ensues. It has been reported that the suppression of the HPA axis induced by exogenous glucocorticoids may persist for 6 to 12 months or rarely even longer, after treatment is withdrawn.

DIAGNOSIS and DIFFERENTIAL DIAGNOSIS

To support the diagnosis of adrenal suppression, several predictors of glucocorticoid-induced HPA axis hypofunction have been suggested the best being the duration and dosage of exogenous glucocorticoid administration (Table 2,3). A strong correlation has been found between prednisone maintenance doses above 5mg/d and a subnormal ACTH-stimulation test result. Hence, patients who are more likely to develop HPA axis suppression are those receiving high doses of glucocorticoids (>20-30mg hydrocortisone or equivalent) (Table 4) for a period longer than 3 weeks and patients who have developed overt Cushingoid features. In addition, the timing of drug administration may affect the degree of adrenal suppression. Thus, prednisolone in a dose of 5mg given at night before bedtime and 2.5mg in the morning will produce more marked HPA axis suppression compared to 2.5mg at night and 5mg in the morning. Higher evening doses block early morning ACTH surge whereas tissues sensitivity to glucocorticoids is increased in the evening and early night hours.

Table 2: Predictors of Glucocorticoid-Induced HPA Axis Suppression
Predictor	Etiology/Risk of HPA Suppression
Type of steroid and potency	Long-acting GCs lead to longer tissue life and longer suppression
Route of administration	Systemic GC therapy (parenterally): increased risk
Timing of administration	Decreased risk in alternate days scheme (from outset or converted before suppression); Increased risk: different doses scheme during day:
Duration and cumulative dose	Decreased risk in treatment ≤1week
Clinical features	Patients with Cushing's Syndrome: increased risk

HPA: hypothalamic-pituitary-adrenal; GC: glucocorticoid.

Table 3: Examples of Different Glucocorticoid-Induced HPA Axis Suppression

HC/cortisone acetate: least potent/suppressive;

prednisone/prednisolone, methylprednisolone, triamcinolone: moderately suppressive;

dexamethasone: strongest suppression

Topical GCs: increased risk but infants at increased risk;
Inhaled GCs: increased risk versus oral/systemic GCs >risk children
Fluticasone proprionate (ciclesonide: recent drug, decreased risk);
Intraarticular GCs: transient suppression

Once-a-day dosing decreased risk intermediate/long acting GCs (prednisone/triamcinolone/dexamethasone);
Short-acting HC/ cortisone acetate: twice-a-day (at waking 2/3; 5PM 1/3 total daily dose); evening doses suppress normal early morning ACTH surge leading to increased suppression, treat with single morning dose

"Short-term" 14 days course systemic GCs decreased risk

ACTH: adrenocorticotropin; HC: hydrocortisone; GC: glucocorticoid

Table 4: Glucocorticoid Equivalent Dose Compared to Cortisol
	equivalent dose (mg)
Short-acting, low potency
Cortisol	20
Cortisone	25
Intermediate-potency
Prednisolone	5-7.5
Methylprednisolone	4
Long-acting, high potency
Dexamethasone	0.75

Clinical awareness is crucial to identify patients with impending adrenal crisis. It is important to consider all patients with unexplained symptoms after glucocorticoid- withdrawal as candidates for possible AI and test them accordingly. An important feature that will raise suspicion of TAI (and SAI) besides drug history is the absence of skin pigmentation. Such patients have an intact renin-angiotensin-aldosterone system (RAAS) accounting for the differences in salt and water balance and clinical presentations compared to primary adrenal insufficiency.

Serum cortisol secretion at 08:00h if diagnostic tests are not feasible and until confirmatory testing is available can be considered a valuable screening method when AI is suspected. In patients with a low index of suspicion obtaining an 8AM cortisol and if the serum cortisol is > 15μg/dL, no further testing is needed. Similarly, a serum cortisol value <5 μg/dL suggests AI.

DIAGNOSTIC TESTS NEEDED TO DOCUMENT AI

Drug history and clinical features cannot be considered reliable tools for the evaluation of HPA axis function in patients treated with synthetic glucocorticoids. Several tests are commonly used in order to assess the degree of glucocorticoid-induced AI or HPA axis recovery (Table 5,6). Both the insulin tolerance test (ITT) and the metyrapone test have been employed as they are both highly sensitive. However, the risks involved with these tests do not justify their use compared to the rapid ACTH stimulation test or short synacthen test (SST) that can safely distinguish almost all cases of clinically significant adrenal suppression.

To evaluate the adequacy of HPA axis recovery, the SST is used to assess the capability of the adrenal cortex to respond to ACTH. However, because of the supraphysiologic ACTH levels achieved with the conventional SST (250 mcg of ACTH administered), if adrenal suppression is of recent onset, the adrenal gland may have not yet atrophied, and is still capable of responding to ACTH stimulation. In these cases, the low-dose SST (1 mcg of ACTH administered) has been proposed as an alternative as it results in lower plasma ACTH levels and thus less pronounced adrenal stimulation. It has recently been suggested that the low-dose SST is the best test to establish the diagnosis of SAI and TAI, whereas the high SST should be used for cases of primary AI. The use of salivary cortisol is also an effective alternative to serum cortisol when assessed in the high-dose ACTH test. Incremental cortisol response at the first SST was suggested as an important predictive factor of adrenal function recovery in SAI after exogenous glucocorticoid administration.

The CRH test can also be used in patients receiving glucocorticoids for prolonged periods, as it can assess both the ACTH and cortisol responses and can distinguish between SAI and TAI. In both conditions, cortisol concentrations are low at baseline and remain low after CRH administration. In patients with SAI, there is little or no ACTH response, whereas in patients with tertiary disease there is an exaggerated and prolonged response of ACTH, which is not followed by an appropriate cortisol response. On the contrary, patients with primary AI have high ACTH levels, which rise further following CRH while patients with hypothalamic disease show a steady rise in ACTH levels.

The prolonged ACTH stimulation test (depot or iv infusions 250µg cosyntropin over 8 hrs or over 24hrs) was suggested as a mean to differentiate between the different types of AI but is now rarely used in routine practice. In SAI or TAI, the adrenal glands display cortisol secretory capacity following prolonged stimulation with ACTH whereas in primary AI, they do not respond to ACTH being partially or completely destroyed.

In a recent systematic review of AI assessment after systemic glucocorticoid therapy, SST (conventional or low-dose) was the most frequently employed, but other tests were also used, including the insulin tolerance test (ITT, the “gold-standard”), the ACTH infusion, and the CRH tests.

Table 5: Diagnostic Tests Used to Diagnose Adrenal Insufficiency
Test / Sampling	Cortisol Response
Short Synacthen test 250mg iv or im cosyntropin; samples at 0/30’/60’	Physiologic response:>500-550nmol/L (18-20µg/dL)
Low-Dose Synacthen Test 1μg ACTH iv at 14:00: samples 10’ 15’ 20’ 25’ 30’ 35’ 40’ 45’	Physiologic response: >18 µg/dL (500nmol/L)
CRH stimulatory test iv bolus 1 or 100µg/kg or 100µgh-CRH/o-CRH	TAI: steady rise in ACTH not followed by appropriate cortisol response;SAI: no ACTH or cortisol response

ACTH: adrenocorticopic hormone; CRH: corticotropin-releasing hormone; im: intramuscular; iv: intravenous; PAI: primary adrenal insufficiency; SAI: secondary adrenal insufficiency; TAI: tertiary adrenal insufficiency

Table 6: Diagnostic Tests Not Commonly Used to Diagnose and Differentiate Adrenal Insufficiency
Test / Sampling	Cortisol Response
Prolonged ACTH stimulation test Depot or iv infusions 250µg cosyntropin over 8hrs(A): cortisol/24hr urinary cortisol/17OHCS before and after infusion or over 24hrs on 2(or3) consecutive days(B)	Physiologic response: A:24hr urinary 17-OHCS excretion increase 3-5-fold; serum cortisol>20μg/dL (550nmol/L) at 30’ and 60’; >25μg/dL (690 nmol/L) at 6-8hrs post-initiation infusion;B: at 4hrs >1000nmol/L (36μg/dL) beyond this time, no further increase; SAI: delayed response at 24 and 48hrs than 4hrs; PAI no response at either time
ITT iv insulin (0.1-0.15U/kg); Samples 0 30’45’ 60’90’120’ with adequate clinical and biochemical hypoglycemia	Physiologic response: >500nmol/L (18μg/dL)
overnight metyrapone test 30 mg/kg (max 3g) at midnight; cortisol/ 11-deoxycortisol measured at 8.00h the following morning	Physiologic response: Increased ACTH plus peak 11-deoxycortisol >7 mg/dL.

ACTH: adrenocorticopic hormone; iv: intravenous; ITT: insulin tolerance test, PAI: primary adrenal insufficiency; SAI: secondary adrenal insufficiency; SST: short synacthen test; 17OHCS: 17-hydroxycorticoids, TAI: tertiary adrenal insufficiency

THERAPY

Glucocorticoid withdrawal is indicated when the use of the steroid is no longer needed or when significant side effects develop. The suggested method of glucocorticoid withdrawal is dose tapering to avoid the occurrence of AI.

Adrenal insufficiency is a potentially life-threatening medical emergency when presenting as adrenal crisis, which requires prompt treatment with hydrocortisone and fluid replacement. Once, clinically suspected, treatment should be initiated and not be delayed while waiting for definitive proof of diagnosis. Blood samples should be obtained for measurement of cortisol concentrations later, and the management approach should be similar to the resuscitation of any critically ill patient.

There is currently no consensus regarding rapid or slow tapering of glucocorticoids and exacerbation and/or relapse rates of the underlying diseases. The key action is that glucocorticoid withdrawal should not be abrupt. In clinical practice, patients being on any steroid dose for less than 2 weeks are not likely to develop adrenal suppression and are advised to stop therapy without tapering. The possible exception to this is the patient who receives frequent "short" steroid courses, as in asthma treatment. In longer regimens, the objective is to rapidly reduce the therapeutic dose to a physiologic level of cortisol (equivalent to 10-15 mg/ms/d) (Table 7). However, a recent systematic review of 73 studies demonstrated evidence of AI following low doses and short durations of glucocorticoid administration at less than 5 mg prednisolone equivalent dose/day, less than 4 weeks of exposure, cumulative dose less than 0.5 g, and following tapered withdrawal.

Table 7: Tapering After a Long-Term Glucocorticoid Regimen
1. Reduction by 2.5mg prednisolone or equivalent	every 3-4 days over few weeks
2. Slower withdrawal	until physiological level achieved (5-7.5mg of prednisolone)
3a. Decrease by 1mg/d prednisolone or equivalent	every 2-4weeks (depending patient's general condition) until medication cessation
Or
3b. Switch to 20mg/d HC+ Decrease by 2.5mg/d	every week until the dose: 10mg/d
4. After 2-3months on same dose	SST or ITT
5a. Pass Response	discontinuation of GC
Or
5b. No HPA axis recovery	Treatment continuation+re-assessment

GC: glucocorticoid; ITT: insulin tolerance test; SST: short synacthen dose

Other tapering regimens suggest switching the patient to an alternate day administration of intermediate action glucocorticoids before cessation of treatment. Irrespectively of the tapering regimen used, if a glucocorticoid withdrawal syndrome, AI or exacerbation of the underlying disease develops, the dose being given at the specific time should be increased or maintained longer. Recent systematic reviews implied that the evidence for the tapering regimens used nowadays is not robust, despite the fact that rapid reduction to a physiologic glucocorticoid dose (5-7.5 mg prednisolone daily or equivalent), and the slow reduction thereafter, is the most frequently used regimen for clinicians.

Care should be given during the tapering regimens period on the interpretation of laboratory tests for cortisol levels measurement. The steroid dose before the test should be omitted (hold off evening and morning dose for hydrocortisone or prednisolone, longer for the other synthetic glucocorticoids); if serum cortisol secretion at 08:00h is > 15μg/dL, the tapering regimen changes to a rapid tapering off of exogenous glucocorticoids. Moreover, there are conditions that affect cortisol-binding globulin concentration (CBG) (↓: inflammation, nephrotic syndrome, liver disease, immediate postoperative period or requiring intensive care, rare genetic disorders; ↑: estrogen, pregnancy, mitotane). Systemic estrogens should be discontinued at least for 4 weeks prior to testing; estrogen patches are preferred since they do not affect CBG. Different criteria may apply according to the cortisol assay.

FOLLOW-UP

Since, there is evidence that AI may persist in 15% of patientsfor more than 3 years after glucocorticoid withdrawal, careful monitoring of patients and gradual glucocorticoid withdrawal should always be performed to avoid manifestations of adrenal suppression and/or an adrenal crisis or reactivation of the underlying disease. In general, plasma ACTH concentrations are not helpful in estimating the optimal glucocorticoid dose whereas mineralocorticoid replacement is not required.

All patients treated with glucocorticoids long-term should receive detailed instructions for glucocortiocoid supplementation equivalent to 100-150mg of hydrocortisone during major stresses (surgery, fractures, severe systemic infections, major burns) until their HPA axis fully recovers and to carry means of identification (medical alert bracelet).

Since full HPA axis recovery may take as long as one year or even longer, abrupt cessation of glucocorticoid treatment or quick tapering can precipitate an acute AI crisis. The diagnosis is a medical emergency, and treatment should be the immediate administration of fluids, electrolytes, glucose, and parenteral glucocorticoids.

GUIDELINE

Joint Formulary Committee, Glucocorticoid therapy. British National Formulary. London: BMJ Group and Pharmaceutical Press; 2013, 462.

National Institute for Health and Care Excellence.Corticosteroids—oral. http:// cks.nice.org.uk/corticosteroids-oral topic summary [accessed 15.09.20]

REFERENCES

Alexandraki KI, Kaltsas GA, Isidori AM, Storr HL, Afshar F, Sabin I, Akker SA, Chew SL, Drake WM, Monson JP, Besser GM, Grossman AB. Long-term remission and recurrence rates in Cushing's disease: predictive factors in a single-centre study. Eur J Endocrinol. 2013 Mar 20;168(4):639-48. doi: 10.1530/EJE-12-0921. Print 2013 Ap

Bansal P, Lila A, Goroshi M, Jadhav S, Lomte N, Thakkar K, Goel A, Shah A, Sankhe S, Goel N, Jaguste N, Bandgar T, Shah N. Duration of post-operative hypocortisolism predicts sustained remission after pituitary surgery for Cushing's disease. Endocr Connect. 2017 Nov;6(8):625-636.

Broersen LH, Pereira AM, Jørgensen JO, Dekkers OM. Adrenal Insufficiency in Corticosteroids Use: Systematic Review and Meta-Analysis. J Clin Endocrinol Metab. 2015 Jun;100(6):2171-80.

Joseph RM, Hunter AL, Ray DW, Dixon WG. Systemic glucocorticoid therapy and adrenal insufficiency in adults: A systematic review. Semin Arthritis Rheum. 2016 Aug;46(1):133-41.

Magnotti M, Shimshi M. Diagnosing adrenal insufficiency: which test is best--the 1-microg or the 250-microg cosyntropin stimulation test? Endocr Pract. 2008 Mar;14(2):233-8,

Neidert S, Schuetz P, Mueller B, Christ-Crain M. Dexamethasone suppression test predicts later development of an impaired adrenal function after a 14-day course of prednisone in healthy volunteers. Eur J Endocrinol. 2010 May;162(5):943-9.

Nicolaides NC, Chrousos GP, Charmandari E. Adrenal Insufficiency. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2017 Oct 14.

PMID: 25905309

Chrousos G, Pavlaki AN, Magiakou MA. Glucocorticoid Therapy and Adrenal Suppression. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2011 Jan 11. PMID: 25905379

Alexandraki KI, Grossman A. Adrenal Insufficiency. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2018 Aug 20. PMID: 25905345

Kidney Stone Emergencies

CLINICAL RECOGNITION

The acute passage of a kidney stone is the 9^th most common cause of emergency room visits. Approximately 7-8% of women and 11-16% of men will have stone disease by age 70. The acute syndrome complex called renal colic implies obstruction of the collecting system or ureter, and the most common cause of obstruction is a kidney stone. Kidney stone colic is relatively constant in contrast to intestinal or biliary colic, which waxes and wanes or comes in waves. The onset of pain heralds the entrance of a stone into the collecting system and the ensuing obstruction. The intensity and location of the pain may vary with stone size, stone location, degree of luminal obstruction, and the suddenness of the obstruction but flank pain is very common. Referred genital pain is common with distal ureteral stones. Symptoms typically begin at night or the early morning hours with abrupt onset and awakening the patient from sleep. During the day, the onset of symptoms may follow heavy exercise and may be more gradual with an occasional prodrome of unilateral discomfort in the flank, testis or vulva on the side of the obstruction. The pain then becomes continuous, steady; and progressively more severe as it approaches a peak. For some, there are acute paroxysms of increasingly intense pain. Anorexia, nausea and vomiting commonly appear with the pain, and gross hematuria may be present. Overall, one-third of patients have a relatively rapid onset and reach peak pain in 30 minutes or less. Untreated, the pain may last for 4 to 12 hours, but most patients have presented to the emergency room by the time the pain becomes continuous, usually by two hours into the colic. Upon presentation, the pain is described as a 9 or 10 out of a scale of 1 to 10. Chills and fever may be present as well and should raise concern for infection as these symptoms are usually not present in uncomplicated urolithiasis. Similarly, hypotension also raises the likelihood of infection as the pain associated with renal colic typically induces hypertension and tachycardia.

PATHOPHYSIOLOGY

Stone-induced renal colic refers to an intraluminal cause, but non-stone related external compression of the ureter can induce the same symptom complex and be confused with the intraluminal presence of a stone. Renal colic can arise from three mechanisms: urinary obstruction, the most common cause, is due to a direct increase in intraluminal pressure and stretch of the nerve endings in the mucosa; local ureteral mucosal or collecting system irritation from direct contact of the stone; and interstitial edema and stretch of the renal capsule, particularly when there is a concomitant pyelonephritis. Stones are more likely to hang up and obstruct at naturally narrow regions of the upper urinary tract including the ureteropelvic junction, crossing of the iliac artery and vein, pelvic brim, and the ureterovesical junction.

DIAGNOSIS and DIFFERENTIAL

Diagnosis

The diagnosis is strongly suspected by the symptom complex. The examination reveals costovertebral angle tenderness with dysesthesia of the skin overlying the area along the flank, lower abdomen, groin, or genitalia. Gross or microscopic hematuria is present in 60% to 90% of patients with renal colic but is not required for the diagnosis.

Children with renal stones may present with more vague abdominal symptoms compared to the symptom complex in adults. Therefore, abdominal pain in children and adolescents should call for a urologic evaluation if no diagnosis has been reached.

Differential Diagnosis

Acute renal colic may be caused by non-kidney stone events listed in Table 1.

Table 1. Causes of Acute Renal Colic

Intrinsic to the Collecting System

Kidney stones

Gross hematuria with clot formation

Tumor emboli

Renal papillary necrosis

Extrinsic to the Collecting System

Calyceal obstruction

Calyceal diverticula

Congenital ureteropelvic obstruction

Retroperitoneal fibrosis

Endometriosis

Dilation of the ovarian veins (pregnancy)

Mass lesions of the uterus

Acute onset of continuous, aching or dull pain that is non-colicky or flank pain without radiation to or toward the groin suggests a non-stone etiology. Common causes of acute non-colicky pain are listed in Table 2.

Table 2. Differential Diagnosis of Acute Non-colicky Renal Pain

Renal vein thrombosis

Pyelonephritis

Renal cortical abscess

Poststreptococcal glomerulonephritis

Rapidly progressive glomerulonephritis

Polycystic kidney disease

Medullary sponge kidney

DIAGNOSTIC TESTING

Imaging

The definitive diagnosis of acute renal colic relies upon radiographic imaging of the kidney and urinary tract to demonstrate the location, number, and size of the stones as well as the degree of obstruction. Non-contrast CT (NCCT) has become the imaging study of choice when evaluating patients with acute flank pain and suspected ureterolithiasis. It has both a high sensitivity and specificity for demonstrating the presence of stones and the ability to detect other abnormalities that maybe accounting for the symptoms. In addition, it has the advantage of providing information regarding stone number, location, size, and in some instances stone composition. It can also reveal signs of obstruction. The majority of patients evaluated by NCCT require no further imaging to determine the need for urological intervention. Many now advocate the use of low dose NCCT for the diagnosis of renal stones to reduce radiation exposure, particularly if the BMI is less than 30kg/m².

Ultrasound is also a sensitive method for detecting ureteral stones in patients with renal colic and can be used as the initial imaging method in investigating these patients. However, the quality of ultrasound information is operator dependent and ultrasound has decreased diagnostic sensitivity. Kidney stones are common during pregnancy. Because fetal radiation exposure should be avoided, ultrasound is the primary radiologic procedure followed by MRI if necessary in pregnant women. NCCT should be used only in rare instances in pregnancy. In children ultrasound is the initial imaging procedure followed by low dose NCCT if needed.

A radiographic study done while the patient is in the emergency room will establish a definitive diagnosis, especially if it can exclude other causes of acute abdominal pain; will avoid a prolongation of the painful episode; avoid delay in treatment; and reduce the risk of loss of renal function when complete obstruction is present.

Laboratory Studies

The laboratory studies that should be obtained are shown in Table 3.

Table 3. Laboratory Studies
Complete Blood Count (CBC)	Increased neutrophils may be due to a stress response or infection
Electrolytes
Creatinine	Usually not markedly increased. A marked increase suggests solitary kidney, baseline kidney disease, or pre-renal injury due to dehydration
Calcium	Hypercalcemia suggests the mechanism of stone formation and requires further evaluation
Uric acid	Elevated uric acid levels suggest the mechanism for stone formation and requires further evaluation
Pregnancy testing in females of reproductive age
U/A	Hematuria very common. WBCs if > 5/high powered field suggest infection
Urine culture and sensitivity if U/A abnormal or other signs of infection

Patients should be instructed to filter their urine in the hopes of retrieving a stone for analysis. Knowing the stone composition will help guide future preventive therapy.

TREATMENT

The goals of management during the acute phase of stone obstruction and renal colic includes: pain control and diagnostic procedures to determine the presence of a kidney stone in the collecting system and the extent of obstruction.

Pain management should be started soon after the patient arrives in the emergency room and should be continued until the episode has resolved. Nonsteroidal anti-inflammatory drugs (NSAIDS) (for example diclofenac, indomethacin or ibuprofen) are effective first line agents for acute pain treatment. If the pain persists or NSAIDS are contraindicated, narcotics, such as morphine sulfate 0.1 mg per kg body weight IM every four hours or meperidine (Demerol) 1.0 mg per kg body weight IM every three to four hours, may be used. Intravenous lidocaine (1.5mg/kg) is another option that has been shown to be effective in reducing renal colic. Anti-emetic agents may be given along with the narcotics as nausea and emesis may occur with stone passage and commonly complicate narcotic use. If medical treatment is not sufficient consultation with urology and consideration of drainage or stone removal is indicated.

Alpha blockers, such as tamsulosin, may be used to facilitate the clearance of kidney stones. In a Cochrane review of 67 studies with 10,509 participants it was concluded that “alpha-blockers likely increase stone clearance but probably also slightly increase the risk of major adverse events (hypotension, syncope, palpitations, tachycardia). Subgroup analyses suggest that alpha-blockers may be less effective for smaller (5 mm or smaller) than for larger stones (greater than 5 mm)”. Smaller stones are more likely to spontaneously pass and therefore the advantages of alpha blockers are minimized but they may induce more rapid clearance. Additionally, alpha blockers also reduce renal colic.

The size of the stone is a major determinant of the need for surgical management vs. conservative management. Stones vary from less than 2 mm to greater than 2 cm in diameter. The majority of stones are less than 4 mm in width, small enough to pass spontaneously in most patients. A stone’s size is an important factor together with symptom severity, degree of obstruction, presence or absence of infection, and level of renal function in deciding whether to manage the stone initially by observation, awaiting spontaneous passage, or to intervene with a surgical procedure. Stones with a width of 5 mm or less have a 50% chance of spontaneous passage if in the proximal ureter and a better chance if in the distal ureter. Overall, for stones ≤5 mm, approximately 68% will pass spontaneously. For stones >5 mm and ≤10 mm, an estimated 47% will pass spontaneously. One study found that stones > 9mm had only a 25% chance of spontaneous passage. Distal stones are more likely to clear than proximal stones (proximal ureter 48%, mid-ureter 60%, distal ureter 75% passage rate). Thus, in many patients with renal colic symptomatic treatment and close follow-up with the anticipation of stone passage is reasonable. The presence of infection, obstruction, refractory or difficult to treat pain, or deterioration of renal function indicates the need to urological consultation and the consideration of surgical intervention.

Urologic consultation should be obtained for possible surgical intervention for a number of reasons including stones with a low likelihood of spontaneous passage (large stones, proximal location), infection, obstruction, renal insufficiency or worsening renal function, and comorbidities that increase the risk of adverse outcomes (for example pregnancy). Depending upon the circumstances a number of procedures are available including ureteroscopic stone lithotripsy and extracorporeal shock wave lithotripsy for stone removal and percutaneous nephrostomy tube and JJ-stent for urinary drainage.

The presence of urinary tract infection increases the risk for development of pyelonephritis and/or pyonephrosis. Urgent intervention is therefore indicated, again regardless of stone size. Near-total or total ureteral obstruction predicts deterioration of renal function that may start within two weeks of presenting with stone disease and therefore indicates the need intervention.

FOLLOW-UP

Follow-up evaluation should be within one to two weeks of the acute event depending on the extent of intervention and whether there is risk for new obstruction from residual stones. Metabolic evaluation using blood and urine tests may be performed after six weeks of recovery to guide specific preventative therapy. Stone analysis, and the results of urine and blood tests can guide decisions on preventive therapy. It should be recognized that after a first stone episode 30-50% of individuals have a recurrent stone within 10 years.

GUIDELINES

Pearle MS, Goldfarb DS, Assimos DG, Curhan G, Denu-Ciocca CJ, Matlaga BR, Monga M, Penniston KL, Preminger GM, Turk TM, White JR; American Urological Assocation. Medical management of kidney stones: AUA guideline. J Urol. 2014 Aug;192(2):316-24. PMID: 24857648

Türk C, Petřík A, Sarica K, Seitz C, Skolarikos A, Straub M, Knoll T. EAU Guidelines on Diagnosis and Conservative Management of Urolithiasis. Eur Urol. 2016 Mar;69(3):468-74.

PMID: 26318710

The EAU Recommendations in 2016. Medical Expulsive Therapy for Ureterolithiasis:

Türk C, Knoll T, Seitz C, Skolarikos A, Chapple C, McClinton S; European Association of Urology. Eur Urol. 2017 Apr;71(4):504-507. PMID: 27506951

REFERENCES

Favus M. Nephrolithiasis. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2016 Dec 11.

PMID: 25905296

Gottlieb M, Long B, Koyfman A. The evaluation and management of urolithiasis in the ED: A review of the literature. Am J Emerg Med. 2018 Apr;36(4):699-706. PMID: 29321112

Fulgham PF, Assimos DG, Pearle MS, Preminger GM. Clinical effectiveness protocols for imaging in the management of ureteral calculous disease: AUA technology assessment.

J Urol. 2013 Apr;189(4):1203-13. PMID: 23085059

Campschroer T, Zhu X, Vernooij RW, Lock MT. Alpha-blockers as medical expulsive therapy for ureteral stones. Cochrane Database Syst Rev. 2018 Apr 5;4. PMID: 29620795

Jung H, Osther PJ. Acute management of stones: when to treat or not to treat? World J Urol. 2015 Feb;33(2):203-11. PMID: 24985553

The Postmenopausal Woman

ABSTRACT

The menopausal transition marks a time of great variability in reproductive hormones, and this variability can be responsible for specific symptoms, such as hot flashes and mood disturbances. Once a woman who is more than 45 years old has gone for 12 months without a menstrual period, she is considered to be menopausal and has consistently low circulating estradiol and elevated gonadotropins. Estrogen is the most efficacious therapy for bothersome vasomotor symptoms. Although estrogen exerts clear-cut protective effects on the cardiovascular system in premenopausal women, medical evidence does not support its use for the prevention of cardiovascular disease. Estrogen is generally not a first line agent for bone preservation in women without concurrent menopausal symptoms, despite its antiresorptive effects. Non-hormonal alternatives to estrogen and new, tissue specific estrogen complexes (TSECs) are now FDA approved and available for clinical use to treat common menopausal symptoms. For complete coverage of this and all related areas of Endocrinology, please visit our FREE on-line web-textbook, www.endotext.org.

INTRODUCTION

Menopause is associated with a constellation of physical changes. Some of these changes are directly attributable to the loss of estrogen, including hot flashes, bone demineralization and vaginal dryness. Though a matter of controversy, an increased incidence of cardiovascular disease and dementia seem to be associated with both menopause and aging. Furthermore, other conditions, such as breast, ovarian and endometrial cancer, are associated primarily with aging but certainly are impacted by ovarian hormones.

This review will address the menopausal transition, its common symptoms, and the risks and benefits of Hormone Therapy (HT), specifically, estrogen therapy and the selective estrogen receptor modulators (SERMs): raloxifene, tamoxifen and bazedoxifene, and other non-hormonal therapies.

DEFINITIONS

The Menopausal Transition

In 2001 [1] and again in 2012 [2], a Stages of Reproductive Aging Workshop (STRAW) was held to describe and define the various stages of the menopausal transition (Figure 1). On average, the menopausal transition lasts 4 years in duration and is divided into early and late phases. It begins when menstrual irregularity first appears, classically defined as either a “skipped” period or by an increase in variability of cycle length by more than 7 days. The menstrual irregularity that characterizes the menopausal transition occurs as the overall ovarian follicular complement decreases. However, the menstrual cycle and hormone changes of the early transition are best explained by a loss of the follicle cohort, rather than insufficient follicles to result in a single ovulation. This decrease in the available pool of growing follicles leads to a decrease in inhibin B production [3]. Reduced inhibin B removes the physiologic restraint on FSH that controls the process of folliculogenesis, and an increase in follicle-stimulating hormone (FSH) secretion is observed. Early in the transition period, FSH levels are not consistently elevated, and may often vary considerably from month to month as the growing follicle cohort itself varies month to month. The follicular phase becomes notably shorter, and as a result, estradiol (E2) production is variable and even elevated at times. Follicle growth is more rapid, but ovulation may occur at smaller follicle diameters [4]. There is evidence that follicles may grow relatively rapidly in the preceding luteal phase, causing very short follicular phases, a phenomenon that has been named ‘luteal out of phase events’ [5]. The Study of Women’s Health Across the Nation (SWAN) collected daily urinary samples for an entire menstrual cycle annually from women at different stages of the menopause transition. A sharp drop-off in the proportion of ovulatory cycles begins at about 5 years before the final menstrual period (FMP) (Figure 2). Moreover, in cycles that appeared ovulatory, luteal progesterone production declines. Gonadotropins rise sharply beginning at 3 years prior to the FMP [6][7]. This early phase of the menopausal transition is associated with an increase in menopausal symptoms such as hot flashes, though initially, the increase may be relatively small as there may not necessarily be a reduction in the amount of circulating E2. By the late transition, prolonged amenorrhea (defined as > 60 days) occurs, and is associated with a persistently reduced follicle pool and failure of folliculogenesis. At this point in the transition, estrogen deficiency begins to dominate, bone mineral density loss begins [8] and menopausal symptoms including hot flashes and vaginal dryness increase sharply in prevalence. Although the median duration of the transition is about 4 years, its duration is longer in women with onset at an earlier age, and can persist as long as 10 years or more in some cases [9].

Figure 1: The Stages of Reproductive Aging Workshop +10 staging system for reproductive aging in women [2].

PNG

Figure 2. Study of Women’s Health Across the Nation (SWAN). Percent of cycles without evidence of luteal activity (ELA). [7]

Menopause

Menopause is defined as the cessation of menstruation for 12 months in a woman over age 45 and occurs at a median age of 52 years [10]. This event represents permanent failure of ovarian function secondary to depletion of the follicular pool. As such, supporting granulosa cells cease to produce estrogen and theca cells cease to produce androgens, and subsequently, ovarian estrogen and progesterone production stops. There is no established relationship between a woman's age at menarche and her age at menopause. However, it is well established that a woman's age at menopause is reflective of her mother's age at menopause [11]. Although few specific linked genes have been identified, there is heritability for age of menopause, and thus, several genes are likely involved in ovarian aging [12]. Menopause is known to occur approximately 1-2 years earlier in tobacco users [10].

Primary Ovarian Insufficiency

Primary ovarian insufficiency (POI), or premature ovarian failure (POF) has been defined as 3-6 months of amenorrhea accompanied by FSH levels greater than 40 IU/L on two separate occasions, at least one month apart in a woman less than 40 years old. POI is diagnosed in 5-10% of women who are evaluated for amenorrhea and the overall prevalence in the general population is thought to be around 1.1% [13]. The designation of “premature menopause” for such patients implies that menses will never happen again and this term should not be used. Rather, many recommend the use of the term “premature ovarian insufficiency (POI)” to describe the syndrome. POI and POF are more neutral terms, as young women with prolonged hypergonadotropic amenorrhea, unlike their older counterparts, are far more likely to have some intermittent ovarian function after the diagnosis has been made.

The treatment for POI usually consists of combined estrogen and progestin replacement. It is important to recognize that the risk to benefit equation of HT for women under age 40 who have ovarian failure differs from that for menopausal women aged 50-79. A preventive cardiovascular benefit for HT appears to be more likely in younger women. Women with early loss of ovarian function are likely to spend more years of their lives exposed to the risk of bone demineralization, and therefore this important protective benefit of hormones is more likely to be realized. There are no current, evidence-based criteria to determine how to best provide hormone therapy to women with POI/POF, but it is widely assumed that hormone treatment up to the mean age at natural menopause should be considered in most cases, with a re-evaluation of risk to benefit once a woman attains the age associated with natural menopause in the population.

PHYSIOLOGIC CHANGES ASSOCIATED WITH AGING AND MENOPAUSE

Cardiovascular System

The largest health threat to women over age 50 is cardiovascular disease (CVD) [14]. In women age 45-49, the incidence of CVD is 3 times lower than men of matched age. However, data from the Framingham study have shown that by age 75-79, a woman's risk of heart disease increases and equals a man's risk for her age [15]. Women are less likely to be diagnosed correctly, less likely to undergo the correct revascularization procedure, and less likely to survive a major cardiac event than are men. It is critical to develop new ways to identify preclinical disease amendable to intervention and prevention. Women appear to have risk factors that differ substantially from men, and include more social/emotional and autoimmune/inflammatory risks, along with more microvascular disease [16]. Further, vascular dysfunction is associated with more bothersome menopausal vasomotor symptoms [17]

Carotid intimal medial thickness (CIMT) has emerged as a strong predictor of subsequent disease and serves as a non-invasive marker of subclinical cardiovascular disease. El Khoudary, et al, have found associations between low endogenous SHBG and estradiol and elevated FSH with increased CIMT in perimenopausal women [18]. Endothelial function is also a predictor for CVD, and has been shown to decrease during the menopause transition. Due to these changes associated with diminishing ovarian function, researchers have been studying the use of hormone therapy to prevent the rise in CVD risk associated with menopause [19].

HT for the secondary prevention of coronary heart disease (CHD) was evaluated in The Heart and Estrogen/Progestin Replacement study (HERS) [20]. This trial included 2763 post-menopausal women with pre-existing CHD followed over 4 years. The objective of this study was to see if initiating HT would alter a woman's risk of future events. All participants were post-menopausal, younger than age 80 with a uterus and established CHD. Women were prescribed conjugated equine estrogen (CEE) 0.625 mg with medroxyprogesterone acetate (MPA) 2.5mg daily or placebo. The primary outcome was the occurrence of fatal or nonfatal myocardial infarction (MI). Secondary outcomes were other cardiovascular events: coronary revascularization, unstable angina, congestive heart failure, resuscitated cardiac arrest, stroke or transient ischemic attack and peripheral arterial disease. The results showed no significant differences in the occurrence of fatal or nonfatal myocardial infarctions by treatment. However, in the first year of the study, there were significantly more CHD events in the HT group, as well as a higher incidence of thromboembolic events (both deep venous thrombosis (DVT) and pulmonary embolus) and gallbladder disease when compared to placebo. The incidence of diabetes mellitus decreased by 3.5% over 4 years in the HT group. Similar to the results of the PEPI Study on intermediate cardiovascular markers [21], the HT group had a decrease in LDL cholesterol and an increase in HDL cholesterol when compared to placebo. These investigators concluded that HT did not reduce the risk of future cardiac events in post-menopausal women with established CHD. In addition, because of the increased incidence of adverse cardiac events in the first year of treatment, initiating HT in women with established CHD is not recommended. Based on the findings of the HERS Study, HT should not be initiated for secondary prevention of cardiovascular disease.

Studies have also focused on the possibility that use of HT during an optimal ‘window of opportunity’ in the early postmenopause could be effective for primary prevention of cardiovascular disease. The Women’s Health Initiative hormone therapy clinical trial did not find primary protection against CVD in women treated for a mean of 5-7 years with either conjugated equine estrogen alone (women with a hysterectomy) [22] or estrogen plus the progestin, medroxyprogesterone acetate [23], and these results were consistent in cumulative 18-year follow-up [24]. When subgroup analyses were performed by age, women in the youngest age group (50-59 at enrollment), did not demonstrate significant benefit from HT. The Kronos Early Estrogen Prevention Study (KEEPS) tested the hypothesis that early intervention with estrogen delays the onset of atherosclerosis using a regimen of either conjugated equine estrogen or transdermal estradiol, both with cyclic administration of oral, micronized progesterone for 12 days each month. KEEPS did not demonstrate any between-group differences in CIMT or coronary calcium scores [25]. The rationale that hormones are protective of the vascular system when initiated early in menopause was supported, however, by the Early versus Late Intervention Trial with Estradiol (ELITE). The ELITE study found that oral estradiol treatment initiated within 6 years of menopause reduced CIMT compared to placebo, and this effect was not seen in women who initiated estrogen 10 or more years from menopause [26]. The long-term effect of time of HT initiation on CVD is therefore not established and HT is not currently recommended for primary prevention of CVD, regardless of age at initiation.

LIPOPROTEIN CHANGES, CARDIOVASCULAR RISK, AND HT

The role of menopause in contributing to dyslipidemia has long been hypothesized. In women, total and low-density lipoprotein (LDL) cholesterol increase with age, and this increase is accelerated by menopause, whereas cardioprotective, high density lipoprotein (HDL) decreases. Moreover, the protective effect of HDL cholesterol appears to be diminished as women progress through menopause—possibly related to denser sub-particle size [27]. A rise in LDL has specifically been associated with the latter part of the menopausal transition and appears to be related to the loss of estrogen at this time of life [28]. In agreement with this finding is the relatively sharp upturn in CIMT observed in association with the late menopausal transition [29]. Through exercise, a low-fat diet, and cholesterol-lowering drugs, patients with high total and LDL cholesterol levels are able to significantly lower these lipoprotein levels and their subsequent risk for heart disease [30].

The Womens Health Initiative (WHI) trials describes a group of randomized, placebo controlled, clinical primary prevention trials that were designed to test the effects of HT, diet modification, and calcium and vitamin D supplements on CVD, fracture risk, and breast and colorectal cancer. The WHI had three overlapping clinical trials. One was to test the effects of a low-fat diet on breast cancer and cardiovascular disease outcomes; one was to test the effect of calcium plus vitamin D on fracture outcomes, and one was to test the effects of hormone therapy in cardiovascular disease outcomes. The hormone therapy trial consisted of three study arms: The Estrogen + Progestin arm (conjugated equine estrogen (CEE) + medroxyprogesterone acetate (MPA) was administered to women with a uterus, the estrogen-alone arm (CEE) was administered to women without a uterus, and a placebo arm involved both women with or without a uterus. The WHI findings suggest that administration of HT does not protect the heart. While the initial analysis showed that CEE + MPA use was associated with a 24% overall increase in the risk of CHD (6 more heart attacks annually per 10,000 women using CEE + MPA) and an 81% increased risk of CHD in the first year alone after starting therapy, 18-year cumulative follow-up showed no difference in CHD and CVD-related mortality [24]. Women who had higher baseline LDL cholesterol levels at the beginning of the study were at particularly high risk of CHD with HT use [31]. Although the expected changes in lipoproteins were observed with hormone therapy (decreased LDL and increased HDL), there was no associated reduction in CHD risk.

The estrogen alone arm (CEE) differed from the CEE + MPA study in that it enrolled women who did not have a uterus, and who therefore did not need progestin. In this trial, 10,739 women with a prior hysterectomy, aged 50-79 years, were assigned to CEE 0.625 mg daily or to placebo. The study was stopped ahead of schedule in February 2004 for ‘futility’. During 7.1 years of follow up, estrogen provided no overall protection against heart attack or CAD in healthy post-menopausal women, most of whom were more than 10 years past menopause when they entered the study. In women 50-59 years of age at study entry, there was a suggestion of lower rates of heart attacks or procedures to revascularize thrombosed coronary arteries; however, these findings could be due to chance [22].

Data from the WHI estrogen-alone arm (CEE) supports the notion that coronary calcium accrual is prevented by early intervention with estrogen. The WHI evaluated the presence of coronary artery calcium (CAC) burden to determine whether or not it differed based on treatment assignment. The WHI Coronary-Artery Calcium Study (WHI-CACS) evaluated 1,064 women aged 50 to 59 years after a mean of 7.4 years. CAC was evaluated by cardiac CT scans, which were performed blindly on patients to measure the CAC in these estrogen-alone participants. CAC scores were lower in women in the (CEE) alone group compared to those in the placebo group. The mean CAC score was 83.1 for (CEE) and 123.1 for placebo. After taking into account other heart disease risk factors, the risk of having mild-to-moderate CAC was 20-30% lower and the risk of severe CAC was 40% lower in the (CEE) group compared to placebo. After the trial ended, the calcium plaque build-up in the coronary arteries was lower in women randomized to estrogen compared to placebo [32].

In conclusion, studies show that most women have minimal CAC and minimal increases in carotid IMT prior to menopause. The findings imply strongly that ovarian hormones exert a protective effect on the cardiovascular system in premenopausal women, even though they do not appear to maintain a protective role after menopause. Despite these data, and secondary findings suggestive that early intervention with hormones may delay the onset of clinical heart disease, prescribing hormones for this purpose cannot be recommended based on the available data. These studies are unlikely to be the last word in this controversial field.

COAGULATION

After menopause, there are noted changes in clotting parameters. There is an increase in procoagulation factors including fibrinogen, plasminogen activator inhibitor-1 (PAI-1), and factor VII, all of which cause a relatively hypercoagulable state. These increases are thought to be another contributor to the increase in cardiovascular and cerebrovascular disease in older women. With the administration of oral estrogen therapy, many procoagulation parameters improve, as evidenced by a decrease in fibrinogen and plasminogen levels; however, there is a higher risk for venous thromboembolism (VTE) due to increased liver metabolism of estrogen given orally [33].

HT in currently used doses is associated with an approximately 3-fold increase in VTE events. Transdermal estrogen preparations bypass liver metabolism and may be associated with the lowest VTE risk. Multiple observational studies have demonstrated fewer VTE and ischemic events with transdermal estrogen preparations compared to oral [34] [35-37]. SERM preparations also increase VTE risk. Tamoxifen increases VTE risk in a manner similar to oral estrogen, whereas raloxifene is associated with fewer VTE events than tamoxifen or estrogen [38, 39]. Bazedoxifene showed similar VTE risk compared to raloxifene in a randomized placebo-controlled trial [40]

Skeletal System

Osteoporosis is a major concern for postmenopausal women, leading to substantial morbidity and mortality. Fifty percent of women over age 65 have a compression fracture. Maintenance of bone mass is critical to prevent the development of osteoporosis. Height loss, up to several inches, and postural changes including kyphosis and lordosis are also caused by vertebral fractures. The mortality rate of women with hip fractures is 20% within the year following the fracture [41].

After peak bone mass is attained, usually around age 30, there is a slow, steady decline during the reproductive years, when approximately 0.7% of total bone is lost per year. At menopause, there is an accelerated rate of bone loss; 5% trabecular and 1.5% of total bone mass, on average, is lost per year. In the first 20 years after menopause, there is a 50% reduction in trabecular bone and 30% reduction in cortical bone, primarily due to the lack of estrogen [42].

Estrogen is responsible for promoting osteoblast (bone-forming cell) activity. It also inhibits bone remodeling and balances osteoblast and osteoclast (bone-resorbing cell) activity. As levels of serum estrogen decline in menopause, there is an increase in the rate of bone loss. As such, increased bone turnover increases serum calcium. This increase in serum calcium, in turn, causes a decrease in parathyroid hormone (PTH) secretion, followed by calcinuria and decreased renal production of 1,25 dihydroxy-vitamin D. Vitamin D is responsible for intestinal calcium absorption and kidney tubular reabsorption. This domino effect causes a postmenopausal woman to lose 20 to 60 mg of calcium daily [43].

OSTEOPOROSIS SCREENING

It is a challenging public health problem to provide a cost-effective approach to identify women who are most likely to fracture, and to preferentially target them for screening and therapy.

An important and sensitive test to identify bone loss is a Dual Energy X Ray Absorptiometry (DEXA) scan. Usually two sites are analyzed-- the lumbar spine and the femoral neck (occasionally the radius is also checked). Scoring systems for evaluating Bone Mineral Density (BMD) are based on the T-score and Z-score. The T-score compares the patient's BMD to young women at peak bone mass whereas the Z-score compares the patient to women her own age. It is the T-score that is used to make a diagnosis.

The World Health Organization (WHO) has established the following definitions:

normal BMD as a T-score => -1 standard deviation (SD) of the mean
osteopenia as BMD between -1 and -2.5 SD
osteoporosis as a T-score =< -2.5 SD

However, BMD via DEXA scan has a precision error of 2 to 6% depending on the site, which can amount to almost 1 t-score unit [44].

Bone density screening is useful, but does not provide all of the desired information about true fracture risk. Low bone density alone will not cause a fracture, unless it is so low that activities of daily living cause bones to break. Rather, women must have a combination of low bone density and a predisposition to falling that increases their risk. All major current guidelines state that BMD screening should begin at age 65 years for women of ‘average risk’ [45]. The rationale for waiting until age 65 to screen is that for most women, therapy will not need to be initiated before this time. Most guidelines also agree that BMD screening can and should be used selectively for women younger than 65 years if they are postmenopausal and have other risk factors for fracture (Table 1). Other considerations for BMD screening include estrogen deficient women of any age, vertebral anomalies and primary hyperparathyroidism.

Table 1. When to Screen for Bone Density Before Age 65 Years

Bone density should be screened in postmenopausal women younger than 65 years if any of the following risk factors are noted:

Medical history of a fragility fracture
Body weight less than 127lb
Medical causes of bone loss (medications or diseases)
Parental medical history of hip fracture
Current smoker
Alcoholism

- Premature ovarian failure
Rheumatoid arthritis

In order to attempt to address the factors beyond bone density that can be used to predict fractures, the World Health Organization (WHO) developed the Fracture Risk Assessment Tool (FRAX) to identify those women who are at the greatest risk for fracture. FRAX was developed to calculate the 10-year probability of a hip fracture and the 10-year probability of a major osteoporotic fracture (defined as a clinical vertebral, hip, forearm or humerus fracture) taking into account femoral neck BMD and the risk factors listed below in Table 2. Clinicians can use the FRAX tool to make clinical decisions regarding BMD testing (http://www.shef.ac.uk/FRAX/index.aspx). FRAX can be used in women younger than 65 years to determine which women should have a BMD scan [46]. Those women with a FRAX 10-year risk of major osteoporotic fracture of 9.3% could justifiably be referred for DXA because that is the risk of fracture found in a 65-year-old Caucasian woman with no risk factors. It is important to note that FRAX does not provide data on fracture risk for women aged 40 or under.

While FRAX has been a highly utilized tool for clinicians, The American College of Physicians (ACP) recently suggested there is little evidence demonstrating effective treatment outcomes [47]. This limitation of the FRAX assessment was based on a randomized, controlled trial that showed that raloxifene significantly reduces clinical fractures in women ages 31-81 years but has similar efficacy regardless of a woman’s degree of fracture risk [48]. Ultimately when using screening tools, it is important for clinicians to take into account not only the age, but also presence of risk factors when deciding whom to screen for BMD testing.

Table 2. WHO Technical Report: Fracture Risk Assessment Model

Risk Factors Included in the Fracture Risk Assessment (FRAX) Model

• Current age

• Rheumatoid arthritis

• Sex

• Secondary osteoporosis

• A prior osteoporotic fracture

• Parental history of hip fracture

• Femoral neck BMD

• Current smoking

• Low body mass index (kg/m2)

• Alcohol intake (3 or more drinks/day)

• Oral glucocorticoids ≥5 mg/d of prednisone for ≥ 3 month

AVOIDING BONE LOSS

Exercise, calcium and vitamin D supplementation can help protect women from bone loss. By engaging in regular weight-bearing exercise, women lose less bone than they would if they remained sedentary [49]. The Institute of Medicine recommends women ingest 1200 mg of dietary calcium and 400 IU dietary vitamin D daily to help protect from menopausal bone loss [50]. Supplementation with calcium and vitamin D if dietary levels cannot be achieved has been recommended. However, concerns have been raised about calcium supplementation including increased risk of renal stones and cardiovascular events [51]. Data from several large clinical trials raise the possibility that a small but statistically significant risk for cardiovascular disease exists (Table 3). This risk does not seem to exist if a woman takes in calcium through dietary sources. It has been speculated that higher serum calcium levels are achieved with supplements but not when calcium is absorbed through consumption of calcium-rich foods, and that this transient; high circulating calcium can cause tissue calcification and dysfunction.

Table 3. Calcium Supplementation and Risk of Heart Disease.
Author	Study	N	Findings
Bostick [52]	Iowa Women’s Health Study	34,486	Decreased risk HR 0.66
Michaelsson [53]	Swedish Cohort Study	61,433	>1400mg/day increased risk HR 2.57
Chung [54]	Meta-analysis	200 articles	No association
Bolland [55, 56]	WHI—CT ONLY	36,282	>1000mg/day Increased risk
Prentice [57]	WHI—CT +OS	>100,000	No association
Xiao [58]	NIH-AARP diet and health study	388,229	No increased risk with supplements
Paik [59]	Nurse’s Health Study	74,245	No increased risk with supplements
Donneyong [60]	WHI – CT + Vit D	35,983	Heart failure reduced in women with highest risk for heart failure

Such cardiovascular risk has not been demonstrated with vitamin D. Two recent controlled trials did not demonstrate increased cardiovascular events with use of high-dose vitamin D supplementation [61, 62]. However, guidelines do not currently support daily supplementation with calcium or vitamin D for primary prevention of fracture in postmenopausal women [45]. Practically speaking, patients should be encouraged to eat as many calcium and vitamin-D rich foods as they can through their diet. Those who have documented vitamin D deficiency should be given supplements.

TREATING OSTEOPOROSIS

Treatment for osteopenia and osteoporosis includes weight-bearing exercise, dietary modification, assuring adequate calcium and vitamin D intake, and the introduction of other medications. There are several different types of medications that can be used to treat low BMD: bisphosphonates, SERMs, calcitonin, hormones, and denosumab are all clinically-proven anti-resorptives (Table 4).

Table 4. Treatments for Osteoporosis
Treatment and Prevention
Bisphosphonates	Alendronate (Fosamax) 10 mg daily tablet, 70 mg weekly tablet, or liquid formulation Risedronate (Actonel) 5 mg daily tablet, 35 mg weekly tablet, or 150 mg monthly (75 mg tablet on 2 consecutive days) Ibandronate (Boniva) 2.5 mg daily tablet, 150 mg monthly tablet, or 3 mg IV therapy every 3 months Zoledronic Acid (Reclast) 5 mg IV therapy yearly
SERM	Raloxifene HCl (Evista) 60 mg daily
Treatment Only
Calcitonin	Calcitonin Salmon (Miacalcin or Fortical) 200 IU daily intranasal spray or 100 IU daily IM or SQ
PTH	Recombinant PTH (1-34) Teriparatide (Forteo) 20 µg SQ daily
RANK-L ligand inhibitor	Denosumab (Prolia) 60mg SC q6months
Prevention Only
HT	Estrogen (see table 10 for detailed information)
TSEC	Conjugated estrogen + bazedoxifene (Duavee) 0.045mg/20mg daily tablet

Although most fractures occur in women with bone density in the osteopenic range, it is

not recommended to treat osteopenia without additional features that carry a more worrisome prognosis for fracture [63]. The approved medications for both treatment and prevention of osteoporosis include bisphosphonates and the SERM, raloxifene. Bisphosphonates have been a mainstay of therapy for many years, and act by inhibiting bone resorption. Although they have a long track record of efficacy and safety, prolonged and high-dose usage has been associated with the rare side effects of osteonecrosis of the jaw and atypical femoral fracture [63]. Recent research indicates that bone density is maintained for several years after discontinuation of treatment, and ‘drug holidays’ may help reduce the risk of developing adynamic bone. Recent guidelines recommend treatment for 5 years and do not recommend additional BMD assessment during this time. Bisphosphonates should be avoided in women of child-bearing potential as they deposit in the bone, have a very long half-life, and accumulate in fetal bone if they are given to the mother.

Raloxifene acts like a pro-estrogen on bone, lipids and liver and acts as an anti-estrogen on both the uterus and the breast. This makes its effects more favorable than tamoxifen, which acts like a mixed estrogen agonist on the uterus. The landmark MORE (Multiple Outcomes of Raloxifene Evaluation) trial evaluated the ability of raloxifene to prevent fractures in women with established osteoporosis. 7705 post-menopausal women were randomized to either 60 or 120 mg of raloxifene versus placebo. The risk of both vertebral and non-vertebral fractures was reduced in the groups treated with raloxifene, and BMD increased in both the hip and the spine in raloxifene treated patients [64]. Furthermore, a substantial decrease in the incidence of breast cancer was noted in raloxifene treated women, and the risk of having estrogen receptor positive invasive breast cancer was decreased when compared to placebo [65]. There was no difference between treatment groups with respect to the development of endometrial cancer.

For prevention of osteoporosis in postmenopausal or hypoestrogenic women, menopausal hormone therapy (when symptoms are present) or bazedoxifene/conjugated equine estrogens are appropriate agents [63]. A disadvantage of HT compared to bisphosphonates is the abrupt decrease in bone density that occurs when HT is stopped. Bazedoxifene is a SERM that has a similar profile to raloxifene, and thus, when combined with estrogen, appears to exert a neutral effect on the endometrium and can therefore be given without a concomitant progestin. This confers a significant advantage over HT for women with a uterus. This combination of SERM with estrogen, such as bazedoxifene with estrogen, is termed a tissue selective estrogen complex (TSEC). Bazedoxifene has a similar profile to raloxifene but has not yet been tested in a large clinical trial for outcomes related to breast cancer [66]. Thus far, clinical studies demonstrate no reports of breast concerns or benefits.

Denosumab is a human monoclonal antibody to the receptor activator of nuclear factor-κB ligand (RANKL) that blocks its binding to RANK, inhibiting the development and activity of osteoclasts, decreasing bone resorption, and increasing bone density. This drug is approved for treatment, but not prevention, of osteoporosis. Denosumab can be given subcutaneously twice yearly to reduce the risk of vertebral, nonvertebral, and hip fractures in women with osteoporosis [67].

Parathyroid hormone (PTH) acts as an anabolic metabolite to stimulate bone production from osteoblasts, and is approved for treatment of osteoporosis. PTH decreases the incidence of new fractures and increases bone density. However, adverse side effects include hypercalcemia and gastrointestinal symptoms. Early rodent studies were concerning for possible bone tumor formation; however, post-marketing studies have not reported any cases. It remains that its approved use in humans is only for 24 months [68].

Calcitonin inhibits bone resorption, though not as effectively as other osteoporotic therapies. It is only available in intranasal or injectable forms as no effectiveness has been shown from oral formulations [69]. This is not generally considered first-line therapy but is a useful alternative when other medications are contraindicated.

Once a patient has been started on therapy, markers of bone turnover can be used to assess a patient's response. Urinary calcium, deoxypyridinoline, pyridinoline, hydroxyproline and N-telopeptides can be checked after 1-3 months of initiating treatment in selected cases [67]. DEXA scans, although they are currently the best method for determining BMD, should not be repeated too frequently since errors in interpretation of trends can occur and lead to inappropriate therapy [70]. It is recommended that DEXA scans be repeated no more frequently than every 2 years.

Central Nervous System

Vasomotor symptoms and “hot flashes” adversely affect the quality of life and functional status of most women during the menopausal transition. Hot flashes can occur in up to 85% of menopausal women. Col et al. estimated the duration of vasomotor symptoms in a longitudinal study on 438 women from the population-based Melbourne Women's Midlife Health Project. The onset and cessation of vasomotor symptoms were reported, and stratified according to whether or not HT was used. They found that the mean (SD) duration of bothersome menopausal symptoms for women who never used HT was 5.2 (3.8) years [71]. A meta-analysis of 35,445 women taken from 10 different studies appeared to confirm a median 4-year duration of hot flashes, with the most bothersome symptoms beginning about 1 year before the final menstrual period and declining thereafter [72]. However, two newer studies that have examined women longitudinally over a longer time frame indicate that the duration of vasomotor symptoms may be far longer than previously appreciated [73, 74]. These studies have found that hot flashes may last as long as 10 years in up to one quarter of women who report them. The earlier in life that they appear, the longer they may last, and among all racial/ethnic groups studied, African-American women appear particularly vulnerable to long duration, bothersome vasomotor symptoms.

The exact etiology of the hot flash has not been elucidated but a resetting and narrowing of the thermoregulatory system is believed to occur. In the past, hot flashes were thought to be related to a withdrawal of estrogen; however, there is no acute change in serum estradiol during a hot flash. Others have related hot flashes to variability in both estradiol and FSH. It is thought that decreased estrogen levels may reduce serotonin levels and thus upregulate the 5-HT2A receptor in the hypothalamus. As such, additional serotonin is then released which can cause activation of the 5-HT2a receptor itself. This activation changes the set point for temperature and results in hot flashes [75]. More recent work has focused on the kisspeptin-neurokinin B-dynorphin neurons of the hypothalamus, the so-called KNDy neurons. Ablation of the neurokin 3 receptor (NK3R) has been shown to abolish cutaneous vasodilatation in oophorectomized rats [76], and use of compounds that selectively block the NK3R have been shown to be effective in humans [77]. These exciting findings bring us closer to an understanding of the etiology of hot flashes and indicate the potential for novel treatments (discussed below).

MOOD

Significantly higher odds of depressive symptoms are reported by women who reach the late perimenopause. In the Study of Women’s Health Across the Nation (SWAN) [78], as well as 2 other longitudinal studies of the menopausal transition [79, 80], risk for depression was most pronounced in women who began the study with a low Center for Epidemiologic Studies Depression Scale score [78], indicating that the depressive symptoms were of new onset and appeared to be directly related to the menopausal transition. Follow-up studies using a Structured Clinical Interview for DSM-IV Axis I Disorders (SCID) confirmed that the late perimenopause is a vulnerable window for new-onset major depression [81]. The late perimenopause is also associated with a higher prevalence of sleep difficulty [82], which in turn is associated with depressive symptoms. Recent examination of anxiety symptoms in perimenopausal women indicate that, similar to depression, those with lower anxiety scores prior to the onset of the menopause transition are most vulnerable to a sudden escalation of anxiety and experience the greatest negative impact from their symptoms [83]. Not surprisingly, women with a lifetime history of anxiety and depressive symptoms during their menopausal transition report the lowest health related quality of life (HRQOL) [84], and poor sleep exacerbates these associations.

COGNITION

Women routinely complain of cognitive deficits around the time of menopause. Certain aspects of cognition appear to be related to a decline in estrogen, but many are simply related to the aging process itself. While some studies have demonstrated improved short term and verbal memory in postmenopausal women taking estrogen [85],others have not found such beneficial effects [86]. Greendale et. al. observed a sub-cohort of 2,362 SWAN participants longitudinally over 4 years to determine the effects of the menopausal transition and HT use on cognitive performance in midlife women. The outcomes analyzed were longitudinal performance in 3 separate areas: processing speed, verbal memory and working memory. The results of the study showed that, consistent with transitioning women's perceived memory difficulties, perimenopause was associated with a decrement in cognitive performance, characterized by women not being able to learn as well as they had during premenopause. Improvement rebounded to near-premenopausal levels once the transition was completed, suggesting that menopause transition-related cognitive difficulties may be time-limited. The initiation of HT prior to the final menstrual period had a beneficial effect, whereas initiation after the final menstrual period had a detrimental effect, on cognitive performance (68) [87]. More recently, the Cognitive Affective Study of the Kronos Early Estrogen Prevention Study (KEEPS-Cog) evaluated the impact of 4 years of HT on mood and cognition in early postmenopausal women. Various cognitive factors were not influenced by HT over 4 years, though a slightly positive effect on mood was observed in patients receiving oral conjugated equine estrogens. Mood and cognition did not differ between women receiving transdermal estrogen or placebo [88].

DEMENTIA AND ALZHEIMER’S DISEASE

The most common form of dementia is Alzheimer's disease (AD), which is 3 times more common in women than in men. Women with preexisting dementia or AD have been noted to have lower serum estradiol levels than women without dementia [89]. In observational studies, less AD has been observed in postmenopausal women who use estrogen and the effect was greater with increasing duration of use [90, 91]. In some trials, women with mild to moderate AD who were given estrogen had improvement in their dementia [92, 93], but this was not observed in all clinical trials [94, 95]. Estrogen has been believed to help prevent AD by regulating synapse formation in the hippocampus and by inducing acetycholinesterase and choline acetyltransferase, both of which are important in memory [96]. Estrogen may also improve cognitive function because of protection against neuronal toxicity caused by oxidation and increasing metabolism of serum amyloid P [97]. However, these molecular findings do not appear to translate into clinical benefits, as the WHI’s Mental Status (WHIMS) Trial demonstrated that hormone treatment with either (CCE+MPA) or (CCE) alone doubled the risk of AD and mild cognitive impairment. These clinical trial findings do not support a long- term role of estrogen in the prevention or treatment of AD. However, considerable controversy remains, as the sensitivity of the testing used in the WHI may not have been adequate to detect early disease. As stated above, the KEEPS Trial did not note cognitive differences among women randomized to 2 types of estrogen plus progesterone or to placebo (86). This is noteworthy because KEEPS used a very detailed cognitive battery of tests.

LIBIDO

Loss of libido is a prevalent complaint in women of all ages and is present in approximately 9% of postmenopausal women [98]. Causes for a menopause-related decline in sexual interest may relate partly to a drop in both estrogen and testosterone with ovarian decline and aging, respectively. It is very important to consider the medication history and to screen for depression when clinically evaluating women with a complaint of diminished libido. In a survey of 35,381 women (the PRESIDE Study) [99], 10% reported decreased sexual desire; when women without concomitant depression or antidepressant medication were accounted for, the prevalence of desire disorder decreased to 6.3%.

Testosterone has long been considered as an agent that might promote libido in women. Several well-conducted, double-blind, randomized trials of testosterone in menopausal women with decreased libido have demonstrated small, but clinically and statistically improved symptoms [100]. Testosterone has been used as a transdermal formulation in most of these studies and demonstrates efficacy with or without concurrent use of estrogen, in women with and without their ovaries. The APHRODITE study examined transdermal testosterone in 814 menopausal women over 52 weeks. Women were randomly assigned to receive either a patch delivering 150 or 300µg of testosterone per day or placebo. Evaluation at week 24 demonstrated that the women on the 300µg testosterone patch noted a significantly greater increase in their 4-week frequency of satisfying sexual episodes in comparison to placebo, but this was not observed in the group receiving 150 µg per day. Both doses of testosterone patches were associated with significant increases in desire compared with placebo. Androgenic adverse events were greater in the group receiving 300 µg of testosterone per day. Breast cancer was diagnosed in 4 women who received testosterone (as compared with none who received placebo) [101]. The excess cases of breast cancer in women treated with testosterone may be due to chance. However, the possibility of a causal relationship must be considered as several published studies have shown that higher levels of endogenous testosterone and administration of exogenous testosterone are associated with the risk of breast cancer [102, 103]. Clearly, long-term data from large clinical trials using testosterone are lacking and are needed [100]. Of note, a recent trial of a testosterone gel for female libido was discontinued because of lack of efficacy. There are no FDA-approved testosterone preparations available for women.

The only FDA approved medication for treatment of hyposexual desire disorder is flibanserin, marketed as Addyi. Flibanserin is a centrally-acting serotonin agonist/antagonist that increases female sexual desire and number of sexual acts [104]. These effects have been observed in the postmenopausal population, although it is not FDA-approved for this group of women. Adverse effects are generally mild and short-lived, except for a risk for hypotension and sedation if it is taken concurrently with alcohol, a problem that resulted in a black box warning and a need for prescribing clinicians to complete a risk evaluation and management strategy (REMS) certification before prescribing the drug [105]. Its effect size appears similar to that of testosterone (small, but statistically and probably clinically significant) [104].

Breast

After menopause and with aging, breast tissue is gradually replaced with increasing amounts of adipose tissue. This causes an age associated decrease in breast density, which makes mammography more effective in detecting breast disease. Breast cancer becomes more prevalent with advancing age with a lifetime risk of breast cancer in 1:8 women [106].

BREAST CANCER AND HT

Combined estrogen and progesterone treatment increase a woman's risk of developing breast cancer. The WHI trials demonstrated a detectably increased risk of developing invasive breast cancer after 3 years of combined HT use, with an unadjusted hazard ratio of 1.26 over 5.2 years of average follow-up [107]. Technically, the 95% confidence interval included 1.00, thus, the data could be considered ‘not significant’; however, this level of risk is biologically plausible, as it is similar to that seen in many observational studies, and similar to the small, incremental risk for breast cancer that is seen with later onset of menopause. The only risk factor identified in WHI patients for the development of invasive breast cancer was the duration of HT use. Patients taking hormones for 10 or more years were at greatest risk followed by patients using HT for 5 to 10 years. Women who took HT for less than 5 years had only a slight increase in risk. No correlation was noted between other risk factors--a patient's age, ethnicity, the 5-year Gail model risk score, body mass index (BMI), or family history--and the development of breast cancer. In women who had undergone hysterectomy and were randomized to CEE alone, no increase in breast cancer risk was observed; in fact, a decreased risk was observed in this group after 18-year follow-up [24, 108].

One of the ways in which HT might increase breast cancer is by increasing breast density. It has been noted that estrogen with cyclic micronized progesterone resulted in 16.4% more women with increased breast density [109]. A subset of 307 women in The Postmenopausal Estrogen/Progestin Interventions (PEPI) trial was studied to examine the effect of HT on mammograms. Of the group of women taking unopposed estrogen, 3.5% had an increase in breast density. Of the women taking both estrogen with progestin therapy, a 19.4-23.5% increase in breast density on mammography was noted, depending upon whether they took cyclic versus continuous MPA. Increased mammographic breast density is a strong independent risk factor (6-fold) for the development of breast cancer [110].

Case series and case-controls studies have suggested that patients taking HT who are diagnosed with breast cancer have a better prognosis than women not taking hormones, even when matched for stage of disease [111]. It has also been suggested that women who develop breast cancer while taking HT have their cancers detected at a more favorable stage and have less malignant disease [112]. These notions were disproven by the WHI Clinical Trial. Women randomized to combined HT with (MPA + CEE) had a higher risk of invasive breast cancer and mortality from breast cancer. Tumors in the women taking combined HT were comparable in histology and grade to the placebo group but were at a more advanced stage [107].

In contrast to combined E+P HT, E alone HT given to women without a uterus in the WHI, led to a decrease in breast cancer risk, which persisted after discontinuation of treatment and became statistically significant in the post-trial follow up study. After a median follow-up of 11.8 years, E alone treated women still had a lower incidence of invasive breast cancer (151 cases, 0·27% per year) compared with placebo (199 cases, 0·35% per year; HR 0·77, 95% CI 0·62—0·95; p=0·02 [113].

SCREENING FOR BREAST CANCER

The lifetime risk of developing breast cancer is 12%. Various organizations recommend breast cancer screening for average-risk women (Table 5). These guidelines all suggest an individualized approach with patients that includes consideration of a patient’s risk factors, as well as shared decision making based on a discussion of risks and benefits of screening. For average-risk women, mammography is the recommended screening modality.

Table 5. Breast Cancer Screening Guidelines*
ACOG[114]	Offer annual or biennial mammogram starting age 40, start no later than age 50. Continue until age 75.
ACS[115]	Offer annual mammogram starting at 40, start no later than age 45. Can offer biennial mammogram at age 55. Continue until within 10-years of life expectancy.
USPSTF[116]	Biennial mammogram starting age 50. Continue until age 75.
*For average-risk females ACOG = American College of Obstetricians and Gynecologists; ACS = American Cancer Society; USPSTF = United States Preventative Services Task Force

ASSESSING BREAST CANCER RISK

The Gail Model was developed to help clinicians determine if a patient was at higher risk than the general female population for the development of breast cancer [117].

The Gail Model takes into account the following characteristics:

Age
Age at menarche
Age at first live birth
Number of first degree relatives with breast cancer
Number of previous breast biopsies
Number of breast biopsies that were hyperplastic
Race/ethnicity

This model provides an individualized risk for developing breast cancer over the next 5 years and over a lifetime. Other prospective scoring systems have been developed, but as of this writing there is no other dominant system that has proven to be superior to the Gail Model. By calculating a woman's risk of breast cancer with this model, a clinician can use the information to determine if a woman should consider chemoprophylaxis to reduce her risk of breast cancer. Note that the Gail model does not factor into account breast density or HT use. It also does not account for mutations, such as BRCA1 or 2, which have a profound effect on a woman’s risk of contracting breast cancer. Other risk factors not included are history of chest radiation prior to age 30 and extreme breast density.

CHEMOPREVENTION

In women who are considered high risk for breast cancer, chemoprevention therapies are approved to reduce breast cancer incidence. These therapies include SERMs (Tamoxifen and Raloxifene) as well as aromatase inhibitors (Exemastane and Anastrazole) [118].

Tamoxifen is indicated as adjuvant treatment for breast cancer. It is also prescribed for chemoprevention of breast cancer in high-risk women. Because tamoxifen is a SERM, it has both estrogenic and anti-estrogen actions. In the breast, it acts as an anti-estrogen. In the bone, on lipids and in the uterus, it acts like estrogen. Raloxifene is also a SERM, but has the advantage of acting as an anti-estrogen at the level of the uterus. Tamoxifen was found to be effective in breast cancer prevention in a trial that included 13,388 women who were at high risk for developing breast cancer because of 1) advancing age (>60 years old), 2) increased risk based on a Gail Model predicted risk of 1.66% over the next 5 years and age 35-59, or 3) a history of lobular carcinoma in situ. Women who were randomly assigned to tamoxifen experienced a 49% decrease in the incidence of invasive breast cancer compared to those who received a placebo. In addition, there was a decrease in the risk of estrogen receptor positive breast cancer and nodal involvement in those with breast cancer. Women randomized to tamoxifen also had fewer diagnoses of non-invasive breast cancer, such as ductal carcinoma in situ (DCIS) [119].

The STAR trial investigated the ability of tamoxifen compared to raloxifene in preventing breast cancer in women at high risk for disease. All participants received either tamoxifen or raloxifene and took the drug for 5 years. In 2006, the results of STAR showed that both raloxifene and tamoxifen were equally effective in reducing breast cancer risk in post-menopausal women at increased risk of the disease. Women in the tamoxifen group and women in the raloxifene group had statistically equivalent numbers of invasive breast cancers (163 cases in 9,726 women in the tamoxifen group versus 167 cases in 9,745 women in the raloxifene group). Tamoxifen is known to be able to reduce breast cancer risk by 49%, and this study showed that raloxifene can also reduce breast cancer risk by half as well. As a result of this study, the FDA approved raloxifene as a second agent to help prevent invasive breast cancer in high-risk, post-menopausal women [120]. On an update of STAR trial, the risk ratio (RR; raloxifene: tamoxifen) for invasive breast cancer was 1.24 (95% confidence interval [CI], 1.05–1.47) and for noninvasive disease, 1.22 (95% CI, 0.95–1.59). Compared with initial results, the RRs widened for invasive and narrowed for noninvasive breast cancer. Toxicity RRs (raloxifene: tamoxifen) were 0.55 (95% CI, 0.36–0.83; P = 0.003) for endometrial cancer (this difference was not significant in the initial results), 0.19 (95% CI, 0.12–0.29) for uterine hyperplasia, and 0.75 (95% CI, 0.60–0.93) for thromboembolic events. There were no significant mortality differences [121].

To become active, tamoxifen must be metabolized by the hepatic cytochrome P450 enzyme system, specifically cytochrome P450 2D6 (CYP2D6), to its active metabolite, endoxifen. Consequently, therapy with drugs that inhibit CYP2D6 may reduce the clinical benefit of tamoxifen by interfering with its bioactivation, particularly when these drugs are used for an extended period. A significant percentage of patients with breast cancer experience a depressive disorder and are prescribed an anti-depressant, most commonly one in the selective serotonin reuptake inhibitor (SSRI) category. This is clinically relevant in the context of tamoxifen therapy, because SSRIs inhibit CYP2D6 to varying degrees. Paroxetine is an irreversible inhibitor of CYP2D6, and therefore has the greatest potential to disrupt the biological activity of tamoxifen. A population-based cohort study was performed on 2430 women treated with tamoxifen and a single SSRI from 1993-2005. Of the group studied, 374 (15.4%) women died of breast cancer during follow-up. After adjustment for age, duration of tamoxifen treatment, and other potential confounders, absolute increases of 25%, 50%, and 75% in the proportion of time on tamoxifen with overlapping use of paroxetine were associated with 24%, 54%, and 91% increases in the risk of death from breast cancer, respectively (P<0.05 for each comparison). No such risk was seen with other anti-depressants [122].

The effectiveness of aromatase inhibitors for reduction of breast cancer incidence has also been demonstrated. The MAP3 trial investigated the incidence of invasive breast cancer with exemestane versus placebo in 5,560 high-risk postmenopausal women for up to 5 years [123]. Exemestane significantly reduced invasive breast cancer by 65% compared to placebo (95% CI, 0.18-0.70). There were no cardiovascular or thromboembolic side effects. However, follow-up study demonstrated worsened BMD after 2 years in the treatment group regardless of calcium and vitamin D supplementation [124]. Thus, for women receiving this therapy, close BMD screening is important. Anastrazole for prevention of breast cancer in high-risk postmenopausal women was studied in the IBIS-II trial [125]. One thousand nine hundred twenty women were randomized to anastrazole vs placebo for 5 years. A 53% reduction in invasive cancer was seen in the anastrazole group (95% CI, 0.32-0.68). Women who were concurrently treated with a bisphosphonate did not have significant bone loss, but the anastrazole-only group demonstrated worsened BMD after 3 years [126].

Thyroid Gland

As women age, the cumulative risk of hypothyroidism increases. Frequently, symptoms are ignored or misattributed to other causes, making the diagnosis difficult. It is recommended by ACOG that all women, even asymptomatic females, have a thyroid stimulating hormone (TSH) level measured beginning at age 50 years and every 5 years thereafter [127]. The American College of Physicians (ACP) also recommends periodic screening beginning at age 50 [128], while the American Thyroid Association (ATA) recommends that screening begin at age 35 [129].

Lower Reproductive Tract

The entire gynecologic tract contains estrogen receptors. As women become menopausal, the pelvic organs may be affected by the loss of estrogen resulting in vaginal atrophy, narrowing and shortening of the vagina and uterine prolapse, leading to high rates of dyspareunia. Furthermore, the urinary tract contains estrogen receptors in the urethra and bladder, and as the loss of estrogen becomes evident, patients may experience urinary incontinence (UI). Collectively, these symptoms, previously called vulvovaginal atrophy, have recently been renamed ‘genitourinary syndrome of menopause’ (GSM) [130]. While HT is effective in reversing changes associated with GSM [131, 132], it does not consistently help with symptoms of UI. The WHI Clinical Trial found that women who received HT and who were continent at baseline demonstrated an increase in the incidence of all types of UI at 1 year. The risk was highest for women in the CEE alone arm. Among women experiencing UI at baseline, the frequency of symptoms worsened in both arms and these women reported that UI limited their daily activities. This clinical trial evidence strongly suggests that HT should not be prescribed as part of a regimen for UI alone [133]. However, HT is highly effective in the treatment of vaginal dryness. Systemic or vaginal estrogen can be used for GSM, though locally applied estrogen is preferable if there are no systemic symptoms that need to be treated. Very low doses can be used for this purpose. These low doses are believed to be safe for the uterus, even without concomitant use of a progestin. The data are currently insufficient to define the minimum effective dose, but vaginal rings, creams, and tablets have all been tested and demonstrated to reduce vaginal symptoms [134]. Ospemifene is a SERM that is FDA approved for the treatment of GSM symptoms [135]. It has a track record of endometrial safety [136] and in pre-clinical testing, was an effective antiresorptive agent for bone and may even have breast-protective effects [137]. These latter benefits remain to be proven in clinical trials. In 2016, prasterone, a formulation of dehydroepiandrosterone (DHEA), was FDA approved for the treatment of dyspareunia related to vulvar and vaginal atrophy. In a randomized controlled trial, 12-weeks of daily vaginal prasterone significantly alleviated dyspareunia compared to placebo [138]. The trial also demonstrated a significant drop in the vaginal pH, as well as improvement in vaginal dryness.

Adrenal Gland

The adrenal gland is responsible for producing androstenedione, dehydroepiandrosterone sulfate (DHEA-S) and, indirectly, total testosterone. After the menopausal years, androstenedione levels decrease by 62%, DHEA-S levels decline by 74% and testosterone, produced by the peripheral conversion of androstenedione, decreases by up to 25%. Circulating estrone, which is produced from the peripheral conversion of androstenedione, increases after menopause, whereas estradiol, which is produced from the peripheral conversion of estrone, declines. The menopause-associated drop in estrogen is related to a significant decline in sex hormone binding globulin (SHBG), resulting in a higher free testosterone level [139]. This increase in free androgens may be responsible for the clinical problem of increased facial hair and androgenetic alopecia that accompanies the postmenopausal years for some women.

MENOPAUSAL TREATMENT

Figure 3: The Hormone Health Network has developed a self-administered algorithm for menopausal women to help them determine whether or not hormone therapy is a reasonable option for them. http://www.hormone.org/MenopauseMap.

Non-Hormonal Treatment

SELECTIVE SEROTONIN REUPTAKE INHIBITORS (SSRIs)

When HT is contraindicated, (i.e., history of breast cancer), women with hot flashes may be treated with non-hormonal prescription drugs; one such class is the SSRIs [140, 141]. Once initiated, the relief of vasomotor symptoms usually occurs within a week, more rapidly than the relief of depressive symptoms, which usually takes 6 weeks or longer. The most common side effects of these drugs are nausea and sexual dysfunction but use of the lowest dose may minimize these effects.

Though not as drastic of a reduction when compared to HT, the SSRIs result in a modest improvement in symptoms. A long-acting mesylate salt of paroxetine, 7.5mg, has been FDA-approved to treat hot flashes [142]. Non-approved SSRIs that have been tested and have clinical efficacy include paroxetine (non-mesylate), escitalopram, citalopram, fluoxetine and sertraline [141].

SEROTONIN-NOREPINEPHRINE REUPTAKE INHIBITORS (SNRIS)

Venlafaxine is a combined serotonin and norepinephrine reuptake inhibitor that has shown promise in reducing the severity of hot flashes in symptomatic women. A randomized trial was conducted in 229 women for 4 weeks where women with breast cancer received either varying doses of venlafaxine (37.5, 75 or 150 mg/day) versus placebo. There was a significant reduction in hot flashes in women receiving all doses of venlafaxine in comparison to placebo. Common side effects included nausea or vomiting, which are usually limited to the first 1 to 2 weeks of treatment. Other side effects include lethargy, dizziness, constipation and sexual dysfunction [143].

GABAPENTIN

A randomized, double-blind, placebo-controlled trial was conducted on 197 women aged 45-65 years, who were menopausal and having at least 14 hot flashes per week. These women were randomized to receive either gabapentin 900 mg daily or placebo for 4 weeks. Of women assigned to receive gabapentin, hot flash scores decreased by 51% as compared with a 26% reduction in the placebo group, from baseline to week 4. These women reported greater dizziness, unsteadiness and drowsiness at week 1 compared with those taking placebo; however, these symptoms improved by week 2 and returned to baseline levels by week 4 [144]. A 2009 meta-analysis confirmed consistency across several clinical studies [145]. The dose range of gabapentin is broad, and although many clinical trials use doses of 900 mg, less may work well for individual patients. The chief limiting side effects of gabapentin are drowsiness, dizziness (which can present a hazard for falls), and weight gain.

NEUROKININ B RECEPTOR (NK3R) INHIBITORS

Neurokinin B acting on its receptor, NK3R, at the level of the hypothalamus induces vasomotor symptoms typical of menopause. It is hypothesized that variable expression of NK3-R and interaction with its ligand is responsible for the differences in reported hot flashes experienced by menopausal women. The TACR3 gene codes for NK3-R. Genome-wide association studies performed on 17,695 women from the WHI trial and observational studies demonstrated significant genetic variation in TACR3 in women who reported vasomotor symptoms [146]. An oral NK3R antagonist completed phase II clinical trials and demonstrated a 45% decrease in the number of hot flashes per week as compared with placebo [147]. This drug is not associated with the side-effects of estrogen therapy, and further study will determine its efficacy and safety for use.

NON-PHARMACOLOGIC

Non-pharmacologic options for treatment of menopausal symptoms have yet to show proven benefit in large clinical trials. There are mixed results from trials evaluating the benefits of acupuncture for treatment of menopausal symptoms including vasomotor symptoms, insomnia and mood. As acupuncture is a generally low-risk therapy, it is at the discretion of the patient to pursue this treatment modality, but effectiveness in large trials is lacking. Phytoestrogens, which are estrogen-like compounds found in products such as soy, have no proven benefit in treatment of menopausal symptoms. Chinese herbal remedies likewise have not been shown to significantly alleviate symptoms, with or without acupuncture. The MsFLASH trial is a randomized controlled trial that showed reduction of insomnia in peri- and post-menopausal women with hot flashes who were treated with cognitive behavioral therapy for insomnia (CBT-I) compared to menopause education control [148]. Women who practiced CBT-I had significant reduction of insomnia after the 8-week intervention, and these results persisted at 24-week follow-up despite having no effect on daily hot flash frequency. Practical daily lifestyle habits including exercise, dressing in layers, consuming cold drinks, avoiding caffeine and alcohol may help alleviate symptoms.

Hormonal Treatment

HT is utilized by many women for treatment of bothersome menopausal symptoms. As outlined above, there are specific risks and benefits associated with HT that may not make it suitable for some women. Moreover, many women have a tendency to shun HT because the level of discourse about its true benefits and risks are so fraught with drama! It is important for the menopause care provider to be knowledgeable about the benefits and potential risks of hormonal therapies and to have some facility with non-hormonal alternatives. This approach allows the clinician to engage the patient in truly shared decision making. It is important to maintain clear lines of communication with menopausal patients who are struggling with bothersome symptoms, because their subjective improvement is frequently the sole arbiter of success of treatment, and it is what all risks must be balanced against.

HT is the most effective treatment for vasomotor symptoms and vaginal dryness caused by the loss of endogenous estrogen production. In addition, it acts like an anti-resorptive and is therefore osteoprotective and also has been shown to reduce the incidence of colon cancer by almost 40%. As mentioned earlier in this review, it is well established that HT changes the lipoprotein profile favorably, although these latter changes do not translate into reduced cardiovascular morbidity.

However, unopposed estrogen use in women who have a uterus creates a risk for developing endometrial hyperplasia and cancer. Therefore, estrogen replacement must be accompanied by a progestin. In patients with a uterus who were given estrogen alone in The Postmenopausal Estrogen/Progestin Intervention (PEPI) Trial, 62% developed endometrial hyperplasia over 3 years. By identifying this pathology early, patients were medically treated with high doses of progestins so that no patients developed endometrial cancer [21]. It is the standard of care to give women estrogen with a progestin when they have a uterus.

The decision to prescribe HT must be based on each individual patient, taking into account the risk factors involved and creating a favorable benefit to risk ratio. To date, acceptable reasons to prescribe HT include relief of severe vasomotor symptoms and to address GSM. There is sufficient medical evidence to consider a trial of HT for women with adverse mood or sleep symptoms in association with their menopause [149]. At present, there is no indication for using HT for the prevention of cardiovascular disease, dementia/AD, or osteoporosis, or for the prevention of colon cancer, as the risks outweigh any potential benefits, although as mentioned earlier in this review, there are suggestions that premenopausal HT may have protective effects in some cases.

A key factor in the decision tree for the initiation of HT is the individual risk of breast cancer, which is a real and serious concern. It is contraindicated to prescribe HT to patients with a history of breast cancer and it is not recommended to give HT to those with a high-risk profile. The adverse events demonstrated in patients taking combined estrogen-progestin HT included a 26% increase of invasive breast cancer, with the excess risk starting to be observed after 3 years of combined HT use. It is important to note that estrogen alone treatment of women without a uterus did not increase the risk of breast cancer.

Recommendations for prescribing HT should be based upon the randomized, clinical trial results of the WHI, as highlighted throughout this review, as they currently constitute the best available medical evidence. Although the WHI studied the Prempro® formulation only, it is biologically plausible that other systemic formulations, including the transdermal patch, will carry similar risks and benefits and it should not be assumed that switching HT formulations protects a patient from adverse events.

However, The Estrogen and Thromboembolism Risk study, a multicenter case-control study of thromboembolism among postmenopausal women aged 45-70 years, demonstrated an odds ratio for venous thromboembolism in users of oral and transdermal estrogen to be 4.2 (95% CI, 1.5-11.6) and 0.9 (95% CI, 0.4-2.1), respectively, when compared with nonusers[34]. This has led ACOG, NAMS and the Endocrine Society to recommend that clinicians take into consideration the possible thrombosis-sparing properties of transdermal forms of estrogen therapy [140, 141, 150].

Women with vasomotor symptoms may consider short-term HT use at the lowest effective dose. Women who are currently taking HT and are asymptomatic, should be encouraged to periodically discontinue HT use to see whether or not symptoms return. Finally, women who desire long-term HT use for quality of life reasons (after appropriate counseling) should be evaluated regularly and their decision to continue HT periodically reassessed.

HT REGIMENS: CONTINUOUS COMBINED AND CYCLIC REGIMENS

There are many ways to prescribe HT: oral tablets, patches, creams, sprays (Table 6). Considering the importance of including a progestin, there are several different modalities of administering these medications as well. This includes continuous combined and cyclical administrations. The continuous combined formulation administers both the estrogen and progestin hormones every day. Cyclical administration means that hormones are given in a cycle: 1) unopposed estrogen is given continuously 2) progestin is added. This regimen can be a cycle every 3 days (e.g. Ortho Prefest), every 14 days (e.g. Premphase), or at the discretion of the prescribing physician (e.g. every 3 months). Although generally believed to be safe, if progestins are given less frequently than monthly, the potential for hyperplasia exists and endometrial monitoring should be considered [151].

In women just entering menopause, the cyclical administration of the estrogen and progestin is usually the simplest choice. These patients can easily make the transition from taking a low dose oral contraceptive pill in the menopausal transition (frequently prescribed to control the irregular vaginal bleeding during that time) to the cyclical form of HT. At the onset of HT, most women will experience a withdrawal bleed at the end of the treatment month. Gradually, as the endometrium thins and becomes atrophic, some women will become amenorrheic on this regimen. Although irregular vaginal bleeding is uncommon, any abnormal uterine bleeding should be investigated. Another advantage of cyclical administration is that women will know when to expect bleeding.

Advantages of giving continuous combined therapy is that a lower dose of progestin can be used and patients should not expect a withdrawal flow at the end of the treatment month. Eventually, most women become amenorrheic on this regimen. Some women also develop irregular and inconvenient vaginal spotting or bleeding. This most frequently occurs in women who have recently entered menopause and still have an endometrial lining.

Besides oral preparations, HT can be administered in a variety of other ways. Estrogen can be delivered through a vaginal ring that delivers either 0.05 or 0.1 mg/day of estradiol acetate over a three-month period. It may also be given transdermally as 17β-estradiol with norethindrone acetate or levonorgestrel. Progesterone can be administered through a levonorgestrel-releasing IUD which can be left in place for up to10 years. Finally, vaginal preparations of progesterone are also available. More recently, transdermal estradiol sprays and gels have been FDA approved (Evamist ®, Divigel, and Elestrin). These preparations are relatively short acting and sometimes need to be used more than once a day. All are FDA approved for the treatment of hot flashes.

Table 6. HT FORMULATIONS
Trade Name	Estrogen	Progestin	Dose
Vasomotor Symptom Therapies
Premarin	Conjugated Estrogen	-	0.3 to 1.25 mg PO daily
Cenestin	Synthetic Conjugated Estrogen	-	0.3 to 1.25 mg PO daily
Menest	Esterified Estrogen	-	0.3 to 1.25 mg PO daily
Estrace	17 β-estradiol	-	1-2 mg PO daily
Estinyl	Ethinyl estradiol	-	0.02 to 0.05 mg PO 1-3 x daily
Evamist	17 β-estradiol	-	1-3 sprays daily
Alora, Climara, Esclim, Menostar, Vivelle, Vivelle Dot, Estraderm	17 β-estradiol	-	1 patch weekly-twice weekly
Estrogel	17 β-estradiol	-	1.25 g daily transdermal gel (equivalent 0.75 mg estradiol)
Estrasorb	17 β-estradiol	-	2 foil pouches daily of transdermal topical emulsion
Activella	Estradiol 1 mg	Norethindrone Acetate 0.5mg	1tab PO daily
FemHRT	Ethinyl Estradiol 5 mcg	Norethindrone Acetate 1 mg	1tab PO daily
Ortho Prefest	17 β-estradiol 1 mg	Norgestimate 0.09 mg	First 3 tablets contain estrogen, next 3 contain both hormones; alternate pills every 3 days
Premphase	Conjugated Estrogen 0.625 mg	Medroxyprogesterone Acetate 5 mg	First 14 tablets contain estrogen only and remaining 14 tablets contain both hormones. 1tab PO daily
Prempro	Conjugated Estrogen 0.625 mg	Medroxyprogesterone Acetate 2.5 or 5 mg	1tab PO daily
Combipatch	17 β-estradiol	Norethindrone acetate	1 patch transdermal twice weekly
Climara-Pro	17 β-estradiol	Levonorgestrel	1 patch weekly
Angeliq	17 β-estradiol	Drosperinone	1tab PO daily
Genitourinary Symptom Therapies
Estrace	17 β-estradiol vaginal cream	-	2-4 g daily x 1 week, then 1 g three times weekly
Premarin	17 β-estradiol vaginal cream	-	0.5 g daily for 21 days on, 7 days off or twice weekly
Vagifem	17 β-estradiol vaginal tablet	-	10 mcg per vagina daily x 2 weeks, then 2 times per week
Estring	Estradiol vaginal ring	-	1 ring inserted vaginally every 3 months
Duavee	Bazedoxifene 20mg Conjugated equine estrogen 0.45mg	-	20/0.45mg daily
Ospemiphene	-	-	60mg PO daily
Prasterone	-	-	DHEA 6.5mg inserted vaginally daily

TSECs—TISSUE SPECIFIC ESTROGEN COMPLEXES

The combination of bazedoxifene/conjugated equine estrogens represents yet another novel approach to hormone therapy. The combination of bazedoxifene, a SERM, with estrogen allows the clinician to apply estrogen where it is most beneficial—reducing or eliminating hot flashes, while the SERM bazedoxifene exerts anti-estrogenic effects at the target tissues where estrogen action is unwelcome—the endometrium and the breast [66]. Thus, the combination of bazedoxifene and conjugated equine estrogens is effective as an antiresorptive agent in bone and does not cause endometrial stimulation. With the bazedoxifene/conjugated equine estrogen combination, the clinician can avoid having to give progestin and avoid irregular or breakthrough bleeding.

SUMMARY

In conclusion, this review has highlighted the major health concerns faced by the post-menopausal woman. Cardiovascular disease becomes more prevalent with the loss of estrogen and the decrease in endothelial function and HDL cholesterol levels that occur concurrent with menopause. Osteoporosis is another serious potential problem that the aging woman faces and can be prevented by careful screening and early treatment. Cognitive decline and memory changes occur as aging ensues and AD becomes more prevalent, making it more difficult for aging women to maintain an independent lifestyle. Finally, breast cancer becomes more prevalent with advancing age. The increased risk of breast cancer needs to be considered when choosing a treatment plan for the post-menopausal woman.

There are a variety of treatments available to protect women from developing serious health problems. First and foremost, a healthy lifestyle is the best preventive medicine. HT will control a patient's vasomotor symptoms, prevent bone loss, maintain a favorable lipoprotein profile, and help prevent vaginal and urogenital atrophy. Other benefits of HT include the reduction in the incidence of colon cancer. The SERM, raloxifene, also can be used to treat osteoporosis in menopausal women. The advantage of a SERM compared to HT is its lack of endometrial stimulation and reduction in the risk of breast cancer. The prevention of bone loss and the beneficial effects on lipoprotein levels with SERMs are similar to those seen with HT.

The role of HT has changed over the years as its risks and benefits have been clarified through carefully designed randomized trials, most notably, the WHI. For a low-risk woman with moderate to severe vasomotor symptoms, the introduction of HT is an effective option and patients will improve. However, the clinician needs to evaluate each patient independently and take into account the individual risk profile, including family history, in order to determine which form of treatment is most appropriate. The ability to modulate estrogen action via the development of SERMs provides the hope that a 'perfect' SERM can be produced, which will relieve vasomotor symptoms, protect the bone and the heart, maintain a favorable lipoprotein profile, and be anti-estrogenic to the endometrium and the breast. Until then, non-hormonal alternatives are available for women who cannot or do not wish to take HT. Prudent clinical judgment and an individualized assessment of risks and benefits for patients using the currently available medical evidence remains the most appropriate approach.

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The Normal Menstrual Cycle and the Control of Ovulation

ABSTRACT

Menstruation is the cyclic, orderly sloughing of the uterine lining, in response to the interactions of hormones produced by the hypothalamus, pituitary, and ovaries. The menstrual cycle may be divided into two phases: (1) follicular or proliferative phase, and (2) the luteal or secretory phase. The length of a menstrual cycle is the number of days between the first day of menstrual bleeding of one cycle to the onset of menses of the next cycle. The median duration of a menstrual cycle is 28 days with most cycle lengths between 25 to 30 days (1-3. Patients who experience menstrual cycles that occur at intervals less than 21 days are termed polymenorrheic, while patients who experience prolonged menstrual cycles greater than 35 days, are termed oligomenorrheic. The typical volume of blood lost during menstruation is approximately 30 mL (4). Any amount greater than 80 mL is considered abnormal (4). The menstrual cycle is typically most irregular around the extremes of reproductive life (menarche and menopause) due to anovulation and inadequate follicular development (5-7). The luteal phase of the cycle is relatively constant in all women, with a duration of 14 days. The variability of cycle length is usually derived from varying lengths of the follicular phase of the cycle, which can range from 10 to 16 days. For complete coverage of this and related topics, please visit www.endotext.org.

THE FOLLICULAR PHASE (Fig.1)

Figure 1. Hormonal, Ovarian, endometrial, and basal body temperature changes and relations throughout the normal menstrual cycle. (From Carr BR, Wilson JD. Disorders of the ovary and female reproductive tract. In: Braunwald E, Isselbacher KJ, Petersdorf RG, et al, eds. Harrison's Principles of Internal Medicine. 11th ed. New York: McGraw-Hill, 1987: 1818-1837.

The follicular phase begins from the first day of menses until ovulation. Lower temperatures on a basal body temperature chart, and more importantly, the development of ovarian follicles, characterize this phase. Folliculogenesis begins during the last few days of the preceding menstrual cycle until the release of the mature follicle at ovulation.

Declining steroid production by the corpus luteum and the dramatic fall of inhibin A allows for follicle stimulating hormone (FSH) to rise during the last few days of the menstrual cycle (Fig. 2) (8). Another influential factor on the FSH level in the late luteal phase is related to an increase in GnRH pulsatile secretion secondary to a decline in both estradiol and progesterone levels (9). This elevation in FSH allows for the recruitment of a cohort of ovarian follicles in each ovary, one of which is destined to ovulate during the next menstrual cycle. Once menses ensues, FSH levels begin to decline due to the negative feedback of estrogen and the negative effects of inhibin B produced by the developing follicle (Fig. 2) (8, 10-12). FSH activates the aromatase enzyme in granulosa cells, which converts androgens to estrogen. A decline in FSH levels leads to the production of a more androgenic microenvironment within adjacent follicles to the growing dominant follicle. Also, the granulosa cells of the growing follicle secrete a variety of peptides that may play an autocrine/paracrine role in the inhibition of development of the adjacent follicles.

Figure 2. Inhibin level changes throughout the menstrual cycle. Inhibin B dominates the follicular phase of the cycle, while Inhibin A dominates the luteal phase.

Development of the dominant follicle has been described in three stages: (1) Recruitment, (2) Selection, and (3) Dominance (Fig.3). The recruitment stage takes place during days 1 through 4 of the menstrual cycle. During this stage, FSH leads to recruitment of a cohort of follicles from the pool of non-proliferating follicles. Between cycle days 5 and 7, selection of a follicle takes place whereby only one follicle is selected from the cohort of recruited follicles to ovulate, and the remaining follicles will undergo atresia. Anti-Müllerian hormone (AMH), a product of granulosa cells, is believed to play a role in the selection of the dominant follicle (13, 14). By cycle day 8, one follicle exerts its dominance by promoting its own growth and suppressing the maturation of the other ovarian follicles thus becoming the dominant follicle.

Figure 3. Time course for recruitment, selection, and ovulation of the dominant ovarian follicle (DF) with onset of atresia among other follicles of the cohort (N-1). (From Hodgen GD. The dominant ovarian follicle. Fertil Steril 1982; 38:281-300).

During the follicular phase, serum estradiol levels rise in parallel to the growth of follicle size as well as to the increasing number of granulosa cells. FSH receptors exist exclusively on the granulosa cell membranes. Increasing FSH levels during the late luteal phase leads to an increase in the number of FSH receptors and ultimately to an increase in estradiol secretion by granulosa cells. It is important to note that the increase in FSH receptor numbers is due to an increase in the population of granulosa cells and not due to an increase in the concentration of FSH receptors per granulosa cell. Each granulosa cell has approximately 1500 FSH receptors by the secondary stage of follicular development and FSH receptor numbers remains relatively constant for the remainder of development (15). The rise in estradiol secretion appears to increase the total number of estradiol receptors on the granulosa cells (16). In the presence of estradiol, FSH stimulates the formation of LH receptors on granulosa cells allowing for the secretion of small quantities of progesterone and 17-hydroxyprogesterone (17-OHP) which may exert a positive feedback on the estrogen- primed pituitary to augment luteinizing hormone (LH) release (17). FSH also stimulates several steroidogenic enzymes including aromatase, and 3β-hydroxysteroid dehydrogenase (3β-HSD) (18, 19). In table 1, the production rates of sex steroids during the follicular phase, luteal phase, and at the time of ovulation are presented.

Table 1. Production Rate of Sex Steroids in Women at Different Stages of the Menstrual Cycle
	DAILY PRODUCTION RATE
SEX STEROIDS*	Early Follicular	Preovulatory	Mid-luteal
Progesterone (mg)	1	4	25
17α-Hydroxyprogesterone (mg)	0.5	4	4
Dehydroepiandrosterone (mg)	7	7	7
Androstenedione (mg)	2.6	4.7	3.4
Testosterone (µg)	144	171	126
Estrone (µg)	50	350	250
Estradiol (µg)	36	380	250
From Baird DT. Fraser IS. Blood production and ovarian secretion rates of esuadiol-17β and estrone in women throughout the menstrual cycle. J Clin Endocrinol Metab 38: l009-1017. 1974. @ The Endocrine Society.
*Values are expressed in milligrams or micrograms per 24 hours.

In contrast to granulosa cells, LH receptors are located on theca cells during all stages of the menstrual cycle. LH principally stimulates androstenedione production, and to a lesser degree testosterone production in theca cells. In the human, androstenedione is then transported to the granulosa cells where it is aromatized to estrone and finally converted to estradiol by 17-β-hydroxysteroid dehydrogenase type I. This is known as the two-cell, two-gonadotropin hypothesis of regulation of estrogen synthesis in the human ovary (Fig. 4).

Figure 4. Two-cell, two-gonadotropin hypothesis of regulation of estrogen synthesis in the human ovary. Adapted by Carr, BR. Diseases of the ovary and Reproductive Tract. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR, eds. Williams Textbook of Endocrinology 9th edition. WB Saunders, Philadelphia, p.751-817.

In the ovary, the primordial follicles are surrounded by a single layer of granulosa cells and are arrested in the diplotene stage of the first meiotic division. After puberty, each primordial follicle enlarges and develops into a preantral follicle. The preantral follicle is now surrounded by several layers of granulosa cells as well as by theca cells. The preantral follicle is the first stage of FSH receptivity, as now the follicle has acquired FSH receptors. The preantral follicle then develops a cavity and is now known as an antral follicle. Finally, it becomes a preovulatory follicle on its way towards ovulation. Due to the presence of 5α-reductase, preantral and early antral follicles produce more androstenedione and testosterone in relation to estrogens (20). 5α-reductase is the enzyme responsible for converting testosterone to dihydrotestosterone (DHT). Once testosterone has been 5α-reduced, DHT cannot be aromatized. However, the dominant follicle is able to secrete large quantities of estrogen, primarily estradiol, due to high levels of CYP19 (aromatase). This shift from an androgenic to an estrogenic follicular microenvironment may play an important role in selection of the dominant follicle from those follicles that will become atretic.

As mentioned earlier, development of the follicle to the preantral stage is gonadotropin independent, and any follicular growth beyond this point will require gonadotropin interaction. Gonadotropin secretion is regulated by gonadotropin releasing hormone (GnRH), steroid hormones, and various peptides released by the dominant follicle. Also, as mentioned earlier, FSH is elevated during the early follicular phase and then begins to decline until ovulation. In contrast, LH is low during the early follicular phase and begins to rise by the mid-follicular phase due to the positive feedback from the rising estrogen levels. For the positive feedback effect of LH release to occur, estradiol levels must be greater than 200 pg/mL for approximately 50 hours in duration (21). Gonadotropins are normally secreted in a pulsatile fashion from the anterior pituitary, and the frequency and amplitude of the pulses vary according to the phase of the menstrual cycle (Table 2). During the early follicular phase, LH secretion occurs at a pulse frequency of 60 to 90 minutes with relatively constant pulse amplitude. During the late follicular phase prior to ovulation, the pulse frequency increases and the amplitude may begin to increase. In most women, the LH pulse amplitude begins to increase after ovulation takes place (22).

Table 2. Mean (SEM) Luteinizing Hormone Secretory Burst Characteristics During Phases of the Menstrual Cycle*
NUMBER (24 hr)	PERODICITY (min)	AMPLITUDE^ (mlU/ml/min)**	HALF-DURATIONS (min)	LH HALF-LIFE (min)	TOTAL DAILY SECRETION (mlU/ml/24 hr)
Early follicular 175±1.4a	80 ± 3a	0.43 ± 0.02a	6.5 ± 1.0a	131 ± 13a	49 ± 6a
Late follicular 26.9±1.6b	53 ± 1b	0.70 ± 0.03b	3.5 ± 0.9b	128 ± 12a	56 ± 8a
Midluteal 10.1±1.0c	177 ± 15^#	0.26 ± 0.02c^#	11.0 ± 1.1e	103 ± 7a	52 ± 4a
	395 ± 37d^#	0.95 ± 0.05d^#
Entries in each column identified by a, b, c, d differ significantly (Duncan's multiple-range test, P <.05). Periodicity is intersecretory burst interval. LH, Luteinizing hormone. *Duration of the deconvolution-resolved LH secretory burst at half-maximal amplitude. ^#Maximal rate of LH secretion attained with the deconvolution-resolved LH secretory burst. The midluteal phase has been divided into small (less than 0.65 mIU/ml/min) and large (greater than 0.65 mIU/ml/min) secretory burst amplitudes.
Data from Sollenberger MJ, Carlsen EC, Johnson ML, et al. Specific physiological regulation of LH secretory events throughout the human menstrual cycle. New insights into the pulsatile mode of gonadotropin release. J Neuroendocrinol 2:845, 1990.

There are numerous substances found in follicular fluid, such as steroids, pituitary hormones, plasma proteins, proteoglycans and non-steroidal ovarian factors, which regulate the microenvironment of the ovary and regulate steroidogenesis in granulosa cells. Growth factors such as insulin-like growth factor 1 and 2 (IGF1, IGF2) and epidermal growth factor (EGF) are recognized as playing important roles in oocyte development and maturation (23-25). The concentration of ovarian steroids is much higher in follicular fluid in comparison to plasma concentrations. There are 2 populations of antral follicles: (1) large follicles, which are greater than 8mm in diameter, and (2) small follicles, which are less than 8mm. In the large follicles, the concentrations of FSH, estrogen, and progesterone are high while prolactin concentration is low. In the small follicles, prolactin and androgen levels are higher compared to large antral follicles (26).

OVULATION

Ovulation occurs approximately 10-12 hours after the LH peak (Fig. 5) (27). The LH surge is initiated by a dramatic rise of estradiol produced by the preovulatory follicle (Fig. 6). To produce the critical concentration of estradiol needed to initiate the positive feedback, the dominant follicle is almost always >15mm in diameter on ultrasound (28). The beginning of the LH surge occurs roughly 34 to 36 hours prior to ovulation and is a relatively precise predictor for timing ovulation (Fig. 5) (29). The LH surge stimulates luteinization of the granulosa cells and stimulates the synthesis of progesterone responsible for the midcycle FSH surge. Also, the LH surge stimulates resumption of meiosis and the completion of reduction division in the oocyte with the release of the first polar body. It has been demonstrated in cultured granulosa cells that spontaneous luteinization can occur in the absence of LH. It is hypothesized that the inhibitory effects of factors such as oocyte maturation inhibitor or luteinization inhibitor are overcome at ovulation (30).

Figure 5. The onset of LH surge usually precedes ovulation by 36 hours. The peak, on the other hand preceded ovulation by 10-12 hours.

Figure 6. Changes in gonadotropins and ovarian steroids at midcycle, just prior to ovulation. The initiation of LH surge is at time 0. Abbreviations: E2, estrogen; P, progesterone (From Hoff JD, Quigley ME, Yen SCC. Hormonal dynamics at midcycle: A re-evaluation. J Clin Endocrinol Metab. 57:792, 1983.

Prostaglandins and proteolytic enzymes, such as collagenase and plasmin, are increased in response to LH and progesterone. Although the precise mechanism is not known, proteolytic enzymes and prostaglandins are activated and digest collagen in the follicular wall, leading to an explosive release of the oocyte-cumulus complex (31). Prostaglandins may also stimulate ovum release by stimulation of smooth muscle within the ovary. The point of the dominant follicle closest to the ovarian surface where this digestion occurs is called the stigma. There is no evidence to support the theory that follicular rupture occurs as a result of increased follicular pressure, although precise measurements precisely at rupture have not been performed (32). In a recent report, laparoscopic visualization of human ovulation during an operative procedure was documented. The authors report visualizing a follicular area called the stigma which was protruding like a bleb from the surface, containing viscous yellow fluid evaginating into the peritoneal cavity (33). In humans, ovulation probably occurs randomly from either ovary during any given cycle. Of interest, some studies have suggested that ovulation occurs more commonly from the right ovary and right sided ovulation carries a higher potential for pregnancy (34). The concentrations of prostaglandins E and F series and hydroxyeicosatetraenoic acid (HETE) reach a peak level in follicular fluid just prior to ovulation (35, 36). Prostaglandins may stimulate proteolytic enzymes while HETEs may stimulate angiogenesis and hyperemia (37). Patients treated with high dose prostaglandin synthetase inhibitors such as Indocin, can have a block in prostaglandin production and effectively block follicular rupture (38-40). This gives rise to what is known as the luteinized, unruptured follicle syndrome and it presents in fertile and infertile patients equally (41). Therefore, infertility patients are advised to avoid taking prostaglandin synthetase inhibitors, as well as cyclo-oxygenase (COX) inhibitors, especially around the time of ovulation (40). A schematic diagram illustrating the proposed mechanisms involved in follicular rupture is presented in Figure 7.

Figure 7. Proposed mechanisms involved in follicular rupture. From Tsafriri A, Chun S-Y. Ovulation. In: Adashi E, Rock JA, Rosenwaks Z. Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott-Raven, 1996:236-249.

Estradiol levels fall dramatically immediately prior to the LH peak. This may be due to LH downregulation of its own receptor or because of direct inhibition of estradiol synthesis by progesterone. Progesterone is also responsible for stimulating the midcycle rise in FSH. Elevated FSH levels at this time are thought to free the oocyte from follicular attachments, stimulate plasminogen activator, and increase granulosa cell LH receptors. The mechanism causing the postovulatory fall in LH is unknown. The decline in LH may be due to the loss of the positive feedback effect of estrogen, due to the increasing inhibitory feedback effect of progesterone, or due to a depletion of LH content of the pituitary from downregulation of GnRH receptors (42).

LUTEAL PHASE

This phase is usually 14 days long in most women. After ovulation, the remaining granulosa cells that are not released with the oocyte continue to enlarge, become vacuolated in appearance, and begin to accumulate a yellow pigment called lutein. The luteinized granulosa cells combine with the newly formed theca-lutein cells and surrounding stroma in the ovary to become what is known as the corpus luteum. The corpus luteum is a transient endocrine organ that predominantly secretes progesterone, and its primary function is to prepare the estrogen primed endometrium for implantation of the fertilized ovum. The basal lamina dissolves and capillaries invade into the granulosa layer of cells in response to secretion of angiogenic factors by the granulosa and thecal cells (43). Eight or nine days after ovulation, approximately around the time of expected implantation, peak vascularization is achieved. Figure 8 demonstrates a corpus luteum as seen on transvaginal ultrasound. Note the increased blood flow seen surrounding the corpus luteum as seen with Doppler evaluation. This time also corresponds to peak serum levels of progesterone and estradiol. The central cavity of the corpus luteum may also accumulate with blood and become a hemorrhagic corpus luteum. The life span of the corpus luteum depends upon continued LH support. Corpus luteum function declines by the end of the luteal phase unless human chorionic gonadotropin is produced by a pregnancy. If pregnancy does not occur, the corpus luteum undergoes luteolysis under the influence of estradiol and prostaglandins and forms a scar tissue called the corpus albicans.

Figure 8. Corpus luteum as seen on transvaginal ultrasound. On the right image, note the Doppler flow indicating vascular flow surrounding the structure.

Estrogen levels rise and fall twice during the menstrual cycle. Estrogen levels rise during the mid-follicular phase and then drop precipitously after ovulation. This is followed by a secondary rise in estrogen levels during the mid-luteal phase with a decrease at the end of the menstrual cycle. The secondary rise in estradiol parallels the rise of serum progesterone and 17α-hydroxyprogesterone levels. Ovarian vein studies confirm that the corpus luteum is the site of steroid production during the luteal phase (44).

The mechanism by which the corpus luteum regulates steroid secretion is not completely understood. Regulation may be determined in part by LH secretory pattern and LH receptors or variations in the levels of the enzymes regulating steroid hormone production, such as 3β-HSD, CYP17, CYP19, or side chain cleavage enzyme. The number of granulosa cells formed during the follicular phase and the amount of readily available LDL cholesterol may also play a role in steroid regulation by the corpus luteum. The luteal cell population consists of at least two cell types, the large and the small cells (45). Small cells are thought to have been derived from thecal cells while the large cells from granulosa cells. The large cells are more active in steroidogenesis and are influenced by various autocrine/paracrine factors such as inhibin, relaxin, and oxytocin (46, 47).

In studies looking into the mechanisms regulating the menstrual cycle, LH was established as the primary luteotropic agent in a cohort of hypophysectomized women (48). After induction of ovulation, the amount of progesterone secreted and the length of the luteal phase is dependent on repeated LH injections. Administration of LH or HCG during the luteal phase can extend corpus luteum function for an additional two weeks (49).

The secretion of progesterone and estradiol during the luteal phase is episodic, and correlates closely with pulses of LH secretion (Fig. 9) (50). The frequency and amplitude of LH secretion during the follicular phase regulates subsequent luteal phase function and is consistent with the regulatory role of LH during the luteal phase (51). Reduced levels of FSH during the follicular phase can lead to a shortened luteal phase and the development of a smaller corpus lutea (52). Also, the life span of the corpus luteum can be reduced by continuous LH administration during the follicular or luteal phase, reduced LH concentration, decreased LH pulse frequency, or decreased LH pulse amplitude (53-55). The role of other luteotropic factors such as prolactin, oxytocin, inhibin and relaxin is still unclear (56, 57).

Figure 9. Episodic secretion of LH (top) and progesterone (bottom) during the luteal phase of a woman. Abbreviations: LH, luteinizing hormone: P, progesterone E2, estradiol; LH + 8, LH surge plus 8 days. (From Filicori M, Butler JP, Crowley WF Jr. Neuroendocrine regulation of the corpus luteum in the human. J Clin Invest. 73:1638 1984.

The corpus luteum function begins to decline 9-11 days after ovulation. The exact mechanism of how the corpus luteum undergoes its demise is unknown. Estrogen is believed to play a role in the luteolysis of the corpus luteum (58). Estradiol injected into the ovary bearing the corpus luteum induces luteolysis while no effect is noted after estradiol injection of the contralateral ovary (56). However, the absence of estrogen receptors in human luteal cells does not support the role of endogenous estrogen in corpus luteum regression (59). Prostaglandin F2α appears to be luteolytic in nonhuman primates and in studies of women (60, 61). Prostaglandin F2α exerts its effects via the synthesis of endothelin-1, which inhibits steroidogenesis and stimulates the release of a growth factor, tumor necrosis factor alpha (TNFα), which induces cell apoptosis (62). Oxytocin and vasopressin exert their luteotropic effects via an autocrine/paracrine mechanism (63). Luteinizing hormone's ability to downregulate its own receptor may also play a role in termination of the luteal phase. Finally, Matrix metalloproteinases also appear to play a role in luteolysis (64).

Not all hormones undergo marked fluctuations during the normal menstrual cycle. Androgens, glucocorticoids, and pituitary hormones, excluding LH and FSH, undergo only minimal fluctuation (65-68). Due to extra-adrenal 21-hyroxylation of progesterone, plasma levels of deoxycorticosterone are increased during the luteal phase (69, 70).

HORMONAL EFFECTS ON THE REPRODUCTIVE TRACT

Endometrium

The effects of varying concentrations of estrogen and progesterone throughout the course of the menstrual cycle have characteristic effects on the endometrium (Fig. 10) (71). The endometrial changes that occur can be visualized with sonography (Fig. 11). The characteristic endometrial changes also allow for histologic dating. Histologic dating is most accurately accomplished by performing an endometrial biopsy 2-3 days prior to expected menstruation. The proliferative phase is more difficult to date accurately in comparison to the luteal phase. The glands during the proliferative phase are narrow, tubular, and some mitosis and pseudostratification is present. The endometrium thickness is usually between 0.5 and 5mm. In a classical 28-day menstrual cycle, ovulation occurs on day 14. On cycle day 16, the glands take on a more pseudostratified appearance with glycogen accumulating at the basal portion of the glandular epithelium and some nuclei are displaced to the midportion of the cells. In a formalin fixed specimen, glycogen is solubilized resulting in the characteristic basal vacuolization at the base of the endometrial cells. This finding confirms the formation of a functional, progesterone producing, corpus luteum. In the luteal phase, progesterone decreases the biologic activity of estradiol on the endometrium by: (1) decreasing the concentration of estradiol receptors, (2) increasing the enzymatic activity of 17β-hydroxysteroid dehydrogenase type II, the enzyme responsible for the conversion of estradiol to estrone, and (3) by increasing the activity of estrone sulfotransferase (72, 73).

Figure 10. Dating of the Endometrium. From Noyes RW, Hertig AW, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3.

Figure 11. Characteristic sonographic endometrial changes seen throughout the menstrual cycle.

On cycle day 17, the endometrial glands become more tortuous and dilated. On cycle day 18, the vacuoles in the epithelium decrease in size and are frequently located next to the nuclei. Also, glycogen is now found at the apex of the endometrial cells. By cycle day 19, the pseudostratification and vacuolation almost completely disappear and intraluminal secretions become present. On cycle day 21 or 22, the endometrial stroma begins to become edematous. On cycle day 23, stromal cells surrounding the spiral arterioles begin to enlarge and stromal mitoses become apparent. On cycle day 24, predecidual cells appear around the spiral arterioles and stromal mitoses become more apparent. On cycle day 25, the predecidua begins to differentiate under the surface epithelium. On cycle day 27, there is a marked lymphocytic infiltration and the upper endometrial stroma appears as a solid sheet of well-developed decidua-like cells. On cycle day 28, menstruation begins.

In 2004, Chan et al., were the first to confirm that stem cells were present in human endometrium (73A). Subsequent research has involved characterization of the different types of endometrial stem cells (73B). Importantly, menstrual fluid may be an easily accessible source for certain types of endometrial stem cells (73C). This may lead to advancements in the treatment of many gynecologic disorders including endometriosis and Asherman syndrome as well as non-gynecologic disorders such as neurologic and cardiac disorders (73B).

Cervix

The mucous secreting glands of the endocervix are affected by the changes in steroid hormone concentration. Immediately after menstruation, the cervical mucous is scant and viscous. During the late follicular phase, under the influence of rising estradiol levels, the cervical mucous becomes clear, copious and elastic. The quantity of cervical mucous increases 30 fold compared to the early follicular phase (74). The stretchability or elasticity of the cervical mucous can be evaluated between two glass slides and recorded as the spinnbarkeit. If examined under the microscope, the cervical mucous will display a characteristic ferning or palm-leaf arborization appearance. After ovulation, as progesterone levels rise, the cervical mucous once again becomes thick, viscous and opaque and the quantity produced by the endocervical cells decreases.

Vagina

The changes in hormonal levels of estrogen and progesterone also have characteristic effects on the vaginal epithelium. During the early follicular phase, exfoliated vaginal epithelial cells have vesicular nuclei and are basophilic. During the late follicular phase, and the influence of the rising estradiol level, the vaginal epithelial cells display pyknotic nuclei and are acidophilic (75). As progesterone rises during the luteal phase, the acidophilic cells decrease in number and are replaced by an increasing number of leukocytes.

MENSTRUATION

In the absence of a pregnancy, steroid hormone levels begin to fall due to declining corpus luteum function. Progesterone withdrawal results in increased coiling and constriction of the spiral arterioles. This eventually results in tissue ischemia due to decreased blood flow to the superficial endometrial layers, the spongiosa and compacta. The endometrium releases prostaglandins that cause contractions of the uterine smooth muscle and sloughing of the degraded endometrial tissue. The release of prostaglandins may be due to decreased stability of lysosomal membranes in the endometrial cells (76). Infusions of prostaglandin F2α in women during the luteal phase has been shown to induce endometrial necrosis and bleeding (77). The use of prostaglandin synthetase inhibitors decreases the amount of menstrual bleeding and can be used as therapy in women with excessive menstrual bleeding or menorrhagia. Menstrual fluid is composed of desquamated endometrial tissue, red blood cells, inflammatory exudates, and proteolytic enzymes. Within two days after the start of menstruation and while endometrial shedding is still occurring, estrogen produced by the growing follicles starts to stimulate the regeneration of the surface endometrial epithelium. The estrogen secreted by the growing ovarian follicles, causes prolonged vasoconstriction enabling the formation of a clot over the denuded endometrial vessels (78). Also, the regeneration and remodeling of the uterine connective tissue is regulated in part by the matrix metalloproteinase (MMP) system (79).

The average duration of menstrual flow is between four to six days, but the normal range in women can be from as little as two days up to eight days. As mentioned earlier, the average amount of menstrual blood loss is 30 mL and greater than 80 mL is considered abnormal [4].

MENSTRUAL DISORDERS

Apart from conditions of abnormal menstruation, certain disorders are increased in women when compared to men. These conditions are thought to be related to hormone differences as well as hormone changes throughout the menstrual cycle. Increased autoimmune conditions, such as rheumatoid arthritis or systemic lupus erythematosus, are believed to be related to estrogen enhancement of humoral immunity (80). Other researchers also describe higher vulnerability for drug abuse during phases of the menstrual cycle when estradiol levels are high (81).

SUMMARY

The length of a menstrual cycle is the number of days between the first day of menstrual bleeding of one cycle to the onset of menses of the next cycle. The median duration of a menstrual cycle is 28 days with most cycle lengths between 25 to 30 days. The menstrual cycle may be divided into two phases: (1) follicular or proliferative phase, and (2) the luteal or secretory phase. The follicular phase begins from the first day of menses until ovulation. The development of ovarian follicles characterizes this phase. The LH surge is initiated by a dramatic rise of estradiol produced by the preovulatory follicle and results in subsequent ovulation. The LH surge stimulates luteinization of the granulosa cells and stimulates the synthesis of progesterone responsible for the midcycle FSH surge. Also, the LH surge stimulates resumption of meiosis and the completion of reduction division in the oocyte with the release of the first polar body. The luteal phase is 14 days long in most women. If the corpus luteum is not rescued by pregnancy, it will undergo atresia. The resultant progesterone withdrawal results in menses. The average duration of menstrual flow is between four and six days, but the normal range in women can be from as little as two days up to eight days. The average amount of menstrual blood is 30ml, and over 60 ml is considered abnormal.

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Thyroxine Poisoning

CLINICAL RECOGNITION

A massive L-Thyroxine (T4) overdose may be accidentally and unintentionally ingested, most commonly by children and adolescents. It may occur intentionally in young and older adults in an attempt to lose weight, with suicidal intentions, or for undeclared purposes. In some localities thyroxine may be obtained at drugstores without prescription (mostly in the generic form). In some reports thyroxine preparations by a pharmacist had an erroneous LT4 dosage. Thyroid hormone pills used to treat hypothyroid dogs typically contains a much higher dose of thyroid hormone and if mistakenly taken by humans can lead to thyroxine poisoning.

Ingested thyroxine, which is itself probably of modest physiologic significance, is rapidly partially converted to triiodothyronine (T3), the active form of thyroid hormone. Both thyroxine and triiodothyronine levels in serum rise within 1-2 hours of ingestion. Agents that inhibit T4>T3 conversion provide one approach to treatment. In children and adolescents, the clinical course is often very mild. Patients in this age range may ingest a full flask of LT4 with 90-100 tablets (100 or 150 mcg/tablet). Rarely, the overdose is discovered immediately and the patient is brought to the hospital 6-12 hours after the ingestion. At this time, the common clinical signs and symptoms include nervousness, insomnia, mild tremor of hands, tachycardia, mild elevation of body temperature, blood pressure elevation, and loose stools. Rarely more serious late effects occur, including coma, convulsions, and acute psychosis. Cardiac effects aside from tachycardia are seldom seen in young adults but may occur in middle age and older adults, with reported arrhythmias and acute myocardial infarction. However, only one fatality has been reported. Interestingly the onset of symptoms and signs (Table 1) may be delayed for up to 3 to 10 days and does not correlate closely with plasma levels of serum total T4 and total T3. Medical consensus has indicated that serious symptoms are less frequent in children even though children usually have higher mean plasma levels of T4 and T3 than adults for the same overdose of LT4 ingested. One-time ingestion of up to 3 mg thyroxine rarely causes symptoms in adult or children. As already mentioned serious complications are not common, but they can appear days after ingestion, and therefore the patients should be closely monitored.

TABLE 1: SYMPTOMS AND SIGNS AFTER INGESTION OF LT4

Severe toxicity is quite rare in children.

Common-effects:

Nervousness

Insomnia

Mild elevation of temperature

Blood pressure elevation

Loose stools

Rare symptoms:

Comma

Convulsions

Acute psychosis

Thyroid storm

Tachycardia, arrhythmias

DIAGNOSIS and DIFFERENTIAL

Elevated levels of total and free T4 and T3 have been described with suppressed serum TSH levels and otherwise typically a normal biochemical profile (Table 2). The half-life of serum T4 may be shortened. In one study the half-life of LT4 was 5.7 days which is slightly shorter than the usual half-life of L-thyroxine. In one report total serum T3 levels reached the normal range five days after ingestion of 9.9 mg of LT4 (99 tablets of 100 mcg), although free T4 levels were still elevated. In many cases, there is a progressive rise in both serum total T4 and total T3 levels in the first 24 hours following the overdose, caused by continued absorption of the ingested LT4.

Table 2: Biochemical Changes After Ingestion of LT4

Elevated serum total T4 and T3

Suppressed serum TSH

Elevated Free T4 and Free T3

Normal biochemical profile

THERAPY

Therapeutic recommendations are made based only in the review of the available literature concerning a relatively large number of patients, most of them children. Acute levothyroxine overdose is much more common in children compared to adolescents and adults. Therapeutic options are related to the time elapsed after the ingestion of a large number of tablets of L-thyroxine and the actual beginning of emergency therapy (Table 3). Acute massive doses of L-thyroxine typically have a mild clinical course that can be controlled by activated charcoal, or possibly cholestyramine, propranolol, dexamethasone, and supporting measures, with close medical evaluation. Rarely critical cardiac conditions, coma, seizures will follow massive doses of L-Thyroxine.

If more than a few hours of ingestion of LT-4 tablets have elapsed, most probably the tablets have travelled from the gastric cavity to duodenum. Moreover, gastric lavage is difficult to conduct in small children. One way to confirm the presence of LT-4 tablets in the gastric cavity is endoscopy, easily conducted in many hospitals and emergency rooms. LT-4 tablets are dissolved by the gastric juice, but there are no data about the rate of dissolution of a large number of tablets of LT-4. Most probably LT-4 would not be entirely dissolved by the gastric juice and may not be absorbed in the duodenum (normally about 10-15%) but would be absorbed in the jejuno-ileum (normally about 53% of absorption of LT4).

Emetics both local (Ipecac) or central agents (apomorphine) should be avoided.

Administration of activated charcoal is a common practice in many drug overdoses and is an agent that can prevent absorption of several drugs from the gastro-intestinal system. However, in many reports repeated doses of activated charcoal were ineffective in accelerating the elimination of levothyroxine, probably due to high uptake in the duodenum and jejuno-ileum.

Hemoperfusion using activated charcoal is a rather complicated procedure but has been reported to be highly effective in decreasing total serum levels. It should be reserved for adult patients with severe intoxication by very large doses of thyroxine and the same applies to plasmapheresis which has been seldom used.

Cholestyramine, an ion-exchange resin (Questran ®), can be administered in the usual dose of 4 grams every 8 hours orally. This drug binds thyroxine and enhances its elimination.

Glucocorticoids (Dexamethasone 4 mg orally) decrease the conversion of LT4 to T3, the active hormone. Sodium Ipodate (oral cholecystographic agent) has also been used for blocking the conversion of LT4 to T3, but it is no longer generally available.

Beta-blockers such as propranolol, are useful to ameliorate the metabolic effects of thyroid hormone, mostly on the cardiac system (controlling tachycardia, preventing arrhythmias). Seizures may be treated with phenytoin and phenobarbital. Propylthiouracil (PTU) might be used for blocking the conversion of T4 to T3 but may have very limited usefulness in the presence of a large load of LT4.

Hemodialysis has been used in severe cases, but it is probably of limited value since both T3 and T4 are highly protein-bound.

TABLE 3: TREATMENT OF INGESTION OF A MASSIVE DOSE OF L-THYROXINE

Gastric lavage (within hours of ingestion).

Emetic agents (not advised)

Propranolol (10-40 mg 3 times daily)

Activated Charcoal (1g/kg p.o.)

Dexamethasone (4 mg p.o. daily)

Sodium ipodate, if available

Cholestyramine (4g every 8h p.o.)

Propylthiouracil (PTU) (May inhibit conversion of T4>T3)

Activated charcoal hemoperfusion

Plasmapheresis (seldom necessary)

Hemodialysis (probably of limited value)

Thyroid storm: demands treatment in an Intensive Care Unit.

FOLLOW-UP

Patients should be monitored for several days to be sure that serum T4 and T3 levels are falling

GUIDELINES

None applicable.

REFERENCES

Shilo L, Kovatz S, Hadari R, Weiss E, Nabriski D, Shenkman L. Massive thyroid hormone overdose: kinetics, clinical manifestations and management, Israel med Ass J 2002; 4:209-299. http://www.ncbi.nlm.nih.gov/pubmed/12001709

Kreisner E, Lutzky M, Gross JL. Charcoal hemoperfusion in the treatment of levothyroxine intoxication. Thyroid 2010, 20:209-212. http://www.ncbi.nlm.nih.gov/pubmed/20151829 De Luis

DA, Duenas A, Abad L, Aller R. Light symptoms following a high dose intentional L-Thyroxine ingestion treated with cholestyramine. Horm Res 2002; 67:61-62. http://www.ncbi.nlm.nih.gov/pubmed/12006723

De Groot LJ, Bartalena L. Thyroid Storm. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-.

2015 Apr 12. PMID: 25905165

Cellular Action of Thyroid Hormone

ABSTRACT

Thyroid hormones (THs) regulate growth, development, metabolism. This chapter aims to provide a comprehensive overview of the molecular and cellular mechanism(s) for intracellular signaling by TH. At the cellular level, THs bind to thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor family. TRs act as ligand-activated transcription factors that bind to their cognate thyroid hormone response elements (TREs) in the promoters of target genes. TRs regulate gene transcription by employing TR-interacting protein complexes containing coactivators (CoAs) or corepressors (CoRs). Coactivator and corepressor complexes include histone modifying enzymes known as histone acetyltransferases (HATs) or histone deacetylases (HDACs), respectively, that induce epigenetic changes in the chromatin structure of target gene promoters to enhance or repress the transcriptional efficiency of RNA polymerase on TH-responsive genes. TRs also can mediate transcriptional effects indirectly by binding to other transcription factors or activating cell signaling cascades. Additionally, emerging evidence suggests that THs may bind to cell membrane proteins other than TRs to activate cell signaling pathways. The important role of TH on metabolism, has spawned interest the pharmacological use of TH and its analogs/metabolites for the treatment of metabolic diseases such as hypercholestronemia, hypertriglycerimia obesity, and non-alcoholic fatty liver disease (NAFLD).

INTRODUCTION

The thyroid hormones (THs, thyroxine (T₄) and triiodothyronine (T₃)) have important effects on development, growth, and metabolism (1-3). Some of the most prominent effects of TH occur during fetal development and early childhood. In humans, the early developmental role of TH is illustrated by the distinctive clinical features of cretinism observed in iodine-deficient areas. In childhood, lack of TH can cause delayed growth. However, in this latter case, many of the effects of TH may be metabolic rather than developmental, as growth is restored rapidly after the institution of TH treatment. In adults, the primary effects of THs are manifested by alterations in metabolism. These effects include changes in oxygen consumption, protein, carbohydrate, lipid, and vitamin metabolism. The clinical features of hypothyroidism and hyperthyroidism emphasize the pleiotropic effects of these hormones on many different pathways and target organs.

At the clinical level, identification of quantitative markers of TH action has been difficult (4). At the extreme ends of the clinical spectrum, which extends from hypothyroidism to hyperthyroidism, the diagnosis of a thyroid abnormality is usually apparent. Clinical suspicion of a thyroid abnormality can be confirmed using laboratory tests for THs and thyroid stimulating hormone (TSH). However, more subtle forms of thyroid dysfunction, such as subclinical hypothyroidism or hyperthyroidism, pose a greater challenge. Although the level of circulating TSH provides a sensitive and quantitative indicator of TH action at the level of the hypothalamic-pituitary axis, there are few reliable peripheral or intracellular markers of TH action (5,6). The effect of TH on basal metabolism has been re-evaluated using measurements of resting energy expenditure (REE). In hypothyroid patients taking varying levels of TH replacement, there is a strong inverse correlation between REE and the TSH level (6). Nevertheless, TSH remains the most sensitive and useful clinical indicator of TH action. As discussed below, tissue-selective metabolism of THs, and variable tissue sensitivity to their effects, underscores the need to develop additional markers of TH activity in peripheral tissues.

Since the initial description of TH effects on metabolic rate more than 100 years ago (7), many theories have been proposed to explain its mechanism of hormone action. The proposed models include: uncoupling oxidative phosphorylation, stimulation of energy expenditure by the activation of Na+-K+ ATPase activity, and direct modulation of TH transporters and enzymes in the plasma membrane and mitochondria (8). Recently, there has been increasing evidence for non-genomic actions (see later under non-genomic actions of TH) (8); however, the major effects of TH occur via nuclear receptors that mediate changes in gene expression.

In 1966, Tata proposed that TH increased gene expression with attendant increases in protein synthesis and enzyme activity (9). In 1972, high affinity nuclear binding sites for TH were documented (Kd approximately 10-10 M for T₃) (10,11). The receptor-binding affinity of various THs and analogues correlated with their biologic potencies, consistent with the view that most biologic effects are mediated via the nuclear receptor (11– 14). Over the past 25 years, there has been a dramatic surge of new information on TH action resulting from the cloning of the TH receptors (15,16), the identification of regulatory DNA elements in TH responsive genes (1-3), the generation of TR isoform knockout mice (17,18), and the discovery and phenotype characterization of patients with mutations in TRα and TRβ (5,19,20). In this chapter, we will focus on our current understanding of nuclear TH receptor action.

BINDING OF THs TO NUCLEAR RECEPTORS

In many respects, T₄can be regarded as a prohormone for the more potent hormone, T₃. Most of the TH bound to receptors is in the form of T₃, either secreted into the circulation by the thyroid gland or derived from T₄to T₃conversion by 5' monodeiodinases (see Chapter 3C). There are three distinct deiodinases- type I, type II, and type III (21,22). The distribution and regulation of these enzymes can have important effects on TH action. For example, Type II deiodinase has high affinity for T₄(Kd in the nanomolar range) and is found primarily in the pituitary gland, brain, and brown fat where conversion of T₄to T₃modulates the intracellular concentration of T₃. Thus, tissues that contain type II deiodinase can respond differently to a given circulating concentration of T₄(by intracellular conversion to T₃) than organs that only can respond to T₃(23,24). Additionally, it appears that both type 1 and type II deiodinase regulate the circulating T₄and T₃levels (25). Recently, MCT8, OATP-1, and System L amino acid transporters have been identified as TH transporters which regulate T₄and T₃uptake into cells (26,27). Mutations in the former have been involved in a number of syndromes of x-linked mental retardation and neurologic deterioration (27,28).

T₃binds to its receptors with approximately 10 fold higher affinity than T₄. The dissociation constants for liver nuclear receptors measured in vitro are 2 x 10^-9M for T₄and 2 x 10^-10M for T₃(1,2). Nuclear receptors are approximately 75% saturated with TH in brain and pituitary and 50% saturated with TH in liver and kidney. It is notable that the extent of TH receptor occupancy varies in different tissues, providing a mechanism for alterations in circulating TH levels to alter receptor activity. In contrast to the related steroid hormone receptors, TRs are mostly nuclear both in the absence and presence of TH (1,2,30). In fact, TH receptors are tightly associated with chromatin (1-3,30), consistent with their proposed role as DNA-binding proteins that regulate gene expression.

CLONING, STRUCTURE, AND EXPRESSION OF TH RECEPTORS

Cloning of TRs

TH receptors (TRs) were first cloned in 1986 and belong to the nuclear hormone receptor superfamily that includes the glucocorticoid, estrogen, progesterone, androgen, aldosterone, vitamin D, retinoic acid (RARs), retinoid X (RXRs) and "orphan" (unknown ligand and/or DNA target) receptors (1,2,15,16,30-33). TRs are the cellular homologs of v-erbA, a viral oncogene product involved in chick erythroblastosis. TRs are encoded at two genomic loci (α and β) located on human chromosomes 17 and 3, respectively and their gene products result in two major isoforms, TRα and TRβ .

Structure of TRs

Like other members of the nuclear receptor superfamily, the TRs have a central DNA-binding domain (DBD) and a carboxy-terminal ligand-binding domain (LBD) (Figure 3d-1). The two major TR isoforms have high amino acid sequence homology in their respective DBDs and LBDs. Dimerization domains also are found in both DBDs and LBDs. The amino-terminal regions are more variable between TRα and TRβ (1-3), and contain ligand-independent activation domains. In contrast, multiple sub-regions are located in the LBD for ligand-dependent transcriptional activation and basal repression of target genes.

Figure 1. Functional domains of the TH receptor (TR). The TH receptor (TR) is depicted schematically. The zinc finger DNA-binding domain (DBD) is denoted along with the carboxy terminal ligand-binding domain (LBD). Other functional domains and interaction sites are indicated.

The DNA-binding domains of the nuclear receptors are comprised of two distinct zinc fingers that are separated by a 15-17 amino acid linker sequence. The crystal structure of the DNA-binding domains of the TH receptor and its heterodimeric partner, RXR has been determined. The two heterodimer partners interact with a direct repeat of the receptor binding site in a head-to-tail manner (34-37).

A small stretch of amino acids at the base of the first finger (referred to as the P-box) dictates the DNA sequence specificity of the receptor (35-39). The P-box sequence of the TH receptor is shared by other receptors that bind to similar or identical DNA recognition sites (AGGTCA). The underlined amino acids in the P-box (EGCKG) of the TH receptor are also found in the retinoic acid receptors, the retinoic acid X receptors, the rev-erbA protein, the vitamin D receptor, and NGFI-B. Of note, the steroid hormone receptors have a different P box sequence and bind as homodimers to a different consensus DNA half-site sequence (AGAACA). The region between the DBD and LBD is called the hinge region and contains the nuclear localization signal, typically a basic amino acid‑rich sequence, first described in viral nuclear proteins.

X‑ray crystallographic studies of the liganded rat TRα-1 show that TH is embedded in a hydrophobic "pocket" lined by discontinuous stretches of amino acid sequences within the LBD. Additionally, there are several hydrophobic interfaces within the LBD that contribute to the TR homo- and heterodimerization with RXR (39). There are twelve amphipathic helices in the LBD and specific helices among them provide the critical contact surfaces for protein-protein interactions with co-activators and co-repressors (helices 3,5,6,12 and 3,4,5,6, respectively) (40-43). Ligand-binding to TR causes a major conformational change in the LBD, particularly in helix 12. This, in turn, facilitates TR discrimination between co-activators and co-repressors (see below).

Splicing variants of TRs

The carboxy-terminal hormone-binding domain of the TRα gene is alternatively-spliced to generate several protein products (Figure 3d-2, below). One variant, referred to as α-2, is identical to TRα-1 through the first 370 amino acids, but then its sequence diverges completely, owing to splicing of alternate exons (44-47). Another splicing variant, referred to as TRvII or α-3, is similar to α-2 except that it lacks the first 39 amino acids found in the unique region of α-2 (45). α -2 cannot bind TH because of the replacement of critical amino acids at the extreme carboxy-terminal end of the protein due to alternative splicing (48), and thus cannot mediate ligand-dependent gene transcription (49– 51). The amino acid replacements in α-2 also alter its dimerization properties and reduce DNA-binding affinity (52-55). The α-2 splicing variant is highly expressed in many tissues such as brain, testis, kidney, and brown fat, but its function remains poorly understood (56). The α-2 isoform has been proposed to be an endogenous inhibitor of TH receptor function as it inhibits TRα and β activity in transient gene expression assays (44,54). The mechanism by which α-2 antagonizes TR action is controversial. Some studies indicate that α-2 competes for active receptor complexes at DNA target sites (57,58). Other studies indicate that α-2 inhibits TR activity independent of DNA-binding (59). It is likely that the inhibitory effects of α-2 involve more than one mechanism. Amino acid substitutions in the carboxy-terminal region of α-2 also prevent its interactions with transcriptional corepressors (see below) (55), and may provide an explanation as to why α-2 is not a more potent inhibitor of TR activity. Additionally, the phosphorylation state of α-2 may modulate its inhibitory activity (60). Given the foregoing features, the TRα-1 and α-2 system represents one of the few examples in mammals whereby multiple mRNAs generated by alternative splicing encode proteins that are antagonistic to each other.

Figure 2. TH receptor isoforms. The TH receptors (TR) β and α are expressed from separate genes. Each TR gene can be expressed as distinct isoforms, reflecting the use of alternate promoters and exons. The central zinc finger DNA-binding region is indicated and unique domains are shown by distinct patterns of shading. The TRβ-2 isoform, which is expressed predominantly in the pituitary and hypothalamus, contains a unique amino-terminus. The TRα-2 isoform contains unique carboxy-terminal sequences that eliminate hormone binding. The DNA- and T3-binding properties and transcriptional activity of the various isoforms are shown at the right.

A receptor-like molecule, Rev-erbA, is, surprisingly, encoded on the opposite strand of the TRα gene locus (61, 62). Rev-erbA mRNA contains a 269-nucleotide stretch which is complementary to the α-2 mRNA due to its transcription from the DNA strand opposite of that used to generate TRα-1 and α-2. This protein also is a member of the nuclear hormone receptor superfamily, and is highly expressed in adipocytes and muscle cells. Rev-erbA, contains a DBD that is homologous to the TR DBD. However, Rev-erbA does not bind TH and its putative LBD has minimal homology with other nuclear hormone receptors. Since no cognate ligand has been identified for Rev-erbA, it is categorized as an "orphan nuclear receptor. " It can act as a transcriptional repressor for nuclear hormone receptors and other transcription factors (63-65). Since Rev-erbA shares an exonic segment of the bidirectionally transcribed TRα gene, it is possible that it modulates the expression or splicing of TRα-1 and α-2 (66, 67) as parallel increases in rev-erbA mRNA and TRα-1 mRNA expression occur relative to α2 mRNA expression.

The major variant of the TRβ gene, TRβ-2, has a different amino-terminus than TRβ 1 (Figure 3d-2, above) (68). The distinct amino-terminal region of the TRβ-2 is due to transcription from a tissue-specific promoter. The function of the amino-terminus of the TH receptor is not known, but it likely plays a role in transcriptional control (69,70). The TRβ-1 and TRβ-2 isoforms function similarly in most transient gene expression assays (69, 71), although differences in the transcriptional activities of the TRβ 1 and TRβ 2 isoforms have been noted with respect to certain target genes (69-72). It is likely that tissue-restricted expression of the TRβ-2 isoform contributes to unique patterns of TR expression, which in turn, may modulate target gene regulation.

Recently, short isoforms of TRα and TRβ have been described (73, 74). The novel TRα isoforms arise from translational start sites in the 7th intron and yield shortened TRα 1 and α 2 isoforms that have dominant negative activity on WT TR. Novel short TRβ isoforms arise from alternative splicing of TRβ. It is possible that these isoforms may modulate T₃-responsiveness in a tissue- and/or developmental stage-specific manner.

Tissue- and development-specific expression of TRs

Most studies of TR isoform expression have employed mRNA analyses rather than protein measurement (30). In general, the α and β receptor isoforms are distributed widely and exhibit overlapping patterns of expression (30,75). TRα 1 mRNA is expressed in skeletal and cardiac muscle whereas TRβ-1 mRNA is predominant in liver, kidney, and brain. Α-2 mRNA is most prevalent in brain and testis. In contrast, TRβ-2 mRNA has the most tissue-restricted expression, and is present in the anterior pituitary gland, hypothalamus, and cochlea (75-79).

The TRs also are expressed in specific stages during development, and are subject to regulation by hormones and other factors (78, 79). For instance, TRα-1 mRNA is expressed early whereas TRβ 1 mRNA is expressed later during embryonic brain development. In the rat pituitary gland, TH decreases TRβ 2, TRα-1, and α-2 mRNAs while slightly increasing TRβ-1 mRNA. However, in most other tissues, TH decreases TRα-1 and α-2, but not TRβ-1 mRNA. Isoform-specific knockout mice of each of the TR isoforms display distinct phenotypes (17,18). However, lack of significant TR isoform-specific gene expression was observed in cDNA microarrays of hepatic genes in TR isoform knockout mice (80). Given the apparent redundancy in TR isoform function, it is possible the different KO phenotypes may be due to absolute TR expression levels in critical tissues and developmental stages.

TRANSCRIPTIONAL REGULATION BY TRS

TH receptors bind to TH response elements (TREs) in specific target genes (Figure 3d-3, below). After binding TH, the receptor induces changes in gene expression by either increasing or decreasing the transcriptional activity of target genes. Examples of the target genes that are positively- and negatively-regulated by TH are summarized in Table 1. cDNA microarrays have been employed to study TH regulation of hepatic genes in mice, and led to the identification of a large number of novel target genes (both positively- and negatively-regulated) (81,82). These studies demonstrated that TH affected gene expression in a wide range of cellular pathways and functions, including gluconeogenesis, lipogenesis, insulin signaling, adenylate cyclase signaling, cell proliferation, and apoptosis. Although many of the TH-responsive genes were regulated directly by TRs, others were probably regulated indirectly through intermediate genes. Indirect regulation of TH-mediated transcription is suggested when the time course for induction is slow (hours) and when it is blocked by protein synthesis inhibitors. Although TH acts mainly at the level of transcription, it also can affect mRNA stability, translational efficiency, and miRNA regulation (83,84). Thus, TH acts at multiple levels to alter protein expression.

Figure 3. Mechanism of TH action via its nuclear receptor. TH is transported across plasma membrane and likely diffuses through nuclear membrane to bind to its receptor. The TH receptor (open circle) is localized almost exclusively in the nucleus where it associated with DNA as a homodimer or as a heterodimer with RXR (stippled box). The hormone-activated receptor binds to TH response elements (TREs) to alter rates of gene transcription and consequently levels of mRNA.

Table 1. Examples of Genes Positively-regulated by T3.

     1. Fatty acid synthetase
     2. Growth hormone
     3. Lysozyme silencer
     4. Malic enzyme
     5. Moloney leukemia virus enhancer
     6. Myelin basic protein
     7. Myosin heavy chain α
     8.Phosphoenolpyruvate carboxykinase
     9. RC3
   10. Spot 14 lipogenic enzyme
   11. Type I 5'-deiodinase
   12. Uncoupling protein

Table 2. Examples of Genes Negatively-regulated by T₃.

     1. Epidermal growth factor receptor
     2. Myosin heavy chain β
     3. Prolactin
     4. Thyroid-stimulating hormone α
     5. Thyroid-stimulating hormone β
     6.Thyrotropin-releasing hormone
     7. Type II 5’-deiodinase

TR binding to TH response elements (TREs)

Detailed analyses of thyroid response elements (TREs) have led to the identification of a canonical TRE half-site sequence (1, 2,85) (Figure 3d-4). The TRE half-site is generally considered to be a hexamer (AGGTCA), but TR binding is optimal with a more extended binding site (37,88,89). Specifically, the sequence TAAGGTCA is optimal for TR binding and T₃-responsiveness. However, inspection of TREs from many different target genes reveals there is a relatively low degree of sequence conservation among these elements. This finding suggests the possibility that naturally-occurring TREs may have diverged from an ideal consensus element during evolution as a means to modulate the degree of TH responsiveness.

TR interactions with DNA are quite different from those observed with steroid receptors, which bind to palindromic DNA sequences as homodimers. Although TR also can bind to certain TREs in vitroas a homodimer, it binds preferentially to most TREs as a heterodimer with the retinoid X receptors (RXRs) (1-3,30). The TR-RXR heterodimer binds to half-sites that are arranged in several different configurations. These include palindromic arrangements (head-to-head), direct repeats (head-to-tail), and inverted repeats (tail-to-tail). Most naturally occurring TREs are direct repeats (Figure 3d-4), typically separated by four nucleotides. The ability of TR dimers to bind to TRE’s in different configurations suggests a flexible protein structure, or the possibility that distinct protein surfaces are involved in the formation of dimers (34,39,90). Taken together, the specificity and affinity for the TR-RXR heterodimer is primarily determined by sequences within the half-site, the length of the spacer region between the half sites, and the sequence context within the spacer region.

Figure 4. Consensus thyroid response element (TRE). Studies of TRE’s in many different promoters has allowed the derivation of a "consensus" TRE comprised of a direct repeat of the hexameric sequence, AGGTCA, spaced by four nucleotides (n). Of note, there is considerable diversity in the sequences of half-sites, orientation of half-sites, and bases that form the spacers between half-sites (see text).

Although TR can interact with a wide variety of other nuclear receptors and transcriptional adaptor proteins (see below), the RXR proteins (α, β, and g) represent its most important heterodimeric partners (1-3). The RXR proteins enhance TR binding to DNA and reduce the rate of receptor dissociation from DNA (91). RXR binds to the 5’ sequence and TR binds to the 3’ sequence of TREs in which half-sites are arranged as direct repeats (92, 93). The DNA-binding domains interact with the major grooves of the half-sites on the same face of the DNA (34,92,93). The carboxy-terminal end of the TR DNA-binding domain forms an α -helical structure that interacts with the spacer region in the DNA minor groove between the TRE half-sites. Although protein-protein contacts between the RXR and TR DNA-binding domains are important for dimerization, the major sub-regions involved in dimerization reside in the carboxy-terminii of the receptors (34). The dimerization surface of the TR appears to involve residues that lie along the surfaces of helices 10 and 11. T₃binding enhances the formation of TR-RXR heterodimers (94). On the other hand, T₃dissociates TR-TR homodimers (95). These findings raise the possibility that T₃binding might induce disruption of TR homodimers and induce the formation of TR-RXR heterodimers. The RXRs bind a stereoisomer of all trans retinoic acid, 9-cis retinoic acid (96,97). which variably alters transcriptional activity depending on the nature of the TH responsive gene (1,2). Additional studies are required to clarify the functional roles of RXRs and their ligands in TH action and interaction with other transcription factors. Recently, Hollenberg and colleagues recentlyanalyzed the hepatic TRβcistrome of hyper- and hypothyroid mice using Chip-seq technology (98). They found that the majority of TRβ-1 binding sites were not in the proximal promoter region but in other portions of target genes. Interestingly, by comparing the TR binding sites with previous Chip-seq data for RXRa, they found that some target genes may be regulated by TR homodimers rather TR/RXR heterodimers. Additionally, T₃increased TRβ-1 binding to DNA sites that, in turn, was correlated with T₃-induced gene expression. DR-4 and DR-0 motifs were significantly enriched at the DNA binding sites where T₃increased or decreased in TRβ1 binding, and were associated with positive and negative transcriptional regulation by T₃. Interestingly, in another study using Chip-chip methodology (99), some TH-regulated genes were identified that had little TR binding, despite the presence of putative TREs suggesting that other mechanisms such as receptor cross-talk, non-genomic effects, or indirect signaling mechanisms (see below) may be involved in regulating these genes.

TRs can have cross-talk with other nuclear hormone receptors owing to their common abilities to heterodimerize with RXRs. TR crosstalk with peroxisome proliferator-activated receptor (PPAR) and LXR signaling via heterodimerization with RXR is a prominent example. PPARg regulates the expression of its target genes by binding to the PPAR response element (direct repeat 1; DR1) as a heterodimer with RXR. Recently, it was shown that TRβ1 competes with PPARg for binding to DR1 as a heterodimer with RXR in vitroand in vivoto repress the transcriptional activity of PPARg (100). Since PPARg plays a key role in lipid metabolism, carcinogenesis, and cardiovascular diseases (101.102), this mode of TR may exert some of its effects by crosstalk with PPARs. Recently, cell-based studies indicate that TRβ inhibits the activity of LXR-α transcription activity of the CYP7A1 promoter which shares a common DR-4 element with TR (103). These studies show that TR cross-talk with other nuclear hormone receptor-mediated signaling expands TR effects beyond those target genes directly regulated by TRs (104).

Basal repression/Transcriptional corepressors

After binding to DNA, TR alters transcriptional activity by interacting directly or indirectly with a complex array of transcriptional cofactors. These proteins include corepressors (CoRs), coactivators (CoAs), integrators like CREB-binding protein (CBP), and general transcription factors (GTFs) (reviewed in 1-3, 31). Many of these factors have been identified by protein-protein interaction assays such as the yeast two-hybrid and glutathione-s-transferase pull down assays.

In the absence of TH, TR represses basal transcription in proportion to the amount of receptor and the affinity of receptor binding sites in positively-regulated target genes.This phenomenon also is referred to as transcriptional silencing (105-108). (Figure 3d-5, below). The addition of TH reverses basal repression and increases transcriptional activation above basal levels seen in the absence of receptor. Our understanding of the molecular mechanism for basal repression of transcription by unliganded receptor was advanced significantly by the discovery of a family of repressor proteins that bind selectively to unliganded TRs and RARs. This corepressor family includes silencing mediator for retinoid and TH receptors (SMRT) and nuclear receptor corepressor (NCoR) (105,109,110). These corepressors are 270 kD proteins that contain three transferable repression domains and two carboxy-terminal α -helical interaction domains. They are able to mediate basal repression by TR and RAR, as well as orphan members of the nuclear hormone receptor family such as rev-erbAα and chicken ovalbumin upstream transcription factor (COUP-TF). They have little or no interaction with steroid hormone receptors and therefore do not mediate basal repression by these receptors. Another protein, small ubiquitous nuclear co-repressor (SUN-CoR) enhances basal repression by TR and rev-erbA (31). This 16kD protein may form part of a co-repressor complex as it interacts with NCoR.

Within the interaction domains of NCoR and SMRT are consensus LXXI/HIXXXI/L sequences which resemble the LXXLL sequences that enable co-activators to interact with nuclear hormone receptors (40-42) (see below). Interestingly, these motifs allow both corepressors and co-activators to interact with similar amino acid residues on helices 3, 5, and 6 which are part of the ligand-binding pocket of TR. Differences in the length and specific sequences of the co-repressor and co-activator interaction sites coupled with the conformational changes in the LBD upon ligand binding, determine whether corepressor or coactivator binds to TR.

Recently, it has been shown that corepressors can form a complex with other repressors such as Sin 3 and histone deacetylases that are mammalian homologs of well-characterized yeast transcriptional repressors RPD1 and RPD3 (1-3,31). Thus, local histone deacetylation likely plays a critical role in the basal repression by unliganded TR/corepressor complex by maintaining local chromatin structure in a state that decreases basal transcription. Upon T₃binding, TR undergoes a conformational change that dissociates CoRs and recruits an array of coactivators (CoAs). Thus, hormone binding relieves repression and stimulates transcription by altering receptor binding to distinct classes of cofactors. Additionally, DNA-methylation may play a role in basal repression as methyl-CpG-binding proteins can associate with a co-repressor complex containing Sin3 and histone deacetylases (111,112). This repression was relieved by the deacetylase inhibitor, trichostatin A. These findings suggest that two repression processes, DNA methylation and histone deacetylation, may be linked via methyl-CpG-binding proteins.

The fact that TR alters the level of gene transcription in both the absence and presence of T₃has important implications for TH physiology. At low hormone concentrations, such as hypothyroidism, the unliganded receptor is predicted to repress transcription rather than function as an inactive, passive receptor. In some respects, this model is borne out by targeted inactivation of the TRα and TRβ genes. The phenotype of these double knockout mice are, for the most part, much less pronounced than the clinical features of congenital hypothyroidism (113,114). Thus, basal repression of transcription may explain why absence of receptor has less deleterious effects than absence of hormone (80,113,114).

Figure 5. TH receptor-mediated transcriptional silencing and activation. (A) Positively regulated genes. In the absence of hormone, the unliganded TH receptor represses or "silences" transcription in a process that involves TR interactions with a corepressor complex. Binding of T3 releases corepressors, relieving silencing and inducing the recruitment of coactivators that mediate transcriptional stimulation. (B) Negatively regulated genes. In the absence of hormone, the unliganded receptor activates transcription in a process that involves corepressors. Addition of TH dissociates corepressors and recruits coactivators. In the case of negatively regulated genes, this T3-mediated exchange of corepressors and coactivators inhibits transcription.

Figure 6. Role of corepressors and coactivators in the control of T3-regulated genes. In the absence of T3, the RXR-TR heterodimer recruits corepressors (CoR), which in turn, assemble additional components of a repressor complex that includes histone deacetylase (HDAC). Deacetylation of histones induce transcriptional repression. In the presence of T3, the corepressor complex dissociates and coactivators (CoA) bind to TR. The coactivator complex can include steroid receptor co-activators (SRCs)/p160, CREB-binding protein (CBP), p300/CBP associated factor (P/CAF), and proteins with histone acetyltransferase (HAT) activity. Vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP) complex can also interact with liganded TR, and may cycle with SRC/p160 complex. The general transcription factors (GTFs) are also indicated.

Transcriptional activation/Coactivators

A large and growing number of co-factors have been shown to interact with liganded nuclear hormone receptors and enhance their transcriptional activation. These include: steroid receptor coactivator 1 (SRC1); SRC2/transcriptional intermediary factor 2 (TIF2) / glucocorticoid receptor interacting protein 1 (GRIP1); SRC3/ amplified in breast cancer 1 (AIB1)/ receptor associated coactivator 3 (RAC3)/ p300/CBP cointegrator associated protein (p/CIP)/ nuclear receptor coactivator (ACTR)/ thyroid receptor activator molecule 1 (TRAM 1); peroxisome proliferator activated protein binding protein (PBP); TR accesory proteins (TRAPs) /vitamin D receptor interacting proteins (DRIPs); p300/CBP associated factor (p/CAF), and cAMP response element binding protein (CREB) binding protein (CBP)/ p300. among others (reviewed elsewhere (1-3,31).

At present, the precise roles of all these putative coactivators are not known; however, it appears that there are at least two major complexes involved in ligand-dependent transcriptional activation: the steroid receptor co-activator (SRC) complex and the vitamin D receptor interacting protein/TR associated protein (DRIP/TRAP complex) (Fig. 3d-6). SRCs (SRC-1,SRC-2, and SRC-3) are 160 kD proteins that associate with nuclear hormone receptors, including TRs, and enhance their ligand-dependent transcription (115-117). SRCs also interact with the CREB-binding protein (CBP), the co-activator for cAMP-stimulated transcription as well as the related protein, p300, which interacts with the viral co-activator E1A (118-121). Recent studies also have shown that CBP/p300 can interact with PCAF (p300/CBP-associated factor), the mammalian homolog of a yeast transcriptional activator, general control nonrepressed protein 5, GCN5. Like GCN5, PCAF has intrinisic histone acetyltransferase activity (HAT) activity. Both PCAF and CBP interact with TBP associated factors (TAFs) and RNA pol II. Thus, PCAF and CBP possess dual functional roles both as adaptors of nuclear receptors to the basal transcriptional machinery as well as enzymes that can alter chromatin structure by histone acetyl transferase (HAT) activity. SRC‑1 and CBP may coordinate with TRs to synergize further the actions of TH, and also allow for the convergence of plasma membrane and nuclear hormone receptor signaling pathways in the cell.

The DRIP/TRAP complex also interacts with liganded VDRs and TRs (122-125). However, none of the subunits are members of the SRC family or their associated proteins. Instead, several DRIP/TRAP components are mammalian homologs of the yeast Mediator complex, which associates with RNA Pol II. Thus, TR recruits DRIP/TRAP complex which, in turn, may recruit or stabilize RNA Pol II holoenzyme via their shared subunits. It is noteworthy that DRIP/TRAP complex does not appear to have intrinisic HAT activity. Recent chromatin immunoprecipitation assays of proteins bound to hormone response elements (HREs), suggest that there may be a sequential, possibly cyclical recruitment, of co-activator complexes to hormone response elements by liganded nuclear hormone receptors (126-129). Studies of co-activator recruitment to TH-regulated genes showed distinct temporal patterns of recruitment. Last, other co-factors such as SW1/Snf and BRG-1 may be involved in early chromatin remodeling before the co-activator complexes are recruited to the TREs (130,131).

Negative regulation by TRs

In contrast to positively-regulated target genes, negatively-regulated genes can be stimulated in the absence of TH and repressed by TH (Figure 3d-5, above). Regulation of TRH and the TSH α and β -subunit genes have been studied most extensively as models of negatively-regulated genes. From a physiological perspective, negative-regulation of these genes represents a critical aspect of feedback control of the TH axis. The T₃-responsive regions of these negatively- regulated genes have been localized to the proximal promoter regions (132-134). However, TR binding to putative TREs in these promoters is relatively weak in comparison to the binding sites in positively-regulated genes.

There are several different potential mechanisms for negative regulation by TH. Negative regulation may involve receptor interference with the actions of other transcription factors or with the basal transcription apparatus (135,136). For instance, TR can inhibit the activity of AP-1, a heterodimeric transcription factor composed of Jun and Fos. T₃-mediated repression of the prolactin promoter has been proposed to occur by preventing AP-1 binding (137). The TR also interacts with other classes of transcription factors, including NF-1, Oct-1, Sp-1, p53, Pit-1, CTCF, and GATA (138-144). By binding to these, or other positive transcription factors, the TH receptor may be able to inhibit gene expression by protein-protein interactions. Negative regulation may also occur by TR directly binding to DNA. A negative TRE from the TSHβ gene resides in an exon downstream of the start site of transcription (134) raising the possibility that it occludes the formation of a transcription complex. (Figure 3d-6, above) Additionally, liganded TRs may potentially recruit positive cofactors off DNA (squelching), which in turn, could lead to decreased transcription of target genes.

Transcriptional CoRs and CoAs, or even novel co-factors, may be involved in the control of negatively regulated genes. In contrast to the basal repression by unliganded TR in the case of positively regulated genes, CoRs cause basal activation of the TSH and TRH genes (132-134,145,146). CoAs also play an apparently paradoxical role in T₃-dependent repression of negatively regulated genes (146,147). Moreover, both SRC-1 knockout mice and knockin mice which express a TRβ mutant with a mutation in the helix 12 region (that interacts with CoAs) have defective negative regulation of TSH (148,149). Interestingly, histone acetylation can be increased in the T₃- mediated negative regulation of TSHawhereas it is decreased in regulation of TSHβ and TRH (150,151).

Epigenetic modifications by TRs

Transcriptional regulation by TRs is a multistep process involving: (1) association of TRs with regulatory sites in the genome (usually within the targe gene promoters) in the context of chromatin, (2) ligand-dependent recruitment and function of coregulators to modify chromatin and thereby regulating RNA Pol II recruitment to the target genes, and (3)co-valent modifications of histones to alter chromatin structure, recruit RNA pol II complex, and to mediate transcription. In particular, the site-specific acetylation of histone tails induces local relaxation of chromatin, which enhances the binding of some transcriptional regulators and facilitates the recruitment and functioning of the general transcriptional machinery. Recent studies have demonstarted that thyroid hormone-positively regulated target genes may have distinct patterns of coactivator recruitment and histone acetylation that may enable highly specific regulation (129). However the epigenetic changes associated with negetively regulated gene seems to be much more complex. For instance, histone acetylation of H3K9 and H3K18 sites, two modifications usually associated with transcriptional activation, occur in negative regulation of TSHapromoter. T₃also caused the release of a corepressor complex composed of histone deacetylase 3 (HDAC3), transducin b-like protein 1, and nuclear receptor coprepressor (NCoR)/ silencing mediator for retinoic and thyroid hormone receptor from TSHapromoter in chromatin immunoprecipitation assays. These findings demonstrate the critical role of NCoR/HDAC3 complex in negative regulation of TSHagene expression and show that similar complexes and overlapping epigenetic modifications can participate in both negative and positive transcriptional regulation (150). Of note, histone deacetylation has been observed in T₃-mediated negative regulation of several target genes (150-152). Moreover, abberant histone modification at the TRH and TSHagenes has been implicated in the inappropriate TSH secretion observed in resistance to thyroid hormone (RTH) syndrome (150,151)). Other coregulators may be involved in T₃-mediated regulation as RIP140, a coregulator that can decrease transcription by some nuclear hormone receptors, mediated T₃repression of Crabp1 gene via chromatin remodeling during adipocyte differentiation (153). Interestingly, the use of HDAC inhibitors to counteract the effects of basal repression of target genes have restored some transcriptional activity in hypothyroidism associated with RTH syndrome and hypothyroidism (154,155). Although nuclear CoRs play a prominent role in T3 nuclear action (156,157), NCoR-independent signaling may account for basal repression by unliganded TRs for a significant number of target genes (158).

Another mechanism for T₃-mediated epigenetic signaling is regulation of small non-coding microRNAs. MicroRNAs act as negative regulators of gene expression by inhibiting the translation or promoting the degradation of target mRNAs. Since individual microRNAs often regulate the expression of multiple target genes with related functions, modulating the expression of a single microRNA can, in principle, influence an entire gene network and thereby modify complex disease phenotypes (159). Thyroid hormone have been shown to regulate the levels of microRNA pair miR-206/miR-133b in human skeletal muscles (160), miR 208a in heart (161), miR21 and miR181d in liver (162,163). These miRNAs regulate important cellular events by TH such as differentiation, contractility, and metabolism. MicroRNAs thus are a novel mechanism for thyroid hormone signaling which may regulate mRNA levels of target genes in which TRs are not recruited to their promoters or directly affect their transcription (164). Last, it recently has been reported that miRNA 27a can modulate the expression of target gene, b-MHC in cardiac myocytes by decreasing TRb mRNA expression (165) and multiple miRNAs may also regulate TRb expression in papillary thyroid cancer (166) suggesting direction regulation of TR expression may be another mechanism for modulating target gene expression by TH.

Novel indirect pathways for TH action

It has been assumed that early transcriptional activation of target genes are mediated by direct transcriptional effects by TRs owing to their abilities to bind to TREs and recruit co-activators (167,168). Previous studies have suggested that TH may have non-genomic signaling activation that may result in rapid transcriptional changes (3, see below). However, several groups have shown that TH can activate SIRT-1 activity by a TR-dependent process, that in turn, can lead to deacetylation and activation of transcription factors such as PGC1a and FoxO1a. These findings raise the possibility that TRs can activate some target genes without TREs through activating other transcription factors (169). On the other hand, TRs can interact with SIRT-1 directly so it is possible that it can recruite deacetylase activity that can act on transcription factors as well as modulate transcription through histone modification (170-72).

Thyroid Hormone Receptors and Carcinogenesis

There are many reports providing evidence that reduced TR expression and/or alterations in TH levels are common events in human cancer (173, 174). These alterations include loss of heterozygosity, gene rearrangements, promoter methylation, aberrant splicing and point mutations (173,175). Tumors, including lung, breast, head and neck, melanoma, renal, uterine, ovarian and testicular tumors, present high frequencies of somatic deletions and mutations in both TR alpha/beta loci (176-178). Aberrant TRs have also been found in more than 70% of human hepatocellular carcinomas The tendency for TR expression to disappear as malignancies progress suggests that TR can act as a tumor suppressor in human cancers; therefore, loss of expression and/or function of this receptor could result in cell transformation and tumor development (179). In fact, TR overexpression in hepatoma cell lines shows repression of various tumor promoting genes such as PTTG1 (180), and activation of anti-tumorogenic TGF-beta. However certain mutant TRs like TRb^PV/PVmey even enhance tumor growth by non-genomicaly activating beta-catenin and PI3K pathways (181,182). Last, it recently has been reported that miRNAs can downregulate the expression of dio 1 in renal cell carcinoma, and TRbin papillary thyroid, carcinoma. Clarifying the molecular mechanisms by which TRs influence tumor progression and elucidating the epigenetic modifications ofT₃target genes in cancers would perhaps lead to a better understanding of the treatment regime in humans.

Recently, targeted gene inactivation or knockout (KO) of TR isoforms, and “knockin” of mutant TRs to their native TR genomic locii have provided new information on the mechanisms of TH action (16,17). The ability to disrupt TR genes by targeted mutagenesis has been particularly challenging given there is more than one gene encoding TRs, multiple splicing variants (TRα-1, α-2, TRβ-1, TRβ-2), and an additional transcript (Rev-erbA) derived from the opposite strand of the TRα gene (12,16,17). Two TRα knockout mouse lines have been generated that display different phenotypes (175, 176). It is likely this difference is due to the different sites in the TRα gene locus used for homologous recombination to generate the knockout mice. The TRα gene is complex as it encodes TRα-1, α-2 (which cannot bind T₃), and rev-erbA (generated from the opposite strand encoding TRα) (12, 16, 17). KO mice in which both TRα-1 and α-2 were deleted (TRα ^-/-) had a more severe phenotype with hypothyroidism, intestinal malformation, growth retardation, and early death shortly after weaning (175). T₃injection prevented the early death of pups. KO mice that lacked only TRα-1 (TRα-1^-/-) had a milder phenotype with decreased body temperature and prolonged QT intervals on electrocardiograms (183). The phenotypic effects of the loss of TRα 1 are relatively mild (184-185). Unexpectedly, there is no evidence of resistance to TH, as occurs with the TRβknockout. Disruption of the TRα-1 causes lower heart rates (19% reduced) and prolonged QRS and QT durations. These cardiac effects persist after hormone replacement. No changes were found in the levels of known TH-responsive genes in the heart (e.g., sarcoplasmic Ca²⁺ATPase, Na⁺-K⁺ATPase, β-adrenergic receptors). The bradycardic effect of the TRα-1 knockout may result from alterations in the sympathetic or parasympathetic nervous systems or it could result from an intrinsic defect in cardiac myocytes. The TRα-1-deficient mice also have a 0.5 ^oC reduction in body temperature that is independent of TH levels. The mice have normal amounts of brown adipose tissue.

Samarut and co-workers have reported generation of short TRα isoforms from intronic transcriptional start sites which have dominant negative activity on TR function (73), and it is likely these short TRα isoforms are responsible for the more severe phenotype of the TRα ^-/-mice. In this connection, TRα KO mice which did not express either TRα-1 and α-2 (TRα^o/o), had a milder phenotype than TRα ^-/-mice which expressed only the short TRα isoforms (186). Interestingly, TH stimulation of some target genes was increased, perhaps due to the absence of α-2 which inhibits normal TR-mediated transcription (57,58).

Targeted disruption of the TRβ locus created a mouse deficient in both TRβ-1 and TRβ-2 (16,17). These mice had elevated circulating TSH and T₄levels, thyroid hyperplasia, as well as hearing defects (187,188). These findings are similar to the index patients with resistance to TH who were later shown to have homozygous deletion of TRβ (4,152). Thus, the mouse model appears to faithfully reproduce some of the features seen in humans with resistance to TH who are lack TRβ or express a dominant negative mutant TRβ (189). TRβ-2-selective knockout mice also have been generated and exhibited elevated levels of TH and TSH suggesting TRβ-2 plays the major role in regulating TSH (190). TRβ-2-selective knockout mice also have abnormal color discrimination and suggest TRβ-2 may play a role in cone development of the retina (191).

The relatively mild phenotypes of the TRα-1 and TRβ KO mice suggest the two isoforms have redundant roles in the transcriptional regulation of many target genes. In this connection, microarray studies of TRα and TRβ KO mice showed similar gene regulation profiles in the absence and presence of T₃in liver (82). Recently, a study employing TRα or TRbreceptor over-expressing cell lines also showed that both these receptor isoforms mostly share a common gene repertoire but with varying degrees of induction or repression of target genes (192).

When both TR isoforms were abolished, the resultant double knockout mice (TRα 1^-/-TRβ^-/-) were surprisingly viable (113, 114). Thus, the absence of TRs is compatible with life. These mice had markedly elevated T₄, T₃, and TSH as well as large goiters. They also showed decreased growth, fertility, heart rate as well as bone density and development. Interestingly comparison of cDNA microarrays of double KO and hypothyroid mice showed only partial overlap of their gene regulation profiles, confirming the observation that the absence of receptor can give a different phenotype than lack of hormone. It is likely that basal transcription occurs even in the absence of receptor whereas basal repression of target genes occurs in the absence of hormone. When the phenotypes of TRa, TRb, and double KO are compared, it is apparent that each isoform may have isoform-specific function, perhaps in part due to different expression patterns of the isoforms as well as gene-specific actions by each isoform (114).

Cheng and colleagues have generated a “knock-in” mouse model in which a mutant TRβ from a patient with RTH (PV) was introduced into the endogenous TRβ gene locus (193). These mice have a phenotype similar to patients with RTH, as the heterozygous mice showed elevated serum T₄and TSH, mild goiter, hypercholesterolemia, impaired weight gain, and abnormal bone development. Homozygous mice had markedly elevated serum T₄and TSH, and a much more severe phenotype than heterozygous mice. Wondisford and colleagues also have generated a “knock-in” mouse that expresses mutant TRβ (194). These mice had abnormal cerebellar development and function, and learning deficits. These latter studies suggest that expression of mutant TRβ under the control of endogenous TRβ promoter produces many of the clinical features of RTH in mice. This same group also recently developed a knock-in of a mutant TRβ that cannot bind DNA. This model should be useful in distinguishing signaling and developmental patterns due to protein-protein interactions of TRs (as well as non-genomic pathways) from those that require TR binding to TREs of target genes (195). Knockin mice harboring a TRamutant at the same site as the TRβ^PVmutant gene decreased white adipose tissue (WAT) and liver mass (196). In contrast, TRβPV markedly induced hepatosteatosis and mass of liver but had little effect on white adipose tissue. The expression of lipogenic genes was decreased in white adipose tissue and liver of TRa^PVmice whereas it was increased in liver and normal in TRβ^PVmice. A recent study showed that the phenotype of impaired adipogenesis can be restored by crossing with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit the TR (197). These findings support the notion that the phenotypes in the TRa^PVmutant mice, and perhaps some patients with RTH with mutant TRa, may be due to aberrant repression of target gene (18, 198, 199).

NONGENOMIC PATHWAYS REGULATED BY TH

There is increasing evidence for non-genomic effects by TH (3) in addition to the transcriptional effects mediated by nuclear TRs. There is continuous shuttling of a small amount of TRs between the cytoplasm and nucleus (200), so non-genomic effects may be mediated by cytoplasmic TRs (see below). Recently, a TRavariant from alternative translation was shown to be palmitoylated and associated with the plasma membrane (201). TH binding to this receptor led to increased intracellular calcium, nitrous oxide, and cyclic guanine monophosphate (cGMP), which in turn activated PKGII, Src, ERK, and Akt signaling pathways. Another recent study suggests that non-DNA-binding TRs that cannot stimulate transcription may have “non-canonical” thyroid hormone signaling to regulate important physiological effects such as serum glucose and triglyceride levels, body temperature, and heart rate. (202). However, it appears that many non-genomic effects by TH are likely mediated by cellular binding proteins other than TRs. Evidence supporting this notion comes from the rapid time course of some TH effects (thus precluding transcription and protein synthesis), utilization of membrane-signaling pathways such as kinases or calmodulin, lack of dependence on the presence of nuclear TRs, and structure-activity correlations by TH analogs that are different than those observed for nuclear TRs (3). Several non-nuclear sites for TH binding have been identified in various cell systems although their functional significances are not well characterized. Some of these include: plasma membrane associated T₃transporters, actin, calcium ATPase, adenylate cyclase, and glucose transporters; an endoplasmic reticulum associated protein, prolyl hydroxylase; and monomeric pyruvate kinase (3, 203-207). A useful guideline that describes transcriptional and non-transcriptional signaling via TR and non-TR mechanisms recently has been published (208)

TH also has profound effects on mitochondrial activity and cellular energy state. A 43 kD protein has been described in mitochondria which also could bind to TREs and could be recognized by antibodies against the TRα ligand-binding domain (209). Recently, it has been shown that TRβ can interact with the p85 subunit of PI3K and activate the PI3K-Akt/PKB signaling cascade; thus, the small subpopulation of cytosolic TRβmay be involved in cell signaling (210). This PI3K activation by T₃leads to both direct and indirect effects on the transcription of several genes involved in glucose metabolism (210, 211,) and provides a mechanism for cross-talk between TH and cell signaling pathways.

Recently, integrin α-Vβ-3, has been identified as a plasma membrane TH-binding site (212). Previously, T₄, but not T₃, was shown to promote actin polymerization and integrin interaction with laminin in neural cells (213). Additionally, both T4 and T₃activated mitogen-activated protein kinase (MAPK) activity, and led, among other events, to phosphorylation of TRβ (210). Using a chick chorioallantoic membrane (CAM) system, Davis et al. showed that both T4 and T₃stimulated angiogenesis (214). Since integrin α-Vβ-3 is involved in angiogenesis, T₄and T₃binding to it was examined, and T₄was found to bind to integrin α-Vβ-3 with high affinity. Tetraiodothyroacetic acid (tetrac) and antibodies against laminin blocked T₄binding (213). Moreover, siRNAs against the integrin α-V or β-3 subunits blocked MAPK activation by TH. These findings suggest that TH activates the APK cascade and stimulates angiogenesis via TH binding to integrin α Vβ 3. Additionally, thyroid hormone non-genomically suppresses Src thereby stimulating osteocalcin expression in primary mouse calvarial osteoblasts (215). A direct physical interaction of TRbPV with cellular proteins, namely the regulatory subunit of the phosphatidylinositol 3-kinase (p85alpha), the pituitary tumor transforming gene (PTTG) and beta-catenin, that are critically involved in cell proliferation, motility, migration, angiogenesis and metastasis suggest a novel mode of non-genomic action, whereby mutant TR isoform acts as an oncogene in thyroid carcinogenesis (216).

TH ANALOGS, METABOLITES, AND ANTAGONISTS

Several tissue-specific and TR isoform-specific compounds have been developed as potential treatments for hypercholesterolemia, obesity, and heart failure. An early prototypical compound was 3,5-dibromo-3-pyridazinone-L-thyronine (L-940901) that bound preferentially to the TRs in the liver over those in the heart (217). Although the relative affinity of this compound for the respective TR isoforms has not been reported, the selective action of L-940901 is likely due to tissue-specific uptake of the compound. Interestingly, mice treated with L-940901 had decreased serum cholesterol levels without cardiotoxicity. Recently, several other TH analogs have been described that have isoform-selective affinity for TRβ compared to TRα (218-220). Since TRs in the liver are approximately 90% TRβ whereas those in the heart are mostly TRα, these isoform-selective compounds may serve as novel agents to lower serum cholesterol with minimal cardiotoxicity. N-[3,5-dimethyl-4-(4’-hydroxy-3’isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4(4’-hydroxy-3’-isopropylbenzyl)-phenoxy) acetic acid (GC-1), and 3,5-dichloro-4[(4-hydroxy-3-isoopropylphenoxy)phenyl] acetic acid (KB-141) all have been reported to lower total serum cholesterol and LDL-cholesterol (205-209). CGS 23425 also increases LDL receptor expression in HepG2 cells. Additionally, these compounds can increase serum apoA1 levels; however, the total serum high density lipoprotein (HDL) cholesterol level does not changes or may even decrease. In this connection, GC-1 decreased serum HDL; increased expression of HDL receptor, SR-B1; stimulated the activity of cholesterol 7α hydroxylase; and increased fecal excretion of bile acids in treated mice (221). Thus, GC-1 regulates important steps in the reverse cholesterol transport pathway (221). Recently, KB141 was shown to be a potential treatment for obesity by decreasing body weight via stimulation of metabolic rate and oxygen consumption (222). The TR agonist MB07811, which is converted to an active metabolite in the liver, has proven to be effective in reducing hepatic steatosis in rodents (223). The TRb-specific compound, KB215 recently was shown to be effective in decreasing LDL cholesterol, apoliprotein B, triglycerides, and Lp(a) in humans (224)when used in combination with statins. These findings suggest that TH analogs may be useful in the treatment of a wide range of metabolic disorders (225).

TH analogs and derivatives also bind specifically to proteins other than TRs, and are involved in non-genomic cell signaling pathways, Thyronamines (3-T1AM, T0AM) are endogenous compounds derived from L-thyroxine or its intermediate metabolites. Activities of intestinal deiodinases and ornithine decarboxylase generate 3-T1AM (226). Significantly, this compound bound poorly to nuclear TRs. T1AM has interesting physiological actions as it produced a rapid drop in body temperature and heart rate when injected intraperitoneally in mice. T1AM also decreased cardiac output in an ex vivo working heart model. Although 3-T1AM have a weak affinity towards classical nuclear TH receptors a number of putative receptors, binding sites, and cellular target molecules mediating actions of 3-T1AM have been proposed. Among those are members of the trace amine associated-receptor family (TAR1), the adrenergic receptor ADRα2a, and the thermosensitive transient receptor potential melastatin 8 channel (226). Preclinical studies using animal models are in progress, and more stable receptor-selective agonistic and antagonistic analogues of 3-T1AM are now being synthesiszed exerting marked cryogenic, metabolic, cardiac and central actions and represents a key lead compound linking endocrine, metabolic, and neuroscience research to advance development of new drugs (226).

TH can increase cardiac performance by increasing cardiac contractility and decreasing systemic vascular resistance (2); however, TH excess also can cause cardiotoxicity. 3,5-diiodothyropropionic acid (DITPA) is a TH-related compound with low metabolic activity and low affinity for nuclear TRs (Kd 10^-7M). DITPA was able to increase cardiac contractility and peripheral circulation without significant effects on heart rate in animal studies (227). Moreover, DITPA improved hemodynamic performance in animal models of congestive heart failure after myocardial infarction. Patients with heart failure treated with DITPA showed significant improvement in systolic cardiac index and systemic vascular resistance in preliminary studies (227). Thus DITPA or similar compounds may represent a novel class of drugs for the treatment of heart failure.

Recently, the naturally occurring analogs, 3,5,3'-triiodothyroacetic (TRIAC) and 3,5,3',5'-tetraiodothyroacetic TETRAC) acids decreased heat-induced albumin fibrillation suggesting they may have a protective effect against amyloid formation (228). Additionally, tetraiodothyroacetic acid (tetrac) caused radiosensitization of GL261 glioma cells (229). The mechanisms for these effects are not understood at this time but likely involve non-genomic effects as TRIAC and TETRAC have weak binding affinity for TRs. Similarly, 3,5-diiodothyronine (T₂) has also shown favorable effects in rodent models of fatty liver diseases (230,231).

SUMMARY

We have learned much about molecular mechanisms of nuclear TH action during the past 25 years. In particular, the identification and characterization of TRs, their heterodimeric partners, corepressors, coactivators, and TREs, generation of TR knockout mice, and discovering non-genomic pathways have provided new insight into TH action. It is expected that new information will be obtained from microarray and proteomic studies, structural biology approaches, and in vitro transcriptional systems. Such information should provide an even better understanding of the mechanisms of disease caused by abnormal circulating TH and/or altered intracellular TH levels, and provide targets for the development useful therapeutic agents for not only TH-related conditions but also for metabolic derangements such as hypercholesterolemia, non-alcoholic fatty liver disease, and obesity.

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Prostate Cancer Detection

ABSTRACT

Prostate cancer is the second most common cause of male cancer deaths in Western countries. However, one of the most contentious topics in medicine continues to be whether testing for this very common tumor is in the best interests of individual patients. Although there is a spectrum of progression rates for this tumor, in most instances, prostate cancer replicates and spreads slowly. As this tumor is uncommonly diagnosed before the age of 40 years and the likelihood of clinical detection increases as men age, most patients have comorbidities when diagnosed with prostate cancer. For this reason and because there are not insignificant potential disadvantages with the detection process and its consequences, it is important to determine whether the benefits of detection are likely to be greater than the unwanted effects of leaving a possible prostate cancer undiagnosed. In this Endotext chapter, the likelihood of a detectable prostate cancer being present is placed in context of patients’ ages and co-morbidies before detailing the tests currently used in clinical practice, together with their limitations. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION AND BACKGROUND

Prostate Cancer is an increasingly common diagnosis in Western societies with over 240 000 diagnoses made in the US (1), as well as 196,200 in Asia and 417,100 in Europe each year (2). There is a wide range in the incidence of prostate cancer across the globe with the highest rates in developed countries, being more than four-fold higher than less developed regions for a slightly lower mortality (3), although non-Westernized societies are changing as reported recently in relation to the Asia-Pacific region (4)(Figure 1). These differences are likely multifactorial, including genetic, environmental, detection, and reporting differences. As reported for 2012, Australia and New Zealand now has had the highest age-standardized incidence (111.6) and cumulative risk (13.6% by age 75) of prostate cancer in the world, with a high incidence observed in Northern America (97.2) and Western Europe (94.9).

Figure 1: Prostate Cancer Incidence Rates for Select Registries, 2000–2004 (5)

Mortality rates vary from country to country as well (6-7)with prostate cancer following lung and bowel cancers in Europe and Australia in terms of mortality rates. Worldwide, Caribbean and African populations display the highest prostate cancer mortality rates (Figure 2) (3). This disparity is also likely multifactorial and additionally includes factors related to treatment availability and practices.

Figure 2: Prostate Cancer Age-Standardized Mortality Rates for Selected Registries, 2000–2006 (5)

Despite advances in prevention and early detection, refinements in surgical technique and improvements in radiotherapy and chemotherapy, the ability to cure many patients remains elusive. However, mortality rates are changing albeit slowly as illustrated in blue below for Australia. A 2013 report by the Australian Institute of Health and Welfarepredicts that by 2020 only 26 out of 100,000 Australian men will die from the disease compared with 34 in 1982 (Figure 3) (8).

This phenomenon is not peculiar to Australia. Baade et al reviewed international trends in prostate cancer mortality and reported significant reductions in prostate-cancer mortality in the UK, USA, Austria, Canada, Italy, France, Germany, Australia and Spain with downward trends in the Netherlands, Ireland and Sweden (9). This has subsequently been observed by others (4,10).

Earlier detection of this disease, as a consequence of the introduction of the prostate specific antigen (PSA) blood test, has been acknowledged by the NCI as one factor contributing to lowering the mortality rate over the past few years (11-14). The use of PSA testing has been estimated to provide a diagnostic lead-time of up to 10 years (15-19). In the mid to late 1980s only one third of prostate cancers were diagnosed at curable stages compared with today when 80% are staged clinically as organ-confined and potentially curable (20-22). Unfortunately, however, even when the tumor is thought to be localized, up to 25% of men have non-localized disease which declares itself subsequently (23).

Figure 3: Panel A – Incidence (solid line) during 1982 – 2014 in Australia demonstrating a rise after widespread availability of PSA testing with a dip after the prostate cancer backlog was addressed: mortality (dashed line) has been falling slowly since the mid-1990s. Panel B – 5-year relative survival from prostate cancer, 1984–1988 to 2009–2013 in Australia demonstrated a reciprocal improvement since the mid-1990s https://prostate-cancer.canceraustralia.gov.au/statistics

Since curative treatments are limited to localized tumors (11-12,15,24), extending effective but non-invasive treatments to include both primary and secondary lesions remains a major goal and challenge. Once prostate cancer metastasizes, apart from causing loss of life, the toll it exacts is often considerable with regard to morbidity from both the disease itself and administered therapies.

As a result of increasing numbers of men having their prostate cancers diagnosed earlier, more patients are now eligible for treatment with curative intent. Improved surgical and radiation-based treatments have been developed so that the prognosis of a man diagnosed today with prostate cancer is better than ever before.

ANATOMY AND PHYSIOLOGY

The word "prostate", originally derived from the Greek prohistani which means "to stand in front of," has been attributed to Herophilus of Alexandria who used the term in 355 BC to describe the small organ located in front of the bladder (25). The prostate gland is a small firm structure, about the size of a chestnut, located below the bladder and in front of the rectum (Figure 4). The urethra, the channel through which urine is voided, passes from the bladder through the prostate and penis (Figure 5).

Figure 4: The Normal Prostate and its Relationship to Other Pelvic Structures

The primary function of the prostate gland, which contracts with ejaculation, is to provide enzymes to maintain the fluid nature of seminal fluid and to nourish sperm as they pass through the prostatic and penile urethra to outside the body.

Figure 5: Zonal Anatomy of the Prostate (sagittal depiction)
1 = peripheral zone - the zone where most cancers originate
2 = central zone – zone in which middle lobe develops
3 = transition zone – zone in which BPH ‘lateral lobes’ form
4 = anterior zone
B = bladder
U = urethra

NATURAL HISTORY OF PROSTATE CANCER

Traditionally, prostate cancer was considered to be a disease of "older men." As such, it was generally accepted that "men never died fromprostate cancer, they died of other conditions with prostate cancer." Consequently, treatment was conservative and directed toward palliation and management of any debilitating and painful sequelae. In addition, diagnosis from histopathology from a biopsy was generally made after palpating a rock-hard and nodular prostate on digital rectal exam [DRE] or by symptoms and signs of primary or secondary tumors, such as urinary obstruction, back pain, nerve root or, less commonly, spinal cord compression. In a large majority of cases, tumors had already disseminated at the time of diagnosis and, therefore, were incurable. It was in the mid-1980s, with the introduction of the PSA blood test that prostate cancer began to be diagnosed earlier and in younger men.

Prostate cancer is usually slow in its development and in the majority of cases, slow to progress as is illustrated in Table 1 below from Surveillance Epidemiology and End Results (SEER) registry: SEER collects and publishes cancer incidence and survival data from population-based cancer registries covering approximately 28% of the population of the United States(1).

If autopsy findings are an indication, premalignant and inapparent tumors are very common with one United States study indicating that, of 249 cases examined, 70% of the prostates with the premalignant condition high grade prostate intra-epithelial neoplasia (HGPIN) harbored adenocarcinoma, whereas the frequency of cancerin prostates without HGPIN was 24%. HGPIN was encountered in 0, 5, 10, 41 and 63% of men in the 3rd, 4th, 5th and 7th decades, respectively. The corresponding figures for invasive carcinoma were 2, 29, 32, 55 and 64% respectively (26).

Although methods of diagnosis and treatment of localized disease have become well-entrenched, they are beginning to change. However, both early detection through PSA screening and the management of prostate cancer remain controversial. The tumor has a variable biologic course, the traditional biopsy approach is invasive, costly and clinical staging of tumors is imprecise. Furthermore, there are significant limitations in prediction of the clinical outcome of patients with both organ-confined and extra-prostatic disease - not to mention the morbidity associated with all currently established treatments. It is sobering to muse that, were the unwanted effects of diagnosis and treatment insignificant, the dilemma of whether or not to diagnose and treat would not be issues.

COMPETING MORBIDITIES AND LIFE EXPECTANCY: COMPARISONS

The likelihood of men dying from causes other than from prostate cancer increases with ageing because of competing mortalities (as indicated by Figure 6 below), in particular cardiovascular and cerebrovascular diseases (Figure 6 below): the fact that most prostate cancers progress slowly compared with other cancers needs to be considered in terms of life expectancy from competing causes of death. Life expectancy has been reported to be increasing for Australian men, recently estimated to be 80.4 years from birth, the 7^thhighest worldwide, and 84.5 years at age 65(27). Calculation of life expectancy is difficult; however, use of statistically calculated “life tables”, based on population estimates, may provide the most accurate prediction.

Figure 6: Panel A – life expectancy estimates for Australian men and women since 1890. Panel B – Population pyramid for Australia in 2016, demonstrating the proportion of population for each age group. Available from: https://www.aihw.gov.au/reports/life-expectancy-death/deaths-in-australia/contents/life-expectancy

If death from prostate cancer is compared with the likelihood of death from other conditions, the older a man, the greater is the likelihood that another condition will be the cause of his demise; in Australia in 2009, one in three male deaths was attributed to cardiovascular disease (28).

The following graphs (Figure 7) from the Australian Government website show approximately parallel increases for incidence and death from prostate cancer, estimated to be 23 years apart (Figure 7). Consequently, if death is the endpoint being addressed, the patient’s life expectancy, based on his age and comorbidities, needs to be considered in the context of the natural history of his disease.

Figure 7: Estimated age-specific incidence (solid line) and mortality (dashed line) rates for prostate cancer, 2017 (Panel A), compared with 2007 (Panel B) (https://prostate-cancer.canceraustralia.gov.au/statistics)

TARGETING PROSTATE CANCER AT-RISK POPULATIONS

Major genetic epidemiologic studies published in the last two decades support the notion that prostate cancer may exist as clusters in families. In the 1980s, a Utah Mormon genealogy study found that prostate cancer exhibited the fourth strongest degree of familial clustering after lip, melanoma, and ovarian cancers (29). Prostate cancer, interestingly, had a higher familial association than either colon or breast carcinoma, to which patients are known to be predisposed by genetic or familial components. A later study determined cancer pedigrees in 691 men with prostate cancer and 640 spouse controls and found that men with an affected father or brother were twice as likely to develop prostate cancer as men with no affected relatives (30). Although these findings strongly suggest that familial clustering of prostate cancer risk does exist, they did not address the underlying etiological mechanisms. Indeed, familial clustering can reflect either shared environmental and lifestyle risk factors, or a genetic mechanism, or both.

To determine what might distinguish hereditary prostate cancer from its sporadic counterparts, a number of clinical features of prostate cancer were examined by Carter, et al.(31). Clinical stage at presentation, pre-operative PSA, final pathologic stage, and prostate weight were examined in a series of approximately 650 patients divided among three categories. Individuals were classified as having hereditary disease if 3 or more relatives were affected in a single generation, prostate cancer occurred in each of 3 successive generations in either paternal or maternal lineages, or 2 relatives were affected under the age of 65 years. For the other groups, either no other family members were affected (sporadic disease), or other family members were affected but not to the extent found in families classified as hereditary. In summary, no unique clinical or pathological characteristics distinguished hereditary prostate cancer in this group of patients. This parallel between hereditary and sporadic prostate cancer also extends to the incidence of multifocality found in both of these categories.

These findings were supported by Brandt et al (2011) in an analysis of the nationwide Swedish Family-Cancer Database between 1961 and 2006. They found that the age-specific hazard ratio of prostate cancer diagnosis increased with the number of affected relatives and decreased with increasing age. The highest hazard ratios were observed for men <65 yr. of age with three affected brothers (approximately 23) and the lowest for men between 65 and 74 yr. of age with an affected father (HR: approximately 1.8). The hazard ratios increased with decreasing paternal or fraternal diagnostic age. The pattern of the risk of death from familial prostate cancer was similar to the incidence data (32). A similar study also from Sweden determined that a positive family history was a risk factor for developing prostate cancer and most pronounced in younger men (aged 45-49 years) (33). A vast array of molecular alterations implicated in sporadic and familial prostate cancer have been described (34)and reported to account for 30% of familial risk (35).

However, there are differences between hereditary and sporadic prostate cancers. The onset of hereditary prostate cancer is, on average, 6 years earlier than for sporadic cancer. Although the clinical course is in no way different and the pathological characteristics are the same in most instances (36), patients with a family history of germ-line mutations in the family-susceptibility genes BRCA1 and BRCA2 , in particular the latter, and G84E mutation in HOXB13(37), have a significantly increased susceptibility for developing this malignancy. Furthermore, these patients tend to present at a younger age, have more aggressive and disseminated disease with poorer survival outcomes [31-6](38-44). Targeted screening of at risk men has been performed, with the IMPACT study reporting a higher positive predictive value of PSA and detection of intermediate- or high-risk disease in BRCA2 mutation carriers(45).

TESTS USED IN DIAGNOSING PROSTATE CANCER

In evaluating this issue, it is important to appreciate that the diagnostic approach is a two-step process that begins with the decision about whether or not to have a Prostate Specific Antigen (PSA) blood test (+/- other investigations) and, secondly, to confirm a suspected diagnosis of prostate cancer by biopsy for histopathology. Most men with a PSA level less than 10ng/ml will have a normal feeling prostate on digital rectal examination (DRE), hence the removal of DRE by non-urologists from many guidelines.

The FDA initially approved PSA testing in 1986 for monitoring the disease status of prostate cancer patients and, subsequently in 1994, it was endorsed as a screening method for prostate cancer (46). The PSA blood test is a continuous variable with no cut point (47)so that very low levels don’t completely exclude the possibility that prostate cancer is present(48-50), but the higher the serum PSA the greater the likelihood of prostate cancer being detectable. Importantly, PSA doesn’t distinguish between those who do and do not have cancer or identify those whose cancers will benefit from curative treatment. PSA increases with a number of conditions including prostate cancer, but the most common associated pathology is the non-cancerous condition benign prostatic hyperplasia (BPH) which is the cause, in most instances, of bladder outlet obstruction in men.

Factors Affecting PSA Measurements

Themedicationfinasteridewhich targets the 5-α-reductase type 2 enzyme and the more recently available drug, dutasteride, which inhibits both type 1 and type 2 enzymes, affect theconversion of testosterone to dihydrotestosterone (DHT) in prostatic cells. They reduce prostate volume with comparable effectiveness, with their designated clinical role being to decrease bladder outflow obstruction responsible for lower urinary tract symptoms (LUTS) present in a large number of older men. In reducing the benign prostatic hyperplasia (BPH) component of the prostate, both finasteride and dutasteride also reduce serum PSA levels by ~50% within 6 months of treatment. However, with the influence of the non-cancer BPH component significantly reduced, PSA changes are more likely to indicate prostate cancer. For patients taking finasteride or dutasteride, an increase in PSA of >0.3 ng/ml from nadir is generally regarded as an indication for further investigation based on the findings of Marks et al (2006) who determined that applying this recommendation resulted in a 71% sensitivity and a 60% specificity for prostate cancer being detected in men receiving dutasteride (51). Use of dutasteride may also affect interpretation of multiparametric MRI (mpMRI) and require a reduced biopsy threshold(52).

Concerns with respect to finasteride use and subsequent prostate cancer were addressed by long-term data from the Prostate Cancer Prevention Trial. Results confirmed that finasteride reduced the risk of prostate cancer by about one third but also found that high-grade prostate cancer was more commonly found on biopsy in the finasteride group than in the placebo group. However, after 18 years of follow-up, there was no significant difference between-groups in the rates of overall survival or survival after the diagnosis of prostate cancer (53).

Other non-malignant causes affecting serum PSA levels include prostatic infection and ageing since prostates tend to become larger as men get older (54). Instrumentation of the prostate and urinary tract can also raise PSA levels (55)as can bacterial or severe prostatitis, both of these capable of resulting in sudden rises in this enzyme (Figure 8).

Testosterone supplementation is commonly used for hypogonadism and might intuitively complicate interpretation of serum PSA levels. However, available data suggests that testosterone supplementation does not significantly increase serum PSA (0.1 ng/mL; 95% CI -0.28 – 0.48)(56-57), prostate size, intraprostatic testosterone, or prostate cancer incidence and progression in men with pre-treatment serum testosterone higher than 5 – 7 nmol/L (144 - 202 ng/dl(58). Testosterone therapy also causes minimal changes in lower urinary tract symptoms, with 77.5% of patients on supplementation having similar or improved symptoms for change in PSA of 0.44 (+/- 2.2)(59).

Figure 8: Factors Affecting Levels of Serum PSA(56-57) (60)

Efforts to improve the diagnostic accuracy of PSA have incorporated age-related reference ranges, which vary according to race (61-62)within and between countries. The normal age-related reference ranges are outlined below for European descent, Japanese, Chinese, Taiwanese, Singaporean and Korean men, as well as between Caucasian (Whites) and African-American (Blacks) men from the United States (Figures 9 – 14).

Furthermore, risk-stratification based on PSA for age is a newly adopted concept, recommended by the European Association of Urology and others, include longitudinal PSA testing for men with a PSA level >1 ng/ml at age 40 yr. or >2 ng/ml at age 60 yr. (63).

Figure 9: Age-based PSA Ranges for Men in Western Societies (19,61-62,64-66)

Figure 10: Age-based PSA Ranges for Japanese Men (67-70)

Figure 11: Age-based PSA Ranges for Chinese Men (67,71)

Figure 12: Age-based PSA Ranges for Taiwanese Men (67,72-74)

Figure 13: Age-based PSA Ranges for Singaporean Men (67,75-76)

Figure 14: Age-based PSA Ranges for Korean Men (67,77-78)

Attempts to improve the predictability of serum PSA for prostate cancer have included measuring the rate of PSA change (PSA velocity) and its relationship to the size of the prostate (PSA density) since prostates vary a lot in size and tend to become bigger as men age. This variable, but overall increase, in prostate size with ageing prompted the introduction of age-related PSAvalues by laboratories, based on the populations tested. The free or unbound PSA and its relationship to total PSA (free: total PSA) is another variation with the higher the free component, the lower the likelihood of cancer: most recently, the prostate health index (PHI) has become available and has been promoted. These are discussed in some detail (below).

Total PSA

Of the tests available, total serum PSA is generally regarded as having the greatest utility, maintaining its predictive value for the detection of prostate cancer (79)even after a first biopsy shows no evidence of cancer in which setting its performance characteristics are only slightly decreased (80). However, as stated above, PSA is far from a perfect test with most men with a serum PSA less than 10 ng/ml not having prostate cancer detected with biopsy, while conversely the possibility remains that prostate cancer may be present even with very low PSA levels. In the Tyrol project, Pelzer et al (2005) found that prostate cancers detected in men with PSA levels <4 ng/ml were in younger patients and at lower stages (81).

In terms of reassurance, a PSA<1 ng/ml in a man aged 60 years has been reported to indicate an extremely low risk of clinically important prostate cancer in his lifetime. Although a 25-30 year risk of prostate cancer metastases could not be excluded by concentrations below the median at age 45-49 (0.68 µg/L) or 51-55 (0.85 µg/L), the 15-year risk remained low at 0.09% (0.03% to 0.23%) at age 45-49 and 0.28% (0.11% to 0.66%) at age 51-55 (82). This finding was supported by Aus et al who failed to find a single case of prostate cancer detected in 2950 screened men age 50-66 with a PSA <1ng/ml over a 3-year period (83).

Serum PSA Summary:

Is a continuous variable with no cut point (47)
Lodding et al (1998) found 15% of prostate cancers detected by investigating a PSA between 3 & 4 ng/ml had extraprostatic growth (49)
In the Tyrol project, prostate cancers detected in men with PSA levels <4 ng/ml were in younger patients and at lower stages with smaller prostate volumes (81)
Doesn’t indicate who will benefit from curative treatment (48)
Total PSA remains the single most significant, clinically-used predictive factor for identifying men at increased risk of harboring cancer (79)
For men 50-70 years, a PSA >1.5 ng/ml is a marker for greater than average risk up to 8 years (7.5-times greater risk versus 1.5 ng/ml or less) (79)
Sustained rises in PSA indicate a significantly greater risk of prostate cancer, particularly high-grade disease
A PSA <1 ng/ml in a man aged 60 years has been reported to indicate an extremely low risk of clinically important prostate cancer in his lifetime (50)
Not a single case of prostate cancer was detected in in 3 years in 2950 screened men with a PSA <1ng/ml (83)

PSA Velocity (PSAV)

PSA is a labile enzyme with falsely high readings as a result of ejaculation within the previous 48 hours, vigorous (non-sexual) exercise, urethral instrumentation, and prostatic infections, as well as different assays providing slightly different readings. Therefore, a single PSA level should not be relied upon to indicate an increase in level. A rate of change of PSA (PSAV) >0.75 ng/ml in year in the absence of another contributing cause equates with an increased risk of a patient having cancer (84). Men taking the 5-α-reductase inhibitors, finasteride and dutasteride, have their serum PSA levels reduced by approximately 50% within 6 months. However, as stated above any sustained subsequent increase is more predictive for prostate cancer with an increase in PSA of 0.3 ng/ml from its nadir as a trigger for biopsy reported to provide 71% sensitivity & 60% specificity for prostate cancer for men who were receiving dutasteride (51).

For men not taking 5α reductase inhibitors, PSA increases >3.3% per annum have been reported to be associated with an increased risk of prostate cancer being detected by biopsy (19,65)and Makarov et al (2011) identified apreoperative PSA velocity >0.35 ng/ml/year to be associated with an increased risk of biochemical progression following radical prostatectomy (85). A more sinister association was observed by D’Amico et al (2004) who found that a PSA increase >2 ng/ml in the year before diagnosis conferred a high risk of death from prostate cancer despite radical prostatectomy (86). Loeb et al (2012) confirmed the adverse significance of a rapidly rising PSA, reporting that patients with two PSA velocity measurements of >0.4 ng/mL/year had an 8-fold increased risk of prostate cancer and a 5.4-fold increased risk of Gleason 8-10 disease on biopsy, adjusting for age and PSA level (87). The same author also concluded from an analysis of the Baltimore Longitudinal Study of Ageing that, since PSAV rose continuously with increasing PSA and was significantly higher in cancers than controls for PSA levels <3 ng/mL and 3-10 ng/mL, the PSA level should be taken into account when interpreting PSAV (88).

PSA Velocity Summary:

A PSA increase >0.75 ng/ml per year increases the risk of prostate cancer (84); for men taking 5-α-reductase inhibitors (finasteride & dutasteride) a PSA increase of 0.3 ng/mL per year increases the risk of prostate cancer
An increase in PSA of 0.3 ng/ml from nadir as a trigger for biopsy maintained 71% sensitivity & 60% specificity for prostate cancer in men receiving dutasteride (51)
A PSA increase of >3.3% per annum = an increased risk of cancer (19,65)
A preoperative PSA velocity >0.35 ng/ml/year = increased risk of biochemical progression following radical prostatectomy (85)
A PSA increase >2 ng/ml in the year before diagnosis = high risk of death from prostate cancer despite radical prostatectomy (86)
Men with two PSA velocity measurements of >0.4 ng/mL/year had an 8-fold increased risk of prostate cancer and 5.4-fold increased risk of Gleason 8-10 disease on biopsy, adjusting for age and PSA level (87)
An analysis of Baltimore Longitudinal Study of Aging concluded that, since PSA velocity rose continuously with increasing PSA and was significantly higher in cancers than controls for PSA levels <3 ng/mL and 3-10 ng/mL, the PSA level should be taken into account when interpreting PSAV (88)

Free/Total PSA

This test measures the percentage of free (or unbound) PSA in the blood and compares it with the percentage bound to proteins (α1 anti-chymotrypsin and α2 macroglobulin). Prostate cancer increases the amount of bound PSA. The lower the ratio of free to total PSA or the percentage of free PSA, the higher the likelihood that the patient has prostate cancer. The proportion of free PSA in seminal fluid is much higher than in serum, consistent with its physiological role in liquefaction (89). Although levels of bound-PSA do not significantly correlate with PSA in semen in young men, levels of free PSA do. With ageing, blood levels of complex-PSA, but not free-PSA, increase (90). The free/total PSA blood test can help to discriminate between patients with indeterminate PSA levels (4-10.0 ng/ml) indicating those who are at the greatest risk of having prostate cancer, in particular aggressive disease (91-92). However, as with all these modifications to PSA, the predictability remains less than perfect.

Free/Total PSA Summary:

Men with prostate cancer have a greater fraction of complexed PSA and a lower free PSA than men without prostate cancer
Free: Total PSA can be helpful in the case of a high PSA and a negative prostate biopsy
Free PSA is unstable: the assay must be frozen to -20°C within 3 hours otherwise the free fraction reduces
Chronic prostatitis may also cause a reduced Free: Total ratio

PSA Density

PSA density relates the concentration of serum PSA to the volume of the prostate and is thus a measure of serum PSA in relation to prostatic size (93). Most neoplastic prostate glands produce higher serum PSA levels per unit mass than do non-malignant glands. Consequently, a serum PSA of 5.0 ng/ml in a patient with a 20-gram prostate is more worrisome for cancer than that a PSA of 5.0 ng/ml in a man with a 60-gram prostate, especially if there is a predominance of transitional zone tissue (BPH) in the latter. To determine the PSA density, a PSA level is obtained and is divided by the volume of the prostate, as estimated by transrectal ultrasound (TRUS). Recent adoption of multiparametric (mp) MRI has allowed for determination of prostate volume as a standard reporting item. A value >0.15 ng/ml per gram of prostate tissue is considered worrisome for prostate cancer, and clinically used in nomograms to aid the urologist in estimating risk of prostate cancer. PSA density has been extended to include transition zone measurements in relation to the overall size of the prostate as the transition zone is the site in which BPH develops with ~25% of prostate cancers also arising in this zone. The larger the transition zone in relation to the overall size of the gland, the lower the likelihood of prostate cancer, other things being equal.

PSA Density Summary:

PSA Density = PSA divided by prostate volume determined by TRUS / mpMRI
The larger the transition zone, the lower the likelihood of prostate cancer
PSAD >0.15 ng/ml per gram is considered worrisome for prostate cancer
Problems with PSA density include:

(i) difficulty in defining the outline of the prostate accurately

(ii) variability in shapes not addressed by automated TRUS calculator estimations

Prostate Health Index

A further variation on the PSA blood test is the Prostate Health Index or phi, formulated by having the value of a truncated form of the PSA molecule (proPSA, greater production by most cancers than benign tissue) as the numerator and the free PSA value as the dominator multiplied by the total PSA level to give a phireading. phiis claimed to better predict prostate cancer risk than the total PSA. A phi-based nomogram in an external validation study performed with 75.2% accuracy (94). Furthermore, phi has been reported to aid in predicting pT3 disease (2.3%) and/or pathologic Gleason score ≥ 7 (2.4%) although decision curve analyses deduced these were not of greater clinical net benefit (95). A potential advantage of phiis that it stratifies according to risk. However, health economic analyses to determine clinical benefits of phi are yet to be realized(96).

Prostate health index [phi] = [−2]proPSA / fPSA) × PSA^1/2

For PSA 2–10 ng/ml, sensitivity, specificity and AUC (0.703) of phiexceeded those of total PSA and % fPSA. Increasing phiwas associated with an increased risk of prostate cancer (97). These estimates have been confirmed in multiple studies (AUC 0.67-0.81 c.f. PCA3 0.73, %fPSA 0.60-0.65, tPSA 0.50-0.52)(94,98-99), resulting in a consistent estimate of approximately 20-30% of avoided biopsies if phi is used instead of %fPSA(100-101). A meta-analysis estimated prostate cancer detection with sensitivity of 90% and specificity of 31.6%, and was better overall than PSA and %fPSA for PSA 2 – 10ng/ml(102).
Including the prostate health index in a multivariable logistic regression model based on patient age, prostate volume, digital rectal examination and biopsy history significantly increased predictive accuracy by 7% from 0.73 to 0.80 (p <0.001) (103).
phi0-22.9 = low probability of prostate cancer (8.4%)

23-44.9 = moderate probability of cancer (21%)

>45 = high probability of cancer (44%)

phi-density (PHID), calculated similarly to PSA density, may also improve diagnostic accuracy of clinically-significant prostate cancer (AUC 0.82) compared to phi (0.79), %fPSA (0.79) and PSA (0.70)(104).

Four-kallikrein (4K) Panel

Another recent variation on the PSA blood test is the four-kallikrein (4K) panel, determined by a combination of kallikrein-related peptidase 2 (hK2), intact PSA, and free and total PSA as well as with clinical data (age, DRE findings, previous biopsy results). The 4K score has been shown to predict biopsy outcome to avoid unnecessary biopsies as well as predict distant metastasis at 10 years.

Among European men with serum PSA 3 – 15 ng/ml, 4K score showed similar diagnostic performance (AUC 0.69 any prostate cancer, 0.72 high-grade prostate cancer) to phi (AUC 0.70 any prostate cancer, 0.71 high-grade prostate cancer) and potentially saved 29% of performed biopsies(105). These findings have been confirmed in multiple studies, including a Swedish community cohort(106).
When applied to a USA cohort, 4K score performed better (AUC 0.82) than PCPT clinical risk calculator (AUC 0.74), equating to a potential 30-58% reduction in biopsies for delayed diagnosis of 1.3-4.7% of Gleason≥7tumors (107). Furthermore, accuracy was maintained between African American and Caucasian groups.
The 4K score was applied to patients enlisted in the ProtecT study and showed superior diagnostic accuracy compared with PSA for any cancer (AUC 0.719 vs. 0.634) and high-grade cancer (0.82 vs. 0.74) and potentially reduced unnecessary biopsies by 42%(108).

Summary: Prostate Specific Antigen (PSA) & Derivatives

Is a continuous variable with no cut point (47)
Doesn’t distinguish between those with and without cancer or identify those with cancer who will benefit from curative treatment (48)
PSA Velocity = rate of change of PSA: A PSA increase >0.75 ng/ml in year = an increased risk of having cancer (84)
PDA Density: PSA density = PSA divided by prostate volume determined by TRUS
Free: total PSA: The higher the free component and the ratio of free to total PSA, the lower the likelihood of cancer but chronic prostatitis may also cause a reduced Free: Total ratio
Prostate Health Index (phi):may predict the risk of prostate cancer better compared with total PSA, but its role in prostate cancer screening is not defined.
Total PSA = the single most significant, clinically used predictive factor for identifying men at increased risk of harboring cancer (79)

Digital Rectal Examination

Traditionally, palpation of the prostate by digital rectal examination (DRE) was the manner by which a diagnosis of prostate cancer was suspected. In historical series, up to 50% of palpable masses were attributable to prostate cancer (17,109-110). Although DRE by itself is a poor method for diagnosing this malignancy (111-112), especially when performed by non-urologists, it does still have an important diagnostic role, hence its variable inclusion in prostate cancer guidelines (recommended only for urologists mostly), as up to 25% of tumors are detected in men with normal PSA levels (113). Unfortunately, when a prostate cancer is diagnosed based on a palpable tumor, the risk of the patient already harboring metastatic or locally advanced malignancy is considerable(114-116). However, a PSA-based prostate cancer detection strategy which omits DRE runs the low risk of missing some curable cancers (49).

The PCA3 Test

The non-coding RNA PCA3, originally called DD3, is highly specific to prostate cancer, with over-expression(117-120)in a number of different cohorts. The first part of a voided urine specimen is collected immediately following firm rectal examination or prostatic massage (121-122)and PCA3 RNA measured using a PCR-based assay. One criticism of the PCA3 test is that is unlikely to obtain prostatic fluid from the anterior part of the prostate, mirroring a deficiency with TRUS-guided biopsies obtained via the rectum, which are also posteriorly-focused, especially in large prostate glands. Although the “PCA3 urine test” has been reported to improve identification of serious disease compared with total PSA in a pre-screened population (Table 2), its role in initial assessment of patients suspected of having prostate cancer has yet to be established (123-124). A prospective multicenter validation trial to assess the diagnostic performance of PCA3 determined a positive predictive value of 80% for initial biopsy, and negative predictive value at repeat biopsy of 88%, while the addition of PCA3 to available risk calculators improved risk prediction of overall and high-grade cancer(125).

Table 2: PCA3 Results in Post-Prostatic Massage Urines (118,120,125-129)

Study	Sensitivity	Specificity	Neg Predictive Value	Number
Hessels et al, 2003	67%	83%	90%	108
Fradet et al, 2004	66%	74%	84%	517
Tinzl et al, 2004	82%	76%	87%	158
Van Gils et al, 2007	65%	66%	80%	534
Van Gils, et al 2007	65%	82%	80%	67
Salami et al, 2013	93%	37%	92%	45
Wei, et al 2014 (initial biopsy, PCA3 > 60)	42%	91%	PPV 80%	562
Wei et al 2014 (repeat biopsy, PCA3 <20)	76%	52%	88%	297

Attempts to analyze PCA3 and other biomarkers in prostatic fluids, such as semen(130-131), have shown comparable diagnostic accuracy (132)but patient recruitment and clinician acceptance is challenging.

Recently, data from analysis of the fusion gene TMPRSS2:ERG and PCA3 from prostatic fluid obtained following firm digital rectal examination/prostatic massage, has been combined with serum PSA to produce a test which is being marketed commercially. Published supportive data is limited but preliminary findings indicate that the combination provides an 80% sensitivity and 90% specificity with an AUC of 0.88 for the 3 parameters(129,133-134).

However, Stephan et al.(135)examined PCA3, TMPRSS2:ERGand phiin an artificial neural network. The addition of TMPRSS2:ERG to PCA3 in urine following firm digital rectal examination only marginally improved detection of prostate cancer in110 men compared with 136 with non-cancer. PCA3 had the largest AUC (0.74) which was not significantly different to the AUC of phi(0.68) although the latter showed somewhat lower specificities than PCA3 at 90% sensitivity. A combination of PCA3 and phionly moderately enhanced diagnostic power with modest AUC gains of 0.01-0.04 for prostate cancer at first or repeat prostate biopsies. These findings were not reproduced by Salami and colleagues in the USA, where PCA3 demonstrated high sensitivity (93%, AUC 0.65), while TMPRSS2:ERG had higher specificity (87%, AUC 0.77) compared to serum PSA (AUC 0.72). A multivariate algorithm optimized cancer prediction (AUC = 0.88; specificity = 90% at 80% sensitivity) (129). In a prospective multicenter study of PCA3 and TMPRSS2:ERG prior to biopsy, Sanda and colleagues reported a 33-39% specificity at 93% sensitivity, which was predicted to reduce 42% of unnecessary biopsies and conferred a cost benefit for younger men(136).

It is likely that future clinical practice will integrate molecular markers into predictive calculators, such as the Prostate Cancer Prevention Trial (PCPT) or ERSPC calculators, to improve diagnostic accuracy above crude traditional markers such as family history or clinical examination findings (137).

Magnetic Resonance Imaging (MRI)

MRI use in prostate cancer is rapidly evolving. Potential applications and benefits include are summarized in Table 3.

Table 3. Utility of MRI in Prostate Cancer(138-141)

Scenario

Potential Benefits

1) Triage prior to biopsy

· Avoid unnecessary biopsy

· Provide target(s) for biopsy

· Assessment of anterior and apical areas that are poorly sampled by TRUS biopsies obtained via the rectum

· Increase detection of clinically significant cancer

· Minimize detection of insignificant cancer

2) Patients with prior negative biopsy

· Provide target for biopsy

· Assessment of anterior and apical areas that are areas poorly sampled by TRUS

· Increase detection of clinically significant cancer

· Minimize detection of insignificant cancer

· Decrease unnecessary repeat biopsy

As an initial form of detection, MRI has the potential to improve the sensitivity of detection of intermediate and high-risk prostate cancer, especially in the anterior zone of the prostate, where cancers may not be sampled using transrectal ultrasound guided biopsy techniques. However, in some countries cost is still a handicap to widespread application. Interpretation of prostate imaging with different sequences (summarized in Table 4), using a “multiparametric” approach, requires expertise and collaboration, most commonly according to a structured reporting scheme, prostate imaging-reporting & data system (PI-RADS), which has since been updated to PI-RADS v2 (142-144). A PI-RADS score is assigned to each individual lesion using T2 weighting, diffusion-weighted imaging (DWI), and dynamic contrast enhancement to assign scores based on a Likert (5-point) scale based on the probability of clinically significant malignancy: PI-RADS 1 very low; PI-RADS 2 low; PI-RADS 3 intermediate; PI-RADS 4; PI-RADS 5 very high (145). A summary of the standardized anatomical description map and PI-RADS scoring specifications are shown Figures 15 and 16). For lesions in the peripheral zone DWI is the dominant sequence, and for the transition zone T2 dominant.

Table 4: MRI Imaging Sequences

	Use
T1-weighted	Detection of hemorrhage post biopsy as hyperintense Detection of bone metastasis Detection of abnormal lymph nodes
T2- weighted	Demonstrates zonal anatomy. Sensitive but non-specific as prostate cancer, prostatitis, atrophy, BPH, and changes after treatment (e.g., radiation induced arteritis) are hypointense Dominant sequence for PIRADS scoring of transitional zone.
Diffusion-weighted imaging (DWI)	Prostate cancer demonstrates restricted diffusion, appearing hyperintense at high b values and hypointense on ADC map Dominant sequence for PIRADS scoring of peripheral zone
Dynamic contrast enhancement (DCE)	Prostate cancer shows early enhancement and early washout
Spectroscopy	Requires extra time for acquisition and may not add diagnostic value. Not frequently used. Citrate is reduced, whereas choline is increased in prostate cancer

Figure 15: Prostate Map for Description of Lesions Detected on mpMRI According to PIRADS Classification

Figure 16: Prostate Map for Description of Lesions Detected on mpMRI According to
PIRADS Classification for Peripheral (right) and Transition (left) Zone Lesions.

CLINICAL USES OF MRI IN PROSTATE CANCER

Triage Prior To Biopsy

The diagnostic accuracy of MRI for the detection of prostate cancer varies widely across studies, with sensitivities from 58-96% and specificity from 23-87% (140-141,146-148). Such variability can be explained by different equipment, level of experience, lack of standardization in the early series, different reference standards, different sequencing protocols and definitions of clinically significant cancer. It is evident that accuracy has been improving in the more recent series, and good standardization of reporting has been achieved with the use of PIRADS V2. A meta-analysis of 21 studies including 3857 patients using PIRADS V2 showed a sensitivity of 89% and specificity of 73% (149). Of note, some studies only considered clinically significant prostate cancer while other considered any prostate cancer (149).

The PROMIS trial of 576 men assessed the capacity of mpMRI to identify men with clinically significant prostate cancer prior to prostate biopsy and compared the diagnostic accuracy of mpMRI to a 10-12-core systematic TRUS biopsy using a template transperineal prostate biopsy with cores taken every 5mm as the reference standard (148). mpMRI was significantly more sensitive than TRUS biopsy via the rectum in all 3 definitions of significant cancer used (Table 5). As a triage test mpMRI performed prior to biopsy and reserving it to patients with suspicious findings, mpMRI would have avoided biopsying 27% of patients at a risk of missing 7-12% of significant cancers - depending on the definition used.

Table 5. PROMIS Trial Results (148)

Definition	mpMRI	TRUS 10-12-core
Gleason ≥4+3 or cancer ≥6mm	Sens: 93% Spec: 41%	Sens: 48% Spec: 96%
Gleason ≥3+4 or cancer ≥4mm	Sens: 87% Spec: 47%	Sens: 60% Spec: 98%
Gleason ≥3+4	Sens: 88% Spec: 45%	Sens: 48% Spec: 99%

The PRECISION study was a multi-centre pragmatic trial that randomized 500 patients with elevated PSA and/or abnormal DRE to TRUS prostate biopsy (10-12 cores) or a mpMRI. Patients in the mpMRI group underwent a targeted biopsy only if lesion(s) PIRADS ≥ 3 were identified. The targeted biopsy could be performed with software or cognitive fusion and could be transrectal or transperineal. The detection of clinically significant prostate cancer (defined as Gleason 3+4 or greater) was 26% in the standard biopsy group and 38% in the mpMRI group. This increased detection occurred despite the fact that 28% of patients in the mpMRI group were not biopsied because the study was reported as PIRADS 1-2 but still included in the population/denominator. Conversely, the detection of Gleason 3+3 cancer was 22% in the standard biopsy and 9% in the mpMRI group. Clinically significant cancer was detected in 12% of PIRADS 3, 60% of PIRADS 4 and 85% of PIRADS 5 lesions, similar to that seen in previous studies. Of note, the centers contributing the majority of patients had significant prior experience with prostate mpMRI reporting and targeted prostate biopsies. Whether centers with limited experience can achieve similar results remains to be demonstrated.

Patients with Prior Negative Biopsy

MRI has shown been shown to be useful in the subset of patients with prior negative biopsies. For these patients, the biopsy detection rate is approximately 30%, decreasing with each subsequent biopsy procedure (150-151).MRI followed by MRI-guided biopsies identifies prostate cancer in 41-59% (152-153). A recent review showed that across 16 studies MRI guidance improved the absolute detection of clinically significant prostate cancer between 6-18% in patients with previous negative TRUS biopsy (154).

DEFINITIVE DIAGNOSIS REQUIRES BIOPSIES

Prostate biopsy is required for the definitive diagnosis of prostate cancer. Systematic TRUS-guided biopsies have been the standard for the past decades but concerns about missing significant cancer and the risk of sepsis are changing the landscape. While TRUS imaging permits spatial positioning for systematic sampling, by itself it has low accuracy in detecting suspicious areas.

The number of biopsy cores taken is important with the chance of missing a cancer by standard sextant biopsy estimated to be approximately 25% (155)so that, more recently, the numbers of cores recommended are at least 10-12. In addition, it is advocated that biopsies should be directed laterally and that they should include the anterior horns of the peripheral zone (155-158). Still, recent studies have shown that systematic TRUS biopsies performed via the rectum miss approximately 50% of clinically significant cancers (139,148,159). The introduction of mpMRI is changing this situation at least in terms of missing significant cancer.

One of the problems facing clinicians has been when to stop from recommending biopsying not only in terms of patient age and overall life expectancy but also with respect to the increasing likelihood of a positive histological diagnosis in those biopsied. Indeed, a continually increasing probability of death from prostate cancer was observed among men of all ages with a PSA of 3.0 ng/ml in Baltimore Longitudinal Study of Ageing (160)of 849 men, 122 with and 727 without biopsy-confirmed prostate cancer. However, no participants between 75 and 80 years old with a PSA lower than 3.0 ng/ml died of prostate cancer. And not unexpectedly, the time to death or diagnosis of aggressive prostate cancer after age 75 years was not significantly different between PSA categories of 3 to 3.9 and 4 to 9.9 ng/ml. Of the 108 subjects older than 75 years with a PSA of 3 ng/ml or greater, 10 died of prostate cancer and 18 had high risk disease. In this group, 90 men did not have a diagnosis of high risk prostate cancer, including 75 who were never diagnosed with cancer (median time to censoring 12.5 years) and 15 who were diagnosed with non-high risk cancer (median time to censoring 17 years) (160). Therefore, many guidelines recommend against PSA testing among men older than 70 with a life expectancy of less than 7 to 10 years.

Routine practice for biopsies taken via the rectum involves peri-operative antibiotic prophylaxis. TRUS biopsies can be performed under local anesthesia or sedation. Rectal cleansing with povidone-iodine is recommended to decrease the risk of sepsis (161).

Changing Morbidity of Biopsy Diagnosis

Periprocedural symptoms such as hematuria, rectal bleeding and hematospermia are frequent, being experienced by over 50% of men having TRUS biopsies performed via the rectum but are almost always benign and self-limiting (162-164). Infectious complications following this procedure are less common but are being reported more often, with the causative mechanism believed to be inoculation of the prostate, blood vessels and urine with bacterial flora from the rectal mucosa and subsequent systemic dissemination (162,165-166). There has been concern expressed that hospital admissions due to post-TRUS biopsy may be rising, with one study reporting a 3-fold increase from 0.55% across 2002-2009 to 2.15% across 2010-2011 (162,167-168). Changing bacterial resistance patterns and antibacterial practices have contributed to the spectrum of infectious complications with the infection rate being much higher in certain population groups such as men who have been taking antibacterial drugs prior to the biopsy and people who have been in South East Asia and Mediterranean countries within the past 6-12 months (168-169). Not surprisingly there is wide variation in the reported incidence of overall infectious complications from 0.1% to 7% and of sepsis from 0.3% to 3.1% across studies (166,170).

A prospective New Zealand study reported that drug resistance rates for patients who required intensive care admission for sepsis following TRUS biopsy were 43% for gentamicin, 60% for trimethoprim-sulphamethoxazole (60%) and 62% for ciprofloxacin as well as 19% for all 3 agents in combination. E. coli sequence type 131 clone was implicated as being particularly problematic, accounting for 41% of all E. coli isolates after TRUS biopsy (171). Fluoroquinolone resistance in rectal cultures has been reported to predict infectious complications following TRUS biopsy (172). The changing patterns of drug sensitivities and reports of low resistant rates to drugs such as carbapenems for patients with unresolving sepsis (173)has resulted in some advocating for the use of these drugs as prophylactic agents just prior to TRUS biopsy (174-175). However, adoption of such a strategy runs the risk of decreasing the number and effectiveness of those pharmaceutical agents currently kept in reserve for patients with overwhelming sepsis (175).

The transperineal approach has emerged as an alternative with significantly decreased risk of infections complications, albeit requiring specialized equipment, general anesthesia in most centers, increased operative time, an increased risk of urinary retention and potential nerve damage affecting erectile ability. A notable advantage of the transperineal approach is better sampling of the anterior zone of the prostate (174).

MRI also allows one to perform targeted biopsies, thereby increasing the detection of significant cancer. The main types of guided prostate biopsy techniques following diagnostic imaging with MRI include cognitive fusion (visual estimation from clinician’s interpretation of TRUS and mpMRI images), MRI-guided (biopsy performed under MRI guidance), fusion software (software integrating MR images on to the TRUS screen to guide biopsy needle to target index lesions), and robotic (automatic fusion and alignment for clinician). A particular issue with biopsying performed under real-time MR imaging is cost because this approach uses MR equipment which otherwise would be used for other purposes. These options are summarized in Table 6.

Table 6: Approaches for MRI-guided Targeted Prostate Biopsy

	Description	Characteristics	References
Cognitive fusion (visual estimation)	Manually directed based on MRI TRUS or TP	Low cost Operator dependent	Sciarra 2010 Lee 2012 Panebianco 2015
In-gantry (real time) MRI-guided biopsy	MRI-compatible biopsy gun used and trajectory established. Biopsy gun fired and sampling confirmed TRUS or TP	High cost with each procedure Steep learning curve Highest precision	Overduin 2013 Penzkofer 2015 Schimmöller 2016 Yaxley 2017
MRI-TRUS software fusion biopsy	Software assisted targeting of lesions TRUS or TP	Initial cost outlay Ongoing costs similar Good accuracy	Porpiglia 2016 Siddiqui 2015 Meng 2016
Robotic-Assisted	Potentially less operator dependent TRUS or TP	Initial cost outlay	Tilak 2015

In a meta-analysis of 11 studies Wegelin et al. compared the prostate cancer detection rates of cognitive-fusion, in-gantry, and TRUS software-fusion biopsy. In-gantry biopsy had a higher overall detection rate than cognitive-fusion, but the detection of clinically significant cancer was not different across the 3 techniques. Yaxley et al. performed a retrospective review comparing in-gantry MRI-guided biopsy to cognitive TRUS biopsy. In 595 PI-RADS 3-5 lesions, there was a high prostate cancer detection rate with no difference across biopsy methods (176). While the advantages of obtaining an MRI prior to biopsy are clear, up to 13% of clinically significant tumors can be missed when only targeted biopsies are performed (177-178). Moreover, this figure may be higher for lower volume centers with limited experience in MRI interpretation and MRI-guided biopsies. Consequently, many practitioner’s biopsy both index lesions seen with MRI as well as systematically sampling all parts of the prostate with 12 or more biopsies. In doing so, cognitive or in-gantry approaches are used for index lesions with a template employed to ensure correct special placing of biopsy needles for systematic sampling of the whole of the prostate.

Histopathological Assessment

The biopsy result provides important information for the patient and clinician on which to base management decisions (179). Important prognostic information on biopsy assessment include tumor quantification values (fraction of positive cores i.e. the number of positive cores versus the number of cores submitted and the percentage or length in mm of cancer in intact positive cores), cancer grade (Gleason score in each positive core and ISUP [International Society of Urological Pathology] grade) and presence or absence of perineural invasion, lymphovascular invasion, intraductal carcinoma, and extraprostatic extension (180). Increasing tumor burden and poor histological differentiation are associated with a higher risk of metastatic disease, an increased chance of post-treatment failure, and a worse overall prognosis (181-183).

Histological analysis is the ‘gold standard’ for classifying prostatic adenocarcinoma. Using architectural patterns, the tumor is assigned a Gleason score and ISUP grade between 1 and 5, with higher numbers representing less differentiated, more aggressive tumors (see Table 7). A single prostate can harbor multiple foci of different histologic patterns of adenocarcinoma, and it is possible to have Gleason grade 3, 4 and 5 patterns in the same specimen: 85% of prostate tumors are multifocal. The Gleason score and ISUP grade are generated by combining the values of the first and second most common (dominant and subdominant) grades assessed by the uropathologist using light microscopy. In needle biopsies, the Gleason score and ISUP grade are calculated using the most common and highest grade of cancer (184). These values provide s important prognostic information.

Table 7. The International Society of Urological Pathology (ISUP) Grading System (185)

ISUP grade	Gleason scores	Definition
Grade 1	2-6	Only individual discrete well-formed glands
Grade 2	3+4=7	Predominantly well-formed glands with lesser component of poorly formed/ fused/ cribriform glands
Grade 3	4+3=7	Predominantly poorly formed/fused/ cribriform glands with lesser component of well-formed glands
Grade 4	4+4=8	Only poorly formed/fused/cribriform glands
	3+5=8	Predominantly well-formed glands and lesser component lacking glands (or with necrosis)
	5+3=8	Predominantly lacking glands (or with necrosis) and lesser component of well-formed glands
Grade 5	9-10	Lacking gland formation (or with necrosis) with or without poorly formed/fused/ cribriform glands

The presence of Gleason grade 4 or greater histology carries a significantly poorer prognosis (186-187). It has been shown that Gleason score 4+3 tumors behave much worse than Gleason score 3+4 tumors and that there is a biological continuum within Gleason score 7 tumors with the proportion of pattern 4 cancer that is reflected in clinical outcome (188).

In the large majority of instances, gray-scale TRUS does not permit differentiation between cancer and non-cancer so TRUS and transperineal biopsies are taken blindly. Consequently, there is a possibility that small tumors may be missed, despite careful spatial positioning of biopsy needles with multiple cores taken. Furthermore, in large glands especially, the anterior part of the prostate may be poorly sampled via the transrectal route so, for these reasons, it is not surprising that the histology from biopsies and radical prostatectomies may differ. In these instances, the Gleason score from the radical prostatectomy specimen is usually higher (upgrading) but downgrading is also observed.

Recently, the International Society of Urological Pathology (ISUP) proposed a new Grading system in order to improve prognostication of tumor grade, as well as improve patient education (184,189-191). Although the term “grade groups”has been used for these prognostic categories, it has been shown to be erroneous as they are not groupings of grades but groupings of scores (184,189). Furthermore, these categories were the result of a consensus conference organized by the ISUP for the purpose updating the ISUP modified Gleason scoring system of 2005 and as such, the new ISUP grades are based on the 2005 ISUP modified Gleason scores (Table 7).

These grades have been validated in surgical cohorts and show distinct patterns of recurrence free progression (RFP) depending on the highest Grade within the RP and biopsy histology (190-191).

PROSTATIC INTRAEPITHELIAL NEOPLASIA [PIN]

Prostatic intraepithelial neoplasia [PIN] is believed to be a precursor of prostate cancer, given the strong association between high grade PIN and prostatic adenocarcinoma (192-194). The presence of high grade PIN is often indicative of the presence of prostate cancer. It has been shown that more than 80 percent of prostates with adenocarcinoma also contain high-grade PIN (PIN-11 & III). High-grade PIN has cytologic features resembling cancer and carries many of the genetic alterations of prostate cancer. The finding of high-grade PIN alone in a biopsy has been cited as an indication to proceed with repeat biopsies given the high co-frequency between high-grade PIN and carcinoma. However, in current practice, the predictive value of PIN in finding cancer on subsequent biopsies has declined, probably due to the extended biopsy techniques yielding higher rates of initial cancer detection (195). A diagnosis of PIN by itself is certainly insufficient for a patient to undergo either radical prostatectomy or radiotherapy.

ATYPICAL PROSTATIC GLANDULAR PROLIFERATIONS

Foci of atypical glands, also labeled ‘atypical small acinar proliferation of uncertain significance’, have features suspicious for, but not diagnostic of, cancer. These encompass a variety of lesions including benign mimickers of cancer, HGPIN, and small foci of carcinoma which, for a variety of reasons, cannot be accurately diagnosed. The reported incidence of these lesions on prostate needle biopsies is 1.5% to 5.3% (195). Patients with atypical glands on needle biopsy have a high risk of harboring cancer. The reported incidence of prostate cancer from repeat biopsies has ranged from 34 to 60% (196). Following an atypical diagnosis, biopsies need to be repeated (197).

TNM STAGING SYSTEM

Once a diagnosis of prostate cancer is made, it must be determined whether the patient is a candidate for potentially curative treatment (surgery or radiation). This depends upon several factors, including general health and projected longevity in conjunction with the likelihood that the cancer is still localized within the prostate and has not yet metastasized. The most important factor, however, is the patient’s decision after he has considered the ‘pros and cons’ of the various choices as they relate to him (see below).

Currently, the TNM system is used for staging (Table 8), and prostate cancers can be assigned both a clinical stageand, subsequently should the prostate be removed surgically, a pathologic stage. This differentiation is important with the clinical and pathological stage designated by the letters ‘c’ and ‘p’, respectively, preceding the stage denotation (e.g. cT2a = clinically, tumor is palpably involving one lobe of the prostate or less).

Table 8: TNM Staging Classifications [per American Joint Committee on Cancer (AJCC) 8th Edition 2016)(198)

Primary Tumor
Tx T0	Primary tumor cannot be assessed No evidence of primary tumor
T1	Clinically inapparent tumor not palpable not visible by imaging
T1a	Incidental tumor in < 5% of TUR tissue
T1b	Incidental tumor in > 5% of TUR tissue
T1c	Needle biopsy prompted by elevated PSA
T2	Organ confined
T3	Tumor extends beyond the prostatic capsule
T3a	Extracapsular, unilateral and bilateral or microscopic invasion of bladder neck
T3b	Tumor invades seminal vesicles (s)
T4	Tumor invades external sphincter, rectum, pelvic side wall
Lymph Nodes
Nx N0	Regional nodes were not assessed No regional (below level of bifurcation of common iliac arteries) nodes
N1	Regional node metastases – including pelvic, hypogastric, obturator, iliac, sacral
Distant Metastases
Mx M0	Regional nodes not assessed No Metastases
M1 M1a M1b M1c	No distant Non-regional lymph nodes (outside true pelvis) Bone(s) Other site(s) with or without bone disease

POTENTIAL BENEFITS & HARMS FROM PSA TESTING

One of the most contentious topics in medicine is whether or not to test for prostate cancer. The key question that needs to be answered is whether a diagnosis of prostate cancer is going to benefit the patient with the qualification that the diagnostic process and treatment should not be worse than the unwanted effects of the disease. Determining who will benefit from testing is very difficult as it is impossible to know exactly how long an individual patient will live and generally both patients and clinicians tend to be optimistic in their estimations.

Early Diagnosis and Treatment With Curative Intent And Prevention Of Subsequent Death From Prostate Cancer

In addition to attributing a slow but continuing reduction in prostate cancer mortality in many Western countries to, at least in part, widespread PSA testing, most of the evidence proffered in support is from low-level cohort studies, many of which have been retrospective. One notable, largestudy undertaken prospectively has been in the Tyrol. Unlike in the rest of Austria, PSA testing has been freely available in Tyrol since 1993 for men 45-75 years with 86.6% of eligible men having been tested at least once since its inception (199). Compared with the rest of the country, there has been a decreasing trend in prostate cancer mortality which, in 2005, was significantly greater in the Tyrol compared with the rest of Austria (P = 0.001). Prostate cancer deaths were 54% lower than expected in this region compared with the rest of Austria, with a significant migration to lower stage disease. These better results in Tyrol have been attributed to early detection, consequent down-staging and effective treatment.

However, the evidence for and against PSA screening is usually based on the findings from 6 mass or whole of population screening trials and meta-analyses of their findings. These studies were the Prostate Lung, Colorectal and Ovarian (PLCO) Screening Trial (200-201), the European Randomized Study of Screening for Prostate Cancer (ERSPC)(48) (202), Göteborg (203), Norrköping (204), Stockholm (205)and Quebec trials (206).

The studies were very different in design and in adherence to protocols. For example, men were invited only once in Stockholm Study and a minority of those with screen-detected prostate cancer were treated with curative intent (205). The participation rate was only 24% in the Quebec study (206). The Norrkoping Study commenced in 1987 with DRE as the only screening test performed up to the third (1993) and the final fourth screening time (1996) when PSA was included. Fewer than 500 men had two PSA measurements & none had more than two. Furthermore, final results were adjusted for the large difference in age at randomization between the study groups (204).

Thus, in terms of trials with reasonable rigor, there are only 3 viz. the ERSPC, the Göteborg (which is also included as part of the larger ERSPC study) and the PLCO trial (Table 9). In the PLCO trial only 85% in the screening arm had a PSA test. In addition, more than 80% in the control arm reported having a PSA test, significantly contaminating this arm (200,207). Furthermore, the follow-up for these trials varied greatly with only one (Göteborg) having an adequate median follow-up period, detailed below.

PLCO: median 11.5 years, maximum 13 years (201)

ERSPC: median 9.8 years, maximum 11 years (202)

Göteborg: median 14 years, maximum 14 years (203)

Norrköping: median 6.3 years, maximum 20 years (204)

Stockholm: median 12.9 years, maximum 15 years (205)

Quebec: median 7.9 years, maximum 13 years (206)

Table 7: Comparison of ERSPC, PLCO and Göteborg Trials

	ERSPC	PLCO	Göteborg
Number studied	162 243	76,693	20,000
Recruitment sites	8 countries	10 US centers	one
Age	50-69	55-74	50-64
PSA screening interval	4 yearly	yearly x6 DRE x4	2 yearly
Biopsy trigger	3.0 ng/ml	>4 ng/ml	3.4, 2.9, 2.5 ng/ml
Contamination rate (PSA testing in control group)	15%	52%	3%

Since the studies are so different in so many ways, the validity of including them in a meta-analysis has been questioned (208). Given the long natural history of prostate cancer in comparison with those of other malignancies and the prevalence of the diseasewith increasing age, few would advocate screening each and every member of a population (209-211)i.e. mass population screening as reported in these trials

SUMMARY OF MORTALITY FINDINGS FROM THE THREE MOST RELEVANT STUDIES

None of these trials had adequate statistical power to detect an overall survival benefit with PSA screening
Deaths from conditions other than prostate cancer dominated causes of death undermining ability to show an advantage for PSA screening

PLCO- At a median follow-up of 11.5 years, of 76 685 men randomized (38,340 in the intervention arm and 38,345 in the control arm) (201). Approximately 92% of the study participants were followed for 10 years and 57% for 13 years.

- deaths from all causes other than prostate, lung, and colorectal cancers were 5783/38,340 (15%) in the intervention arm: 5982/38 345 (15.6%) in the control arm

- of those who died, 158/5783 (2.7%) & 145/5982 (2.4%) in the control arm, died from prostate cancer, respectively

- cumulative mortality rates from prostate cancer in the intervention and control arms were 3.7 and 3.4 deaths per 10 000 person-years

ERSPC- At a median follow-up of 11 years, 31,318 of 162,388 (19.3%) of men between 55 & 69 yr. who underwent randomization had died [154] (202)

- 13,917/72,891 (19%) in screening group: 17,256/89,352 (19%) in control group

- of those who died, 299/13,917 (0.4%) & 462/17,256 (0.5%) died from prostate cancer, respectively

- the absolute reduction in mortality in the screening group was 0.10 deaths per 1000 person-years or 1.07 deaths per 1000 men who underwent randomization.

- to prevent one death from prostate cancer at 13 years of follow-up, 781 men would need to be invited for screening and 27 cancers would need to be detected (212)

Göteborg- At a median follow-up of 14 years, 3,963 of 20,000 (19.8%) of men between 50 & 64 who underwent randomization had died (203)

- 1981/10,000 (19.8%) in the screening group and 1982/10,000 (19.8%) in the control group died

- of those who died, 44/1981 (2.2%) & 78/1982 (3.9%) died from prostate cancer, respectively

overall the relative risk reduction in mortality was 44% for men randomized to screening compared with controls at 14 years.
Overall, 293 men needed to be invited for screening and 12 to be diagnosed to prevent one prostate cancer death

Overall, the benefits of early detection of prostate cancer increase with time.

Findings are based exclusively on systematic reviews (meta-analyses) of 6 randomized controlled [RCTs] PSA screening trials with 8 systematic appraisals of these RCTs but

RCTs are not the only form of evidence: absence of RCT evidence does not equal evidence of absence

These were mass population screening trials – no patient selection - as opposed to opportunistic & selective screening (which most people advocate)

Recently the ERSPC and PLCO data were analyzed considering implementation and practice settings between the trials, which estimated a similar effect between the trials and that screening conferred a 7-9% reduction in prostate cancer specific mortality per year and 26-31% lower risk of prostate cancer death with screening (213). This analysis has been reported to conclude that PSA screening reduced prostate cancer mortality; however the optimal screening strategy is yet to be determined or implemented to maximize benefit and reduce risk (214). One recently proposed strategy (as discussed above) has been based on a PSA level at age 60, suggesting that men with PSA <1 ng/mL at age 60 require no further screening, while men with PSA levels ≥2 ng/mL can expect a large reduction in cancer mortality, resulting in an estimated 23 men needing to be screened and six diagnosed to avoid one prostate cancer death by 15 years (215).

Survival Estimation

There are several approaches that can be used to improve a rough clinical estimation of a patient’s life-expectancy. Validated instruments are available such as a modified form of the Total Illness Burden Index for prostate cancer by Litwin (216)and the Charlson Comorbidity Index, which seems to be most useful in men<65 years undertaking initial treatment, in particular radical prostatectomy (217-218). Although these are not used commonly in clinical practice, they do provide one option. Froehner et al (2013) recently examined available comorbidityassessments to determine which may best assist in the treatment choice for elderly men with prostate cancer. A total of 1,106 men aged 65 years or older who underwent radical prostatectomy for clinically localized prostate cancer was examined with overall survival as the study endpoint. They concluded that the American Society of Anesthesiologists (ASA) physical status classification tool, supplemented by a list of more clearly defined concomitant diseases, could be useful in clinical practice and outcome studies (219).

Another approach is to refer to Life Expectancy Tables (such as the Table 10 below modified from the Australian Bureau of Statistics website 2017). Such tables do not take into account an individual’s comorbidities.

Table 10: Life Expectancy Table for Australia

Age	2000-2002	2004-2006	2010-2012	2014-2016	Age	2000-2002	2004-2006	2010-2012	2014-2016
35	44.08	45.17	46.1	46.6	68	15.14	15.97	16.8	17.2
36	43.14	44.22	45.2	45.6	69	14.42	15.23	16.0	16.5
37	42.20	43.27	44.2	44.7	70	13.72	14.51	15.3	15.7
38	41.25	42.32	43.3	43.7	71	13.04	13.80	14.5	14.9
39	40.31	41.37	42.3	42.8	72	12.38	13.10	13.8	14.2
40	39.37	40.43	41.4	41.8	73	11.74	12.42	13.1	13.5
41	38.43	39.49	40.4	40.9	74	11.11	11.76	12.4	12.8
42	37.49	38.55	39.5	39.9	75	10.51	11.12	11.7	12.1
43	36.56	37.61	38.6	39.0	76	9.92	10.50	11.0	11.4
44	35.63	36.68	37.6	38.1	77	9.36	9.90	10.4	10.7
45	34.70	35.74	36.7	37.1	78	8.82	9.32	9.8	10.1
46	33.78	34.82	35.8	36.2	79	8.29	8.76	9.2	9.5
47	32.86	33.89	34.8	35.3	80	7.79	8.22	8.6	8.9
48	31.94	32.98	33.9	34.4	81	7.31	7.70	8.0	8.3
49	31.02	32.06	33.0	33.5	82	6.84	7.21	7.5	7.7
50	30.11	31.15	32.1	32.5	83	6.40	6.75	7.0	7.2
51	29.21	30.24	31.2	31.6	84	5.98	6.31	6.5	6.7
52	28.30	29.34	30.3	30.7	85	5.59	5.90	6.1	6.2
53	27.41	28.45	29.4	29.8	86	5.23	5.50	5.7	5.8
54	26.52	27.55	28.5	29.0	87	4.90	5.12	5.3	5.4
55	25.64	26.67	27.6	28.1	88	4.61	4.77	4.9	5.0
56	24.76	25.79	26.7	27.2	89	4.34	4.45	4.6	4.6
57	23.90	24.92	25.9	26.3	90	4.10	4.17	4.3	4.3
58	23.05	24.05	25.0	25.5	91	3.89	3.92	4.0	4.0
59	22.20	23.20	24.1	24.6	92	3.69	3.71	3.8	3.7
60	21.37	22.35	23.3	23.8	93	3.51	3.53	3.5	3.5
61	20.55	21.51	22.4	22.9	94	3.34	3.37	3.3	3.2
62	19.73	20.69	21.6	22.1	95	3.18	3.24	3.1	3.0
63	18.94	19.87	20.8	21.3	96	3.03	3.13	2.9	2.8
64	18.15	19.07	20.0	20.4	97	2.89	3.04	2.7	2.6
65	17.37	18.27	19.1	19.6	98	2.76	2.94	2.6	2.5
66	16.61	17.50	18.3	18.8	99	2.65	2.84	2.4	2.3
67	15.87	16.73	17.6	18.0

In terms of likelihood of dying from cardiovascular disease, whether or not a man has started to have erectile dysfunction may serve as a surrogate indicator. One recent large study indicated that the median time to death from a cardiovascular cause from the onset of erectile dysfunction (ED) was 10 years (220)since the reason for ED in the majority of cases is impaired arterial flow (221).

Factors To Consider When Deciding To Test For Prostate Cancer

In the Scandinavian randomized trial of Radical Prostatectomy & watchful waiting. At a median follow-up of 13.4 years, 63 in the surgery group and 99 in the watchful-waiting group died from prostate cancer; the relative risk was 0.56 (95% confidence interval [CI], 0.41 to 0.77; P=0.001). The number needed to treat to prevent one death due to prostate cancer was 8 (222)

The benefit of surgery with respect to death from prostate cancer was largest in men younger than 65 years of age (relative risk, 0.45) and in those with intermediate-risk prostate cancer (222)

At a median of 12.8 years of follow-up in an earlier report on this trial, men with more than 2 significant co-morbidities did not benefit from PSA testing (223)

In a follow up analysis of the PLCO study, there was a striking mortality benefit in men with minimal or no co-morbidities a 44% drop in prostate cancer-specific mortality and a number needed to treat of only 5. However, for men with at least one significant co-morbidity, there was no significant difference in prostate cancer mortality (224)

But what constitutes a significant comorbidity? “a condition or complaint either coexisting with the principal diagnosis or arising during the episode of care or attendance at a health care facility” (225). How do you assess it?

Crawford et al chose an expanded definition that included both ‘standard’ Charlson comorbidity index conditions and hypertension (even if well controlled), diverticulosis, gallbladder disease and obesity (224)

But when the analysis was repeated using only validated measures of comorbidity (Charlson comorbidity index conditions only), there was no interaction (226)

A simple patient-reported index, a modified form of the Total Illness Burden Index modified for prostate cancer (216)vs Charlson Comorbidity Index (217-218)

The American Society of Anesthesiologists (ASA) physical status classificationhas been recommended to serve as a basis of assessing suitability for radical prostatectomy in men >65 years (219)

Onset of erectile dysfunctionmay serve as an indicator of limited life expectancy due to cardiovascular death (220-221)

Morbidity of (frequently repeated) TRUS & T/P biopsies TRUS biopsy infections in 4.5%: 48% had rectal swabs showing Ciprofloxacin resistant bacteria (165,167,169)

High over-diagnosis rate: active surveillance, where men diagnosed with low risk prostate cancer may be monitored with serial PSA and biopsies to delay or avoid treatment, may decrease the concern of over detection and over treatment

Psychosocial aspects pervade all aspects of detection & treatment.

Recent studies have reported psychological distress levels severe enough to meet defined criteria close to the time of diagnosis from 10% to 23% (227). Bill-Axelson and colleagues in an eight year longitudinal study reported that although extreme distress was not common in men with localized prostate cancer, 30–40% of men reported ongoing health-related distress and worry about their health, feeling low, and sleep disturbance (228). Risk of suicide may be increased in the first six to twelve months after the diagnosis of prostate cancer (229-230). Screening for distress and referral to appropriate support services is widely accepted and recommended in men diagnosed with prostate cancer (231-232). In addition to distress, contemporary evidence suggests socio-demographic and psychosocial variables to be highly influential on intervention effects (232). Decisional conflicts impact upon continuation of Active Surveillance (233-234). When making decisions about treatment for prostate cancer men tend to rely on lay beliefs about cancer with the opinion of the clinician highly influential (233). A systematic review of psychosocial interventions for men with prostate cancer and their partners found that group cognitive-behavioral and psychoeducational interventions were helpful in promoting better psychological adjustment and quality of life (QOL) for men with prostate cancer (235). Multi-modal psychosexual and psychosocial interventions for men diagnosed with prostate cancer are recommended(232).

Reassurance: PSA level <1ng/ml at the age of 65 years (50)or <3 ng/ml at the age of 75 years have a very low chance of contracting fatal cancer (160)

EARLY DIAGNOSIS AND TREATMENT WITH CURATIVE INTENET AND LESSENING THE LIKELIHOOD OF METASTASES OCCURRING

The recently completed PSA Evaluation Report by the National Health and Medical Research Council (NHMRC) of Australia concluded that, although there was some inconsistency in the definition of prostate cancer metastases across the RCTs, overall, the evidence indicates that PSA testing reduces the risk of having metastases present at the time of diagnosis of prostate cancer. The NHMRC review focused on the RCTs above in its considerations but did not conclude that intervention with curative intent reduces the likelihood of subsequent metastases [163]. However, evaluation of evidence from multiple non-RCTs has reported that PSA testing and intervention with curative intent does reduce the likelihood of subsequent metastases.

There are very few RCTs for prostate cancer treated with curative intent. Bill Axelson et al (2014) (222)recruited patients from 14 centers in Sweden, Finland and Iceland: The trial is noteworthy since the study included patients detected with prostate cancer at a later stage than is currently diagnosed: only 12% had impalpable disease on DRE - detected by what are now outmoded methods. The results are summarized below:

Swedish Trial Of Radical Prostatectomy Versus Watchful Waiting (222,236)

From October 1989 through February 1999, 695 men with ‘early’ prostate cancer were randomly assigned to watchful waiting, where men are “watched” and treated only when symptomatic or with significant concern for complications, or radical prostatectomy
Eligibility required patients to be
<75 yrs. of age and a life expectancy >10 years: mean age was 65 yrs.
Clinically localized disease (T1 or T2, using IUCC 1978 criteria)
Diagnosis by core biopsy or fine needle aspiration cytology
Well or moderately differentiated adenocarcinoma (WHO classification)
PSA <50 ng/ml: mean PSA was 13 ng/ml
a negative bone scan
During a median of 13.4 years, 200 of the 347 men in the radical prostatectomy group and 247 of the 348 in the watchful-waiting group died (222). In the case of 63 men assigned to surgery and 99 men assigned to watchful waiting, death was due to prostate cancer (P = 0.001)
The survival benefit was largest in men younger than 65 years of age and those with intermediate-risk prostate cancer
The number needed to treat to avert one death at 18 years of follow-up was 8 (P=0.001) and 4 for men younger than 65 years of age (222)
Among men who underwent radical prostatectomy, those with extracapsular tumor growth had a risk of death from prostate cancer that was 7 times that of men without extracapsular tumor growth (236)
Distant metastases were diagnosed in 89 men in the radical prostatectomy group and 138 in the watchful waiting cohort resulting in a relative risk of metastases in the RP group of 0.57 (P <0.001) (222)

However, by contrast, in the Prostate Cancer Intervention versus Observation Trial (PIVOT) of radicalprostatectomy versus observation for localized prostate cancer found differently (237). Between November 1994 and January 2002, 731 men with localized prostate cancer (mean age, 67 years; median PSA value, 7.8 ng per milliliter) were randomly assigned to radical prostatectomy or observation and followed to January 2010. The primary outcome was all-cause mortality; the secondary outcome was prostate-cancer mortality

During the median follow-up of 10.0 years, 171 of 364 men (47.0%) assigned to radical prostatectomy died, compared with 183 of 367 (49.9%) assigned to observation (P=0.22). Among men assigned to radical prostatectomy, 21 (5.8%) died from prostate cancer or treatment, compared with 31 men (8.4%) assigned to observation (P=0.09). The effect of treatment on all-cause and prostate-cancer mortality did not differ according to age, race, coexisting conditions, self-reported performance status, or histological features of the tumor. Radical prostatectomy was associated with reduced all-cause mortality among men with a PSA value greater than 10 ng per milliliter (P=0.04 for interaction) and possibly among those with intermediate-risk or high-risk tumors (P=0.07 for interaction). Adverse events within 30 days after surgery occurred in 21.4% of men, including one death. In 2017, Wilt et al updated the results reporting that after nearly 20 years of follow-up among men with localized prostate cancer: surgery, was not associated with significantly lower all-cause or prostate-cancer mortality than observation.

However, there were serious deficiencies with the PIVOT study (238). Although the three Endpoints Committee members were blinded to randomized treatment assignments, reviewed medical records and death certificates when available to assign a cause of death using a primary and a secondary adjudication question, initial disagreements were resolved through discussion. Complete agreement on cause of death by all three committee members before any discussion was achieved in 200/354 (56%) cases on the primary and 209/354 (59%) cases on the secondary. Complete agreement on the primary cause rose to 306/354 (86%) when ‘definite’ and ‘probably’ categories were collapsed, as planned a priori. There was no separate ‘gold standard’ by which to judge the accuracy of the final endpoints committee adjudications, and useful death certificates could not be obtained on about a third of PIVOT participants who died.

U.S. PIVOT – Radical Prostatectomy Versus Observation (237)

Recruitment difficulties and patient compliance issues affected numbers so that only 731 of the proposed 2000 men could be recruited to the trial and hence this study is considered to be underpowered to detect a difference in overall survival (239)
Serious lack of agreement on cause of death by Endpoint Committee Members: Useful death certificates could not be obtained for approximately one third of participants
Differences between histological reporting at participating sites and by a central pathologist affected risk stratification and, consequently, secondary endpoint results
A less predictive pre-2005 ISUP Consensus Gleason classification was used with ~25% of patients with Gleason scores of 7 or higher reported at the peripheral sites compared with 48% with Gleason scores 7 or higher by a central pathologist

Consequently, the answer based on RCT evidence remains uncertain.

Early Diagnosis And Treatment With Curative Intent: Avoiding The Late Clinical Problems Resulting From A Large Pelvic Tumor

There is a paucity of high level evidence that early diagnosis of prostate cancer will prevent or minimize the problems resulting from a large pelvic tumor. Anecdotally, managing patients with disabling symptoms from advanced local prostate cancer constituted a considerable part of a urologist’s workload. Frequent visits to hospital for interventions together with burden of clinical symptoms such as unremitting day and night frequency, incontinence and bleeding, impact significantly on the dignity and quality of life of these men (240).

Figure 17: CT of pelvis showing prostatic tumor extending into the (thick-walled) bladder and spread to involve pelvic lymph nodes: the patient had multiple lower urinary tract symptoms

WHETHER TO TEST FOR PROSTATE CANCER

Prostate cancer is the most common male visceral malignancy in the developed world and the second most common cause of cancer deaths, uncertainties remain about management practices at several points in the illness continuum. For example, owing to controversies regarding the outcomes of screening trials for prostate cancer reducing the death rate from this disease, population-based screening for prostate cancer in asymptomatic men is not currently recommended in most countries (241). Rather, it is suggested that men should be able to access PSA testing as long as they are fully informed of the pros and cons of testing.

The Prostate Cancer Foundation of Australia (PCFA), in partnership with National Health and Medical Research Council (NHMRC) and Cancer Council Australia, published a “PSA Testing for Prostate Cancer in Asymptomatic Men” guideline in 2016, which was commissioned by the Department of Health and comprised a multi-disciplinary expert advisory panel. The guidelines have been endorsed by the Urological Society of Australia and New Zealand (USANZ) and the Royal Australian College of General Practitioners (RACGP). The recommendations are as follows:

A population screening program for prostate cancer (a program that offers testing to all men of a certain age group) is not recommended
Men should be offered evidence-based decision support, including the opportunity to discuss the benefits and harms of PSA testing before making the decision to be tested
Men at average risk of prostate cancer who decide to be tested should be offered PSA testing every 2 years from age 50 to 69
The harms of PSA testing may outweigh the benefits for men aged 70 and older or those with a life expectancy less than 7 years.
Men with a family history of prostate cancer who decide to be tested should be offered PSA testing every 2 years from age 40/ 45 to 69 with the starting age depending on the strength of their family history
Digital rectal examination is not recommended in addition to PSA testing in the primary care setting

The full guideline can be accessed at www.pcfa.org.au/psa-testing-guidelinesor https://wiki.cancer.org.au/australia/Guidelines:PSA_Testing

The approach that is considered to be optimal for achieving high quality patient decisions is shared decision making (242).

Shared decision makingis defined as a process carried out between a patient and his health care professional where both parties share information and the patient understands the risks and benefits of each treatment option, participates in the decision to the extent that he desires and makes a decision consistent with his preferences and values, or defers the decision to another time(243).

Shared decision making may not be easy to achieve for all patients (243). For example, although many patients with cancer indicate a preference for sharing decision making with their clinicians, some, in the case of prostate cancer between 8% to 58% of men, prefer a passive decision-making role where clinicians make treatment decisions on their behalf (244-245). However, clinicians still need to understand patients’ preferences to ensure that they are making quality decisions on behalf of their patients. As well, there is often a gap between the clinical ideal of shared decision making and actual clinical practice where decision complexity and time constraints may make this approach difficult for both parties to achieve (246-247). There are, however, defined strategies and decision aids that can facilitate this process (248).

Supporting Patient Choice About Testing For Prostate Cancer

Many groups advocate an informed decision-making process as an evidence-based approach and necessary precursor to screening for early prostate cancer (241,249). Others have suggested that informed decision-making on this health topic is also necessary as a medico-legal risk management strategy (250-251). While some researchers have suggested a set of information that needs to be communicated to men about this health decision (252-253), there are few explicit guidelines on this subject (254). Problematically, patients and clinicians do not agree on core content, including a basic explanation of a PSA test and the psychological effects of a positive PSA test result (255). It has been advised that, for any screening test, patients need to understand the purpose of the test, the likelihood of false-negatives and false-positives, the uncertainties and risks associated with testing, significant medical, social or financial implications of testing and any possible sequelae and follow up care plans (256)www.ipdas.ohri.ca.

Such information needs to be communicated to patients in a logical and balanced sequence in order to promote better understanding and increased decisional control by men. One approach that has been proposed in primary care in Australia is the use of six decision steps (see Figure 18). Each decision step logically follows to prompt the clinician to overview important health information, with tailoring suggested in Step 1 to ensure the discussion is consistent with the patient’s concerns. For example, for a man with a significant family history of prostate cancer, this factor is likely to be central to the patient discussion (257). Men who experience uncomplicated LUTS often worry about prostate cancer, so addressing this concern first may be priority (258-259). In this regard, resources for patients that explain about male reproductive health problems such as urinary symptoms and sexual dysfunction are available at www.andrologyaustralia.org. As well, National Health and Medical Research Council guidelines are available about the management of LUTS http://www.health.gov.au/nhmrc/publications/synopses/cp42syn.htm

Other overseas websites include:

Figure 18: Six Decision Steps

From this point, checking to ensure the patient has a basic understanding of both the prostate and possible tests is needed and, given many men may be unaware of the location and function of the prostate gland, an anatomical diagram may be a useful teaching tool here. Next, a consideration of individual risk with regard to both the incidence and mortality of prostate cancer is needed. Communicating health risks effectively is a challenge in the provision of effective decision support. In general people find probabilities hard to understand, often estimate their level of risk incorrectly, and tend not to weigh up pros and cons in a systematic way when deciding about treatments (234,260-261). As well, population-based statistics provide data about populations, not individuals, so risk communication needs to acknowledge this as a limitation and, where possible, refer to age-based risk estimates and relevant individual factors such as family history) (262).

There are a number of communication strategies that have been suggested to help patients understand risk. These include

using numbers as well as words to explain risk
where possible providing the absolute risk or benefit
using frequencies rather than single event probabilities
using consistent denominators
putting the risk into context by comparing it to other life events
offering both the possible negative and positive outcomes to balance the message frame (263-265).

However, a quality health decision goes beyond the simple transfer of information and includes consideration and incorporation of each patient’s values and personal preferences (266). Thus, Step 5 in Box 1 prompts the clinician to discuss each man’s individual preferences. A number of strategies can be used to do this, most commonly the use of a pros and cons exercise in which patients are encouraged to explicitly consider the factors that matter most to them personally in this decision, and the direction and leaning of their preferences either for or against each possible option. One approach to support this process for this health topic is the inclusion of a values table within a decision card (see Table 11). A decision aid that incorporates both the six decision steps and this values clarification exercise can be found on the Andrology Australia website at: http://www.prostate.org.au/articleLive/attachments/1/GP%20Show%20Card%20041007.pdf

Table 11: What is most important to you?

*FOR: Is this like you?*	*AGAINST: Is this like you?*
I’m concerned that I might get prostate cancer	I think my chance of getting prostate cancer is low
I want the best chance of finding it early, if I do get it	I am not convinced about the effectiveness of testing
I’m not interested in waiting for all the proof to be in	I am more concerned about avoiding treatment side effects, if there’s no guarantee I’d be reducing my risk of dying from prostate cancer
I want to do everything possible to reduce my risk of dying from prostate cancer

Decision aids are also effective in supporting patients to make informed choices. With regards to PSA testing, patient-focused decision aids and decision counselling or support interventions have been found to be effective in increasing men’s knowledge about PSA testing and decreasing decision-related distress (254,267-270), with a variable effect on actual testing behavior.

A range of aids is freely available from the web (www.prostatehealth.org.au; www.cdc.gov/cancer/prostate;www.cancerbacup.org.uk).

Cancer helplines also often provide such information, for example:

The Cancer Council Australia Cancer Helpline on 13 11 20;
the UK helpline on 0808 800 1234;
the USA Cancer Helpline on 1800 227 2345.

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Hyponatremia

CLINICAL RECOGNITION

Hyponatremia is common, being found in some 15–20% of non-selected emergency admissions to hospitals. It is associated with increased mortality, morbidity and increased duration of hospital stay, independent of the cause of admission. Clinical presentation is diverse, ranging from seizure and coma at one extreme to apparent absence of symptoms. This spectrum reflects a number of influences (Figure 1).

- The rate of development of hyponatremia

- The degree of hyponatremia

- The neurophysiologic adaptive capacity of the individual

- The influence of co-morbidities

The management strategy is stratified, based on an overall assessment of the severity of the clinical presentation.

Plasma sodium concentration reflects the balance between sodium and water content, with each component reflecting the balance of intake and output. This approach can be used to develop a framework for classifying hyponatremia by etiology (Table 1).

Intravascular Volume Depletion (Hypovolemia)

Intravascular volume depletion leads to non-osmoregulated vasopressin (AVP) production and reduced free water excretion. AVP production can persist despite ensuing hyponatremia. Portal hypertension, congestive cardiac failure and hypoalbuminemia can all reduce effective circulating volume (independent of drug treatment), even in the context of excess total body sodium. Long-term diuretic use is a common cause of hyponatremia. Because it may develop slowly, patients can be relatively asymptomatic. Those on thiazide diuretics are particularly at risk as these agents produce solute loss without limiting renal concentrating ability.

Excess Hypotonic Fluid Intake

The administration or absorption of hypotonic fluids at a rate that exceeds renal free water excretion will inevitably result in hyponatremia. This can be seen with oral fluid intake (dipsogenic diabetes insipidus), intravenous fluid therapy and the absorption of hypotonic irrigating fluids following surgery to the lower renal tract or colonoscopy.

Syndrome of Inappropriate Antidiuresis (SIAD)

In SIAD there is a failure to maximally suppress AVP secretion as plasma osmolality falls below the normal osmotic threshold for AVP release. As patients continue to drink, persistent antidiuresis produces dilutional hyponatremia. Diagnosis of SIAD involves the exclusion of volume depletion and other endocrine causes of reduced free water excretion (Table 2).

Table 2. Diagnostic Criteria for SIAD

Hyponatremia

Urine Osmolality >100 mOsm/kg (sub-maximum dilution)

Urine Na+ >20 mmol/L (excluding effective intravascular volume depletion)

Absence of the following:

- hypotension and hypovolemia

- non-osmotic stimuli for AVP release

- oedema

- adrenal failure

- hypothyroidism

The majority of patients with SIAD are euvolemic on clinical examination. Urine sodium concentration is often above 80 mmol/l.

Many drugs cause SIAD. Drug histories are therefore an important part of the clinical assessment of patients presenting with hyponatremia (Table 3).

Table 3. Causes of SIAD
Drugs	- Antidepressants (Tricyclics, SSRIs) - Dopamine agonists (Metoclopramide, Prochlorperazine, Antipsychotics) - Anticonvulsants (Carbamazepine, Phenytoin, Sodium valproate) - Opiates
CNS Disturbances	- Stroke - Haemorrhage - Infection - Trauma
Malignancies	- Small cell lung cancer - Pancreatic, duodenal and head/neck cancers
Surgery	- Abdominal, thoracic or pituitary surgery
Pulmonary disease	- Pneumonia - Pneumothorax
Idiopathic

Central Salt Wasting

This acquired primary natriuresis is a very rare cause of hyponatremia with hypovolemia. The underlying mechanism(s) remains unclear but may involve a number of processes.

- Increased release of natriuretic peptides

- Reduced sympathetic drive

Central salt wasting has been described following a variety of neurosurgical situations. Diagnosis hinges on the natural history of the process.

- The development of hyponatremia, preceded by natriuresis and diuresis

- Clinical and biochemical features of hypovolemia and renal impairment

Central salt wasting is a concern for the neurosurgical patient in whom autoregulation of cerebral blood flow is disturbed such that small reductions in circulating volume can reduce cerebral perfusion. The syndrome of inappropriate antidiuresis (SIAD) can occur in the same group of patients. As management of the two conditions is diametrically opposed, it is important to make the correct diagnosis. Recent data suggest that salt wasting is indeed a very rare cause of hyponatremia in the neurosurgical patient.

Nephrogenic SIAD

The action of AVP on renal water excretion is mediated by the G-protein-coupled type 2 AVP-receptor (V2-R). Loss-of-function mutations of the V2-R are the cause of X-linked nephrogenic diabetes insipidus. The reciprocal phenotype, mutations in the V2-R that are constitutively activating, lead to AVP-independent but V2-R-mediated antidiuresis with persistent hyponatremia. Patients may present in infancy or may remain undetected until adulthood.

DIAGNOSIS AND DIFFERENTIAL

History and examination are key to the clinical approach. They provide insights into the etiology, rate of development, clinical impact, and contributing co-morbidities: all factors important in a patient-focused approach to management (Table 4).

Table 4. Focus of History & Examination in the Patient with Hyponatremia

History

- Fluid loss (e.g. vomiting, diarrhoea)

- Causes of SIAD

- Symptoms of endocrine dysfunction suggestive of hypoadrenalism or hypopituitarism

- Medication/drug use

Examination

- Signs of extracellular volume depletion

- Orthostatic or persistent hypotension.

- Signs of peripheral oedema, or ascites, heart failure, cirrhosis, or renal failure

Laboratory tests provide important information in the differential diagnosis of hyponatremia.

Serum Osmolality

The serum osmolality (which normally ranges from 275 to 290 mOsmol/kg) is primarily determined by the concentration of serum sodium (and accompanying anions). It is reduced in most cases of hyponatremia (hypotonic hyponatremia). In some instances, hyponatremia is not associated with dilute plasma. This is termed non-hypotonic hyponatremia (Table 5).

Urine Osmolality

The normal response to hyponatremia is to suppress AVP secretion, resulting in the excretion of maximally dilute urine with an osmolality below 100 mOsmol/kg. Values above this level indicate an inability to normally excrete free water, indicating secretion and action of AVP.

Urine Sodium Concentration

The urine sodium concentration can be used to distinguish between hyponatremia caused by effective arterial volume depletion (such as in true hypovolemia, heart failure, and cirrhosis) and that caused by euvolemic/hypervolemic hyponatremia (most often due to SIAD).

The combination of urine osmolality and urine Na+ concentration can be used to form a utilitarian algorithm for the investigation and differential diagnosis of hyponatremia (Figure 2).

TREATMENT

While hyponatremia can be life threatening, chronic hyponatremia can be tolerated very well even when profound. This diversity poses a management challenge: clinicians must balance the efficacy of any intervention with that of the potential adverse impact of both the intervention and persisting hyponatremia. Over-rapid correction of hyponatremia can trigger central nervous system osmotic demyelination. If urgent intervention is required, the aim of management should not be to normalise plasma sodium. Rather, it should be achieving a plasma sodium level that reverses or reduces morbidity while minimising the risk of osmotic demyelination. This requires a stratified approach based on clinical presentation: balancing a number of clinical drivers to optimise outcome (Figure 3).

General and Supportive Measures

The appropriate clinical environment for the management of hyponatremia is the one that matches the clinical needs of the patient. Management of hyponatremia in a patient with significant neurological morbidity, in whom plasma sodium is being raised over hours, requires close clinical and biochemical observation. This is best achieved in a high-dependency setting. Cerebral oedema and coma associated with hyponatremia may need supportive management with assisted ventilation.

Patients with Significant Coma or Seizures

Treatment with hypertonic sodium chloride increases plasma sodium concentration both directly and through the ensuing sodium load that increases renal sodium loss with a parallel, obligate renal water loss. 100-150ml boluses of 3% sodium chloride can be used to increase plasma sodium by some 2–4 mmol/L over the initial 2–4 hours reducing intra-cerebral pressure in the acute setting. Thereafter, the increase in plasma sodium should be limited to 10 mmol/L in the first 24 hours. Given the risk of over-correction, a pragmatic strategy is to aim for a rise of 6-8 mmol/L in first 24 hours. After the first 24 hours, the rate of rise of sodium should be limited to no more than 8 mmol/L per day.

Avoidance of over-correction is critical. If over-correction does occur, it is important to seek expert advice and consider actively controlling the rate of rise of plasma sodium with hypotonic fluid. Hypertonic fluid should be stopped when the defined clinical target (such as cessation of seizures) or a sodium concentration of 130 mmol/L is reached, whichever is first. If hypertonic sodium chloride cannot be given safely, it should not be given.

Patients with Mild or Less Severe Symptoms and Signs

The clinician has time to make a diagnosis, identify and address contributing factors if possible, and introduce a cause-dependent intervention. Importantly, the limits on rate of rise of sodium remain the same as for the patient with critical signs and symptoms. The majority of patients will have circulating volume depletion or SIAD. Where these are due to drug treatments, the removal of the causal agent may be sufficient to normalise sodium. There will be some cases where the causal agent cannot be removed or where there is an alternative diagnosis. Plasma sodium may rise faster than expected due to ‘auto correction’ of hyponatremia when an underlying cause has simply been removed e.g. correction of glucocorticoid insufficiency or withdrawal of excess desmopressin. Osmotic demyelination can still occur in these circumstances and so care must be taken to control the rise of plasma sodium such that the rate remains within target.

Fluid Challenge in Hypovolemic Hyponatremia

Mild to moderate hypovolemia can be difficult to diagnose clinically and low urine sodium excretion (<20 or 30 mmol/L) may be unreliable as a diagnostic test in the face of diuretic use or renin-angiotensin system blockade. If volume depletion is suspected, a moderate intravenous fluid challenge with 0.5–1 L N-saline over 2–4 hours may be both diagnostic and therapeutic.

Fluid Restriction in SIAD

Fluid restriction of 0.5–1 L/day is a reasonable initial intervention when excess plasma water is suspected and when the clinical condition is not critical. In patients with primary polydipsia, reduction in fluid intake remains the most reasonable approach. All fluids need to be included in the restriction. As SIAD is associated with negative sodium balance, sodium intake needs to be maintained. Several days of restriction may be required before sodium levels rise and a negative fluid balance needs to be confirmed through appropriate monitoring.

Management of Persistent Hyponatremia

Hyponatremia may persist or recur after initial intervention. It is important that the differential diagnosis is reviewed and the basis for intervention reconsidered.

- In SIAD, fluid restriction may be only partly effective or may prove non-sustainable

- Chronic liver or cardiac dysfunction may persist

- Drug therapy exacerbating hyponatremia may need to continue

Clinical decisions on further management have to balance the merits of incremental intervention with those of tolerating mild, persisting hyponatremia. Treatment aimed at simply raising plasma sodium in the absence of signs or symptoms may be of limited benefit and it is important to consider the clinical ‘trade-offs’ that may be required.

Demeclocycline

Demeclocycline has been used in SIAD. It produces a form of nephrogenic diabetes insipidus and so increases renal water loss, even in the presence of high concentrations of AVP. Treatment is 600–1,200 mg/day in divided doses. There is a lag time of some 3–4 days in onset of action. Dose adjustment needs to take this into account. Photosensitive skin reactions and renal impairment are significant adverse effects and limit clinical utility.

Vasopressin Receptor Antagonists

Vasopressin receptor antagonists (Vaptans) are a rational approach to the management of SIAD. They are classified as either selective (V2-R specific) or non-selective (V2- and V1a antagonism). Both increase renal water excretion without a significant impact on renal electrolyte loss (aquaretic action). To avoid precipitating a rapid rise in plasma Na+, fluid restriction should be relaxed if these drugs are introduced. They should not be used in profound hyponatremia or in patients with severe symptoms. Their role in the management of SIAD in the majority of patients remains unclear.

GUIDELINES

Spasovski G, Vanholder R, Allolio B et al. Clinical practice guideline on diagnosis and treatment of hyponatremia. European Journal of Endocrinology 2014; 170: G1–G47
Verbalis JG, Goldsmith SR, Greenberg A, Korzelius C, Schrier RW, Sterns RH, Thompson CJ. Diagnosis, evaluation and treatment of hyponatremia: expert panel recommendations. The American Journal of Medicine 2014;126: S1-S42

REFERENCES

Hannon MJ, Behan LA, Brien MMC, Tormey W, Ball S, Javadpour M, Sherlock M, Thompson CJ. Hyponatremia following mild/moderate subarachnoid haemorrhage is due to SIAD and glucocorticoid deficiency and not cerebral salt wasting. Journal of Clinical Endocrinology and Metabolism 2014; 99: 291-298
Ward FL, Tobe SW, Naimark DMJ. The Role of Desmopressin in the Management of Severe, Hypovolemic Hyponatremia: A Single-Center, Comparative Analysis. Canadian Journal of Kidney Health and Disease 2018; 5: 1–8
Ball SG. The Neurohypophysis: Endocrinology of Vasopressin and Oxytocin. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2017 Apr 22. PMID: 25905380

ABSTRACT

INTRODUCTION

MOLECULAR MECHANISMS OF GLUCOCORTICOID ACTION

SYNTHETIC GLUCOCORTICOIDS

Table 1: Glucocorticoid Equivalencies (11, 20, 21)

SYSTEMIC GLUCOCORTICOID ADMINISTRATION

Therapeutic Indications

Adverse Effects (AEs)

ADRENAL INSUFFICIENCY (AI)

CARDIOVASCULAR DISEASE

GLUCOSE HOMEOSTASIS

INFECTIOUS EVENTS

OSTEOPOROSIS

NEUROPSYCHIATRIC EVENTS

PEDIATRIC EVENTS

PHEOCHROMOCYTOMA CRISIS

Table 2: Common AEs of Glucocorticoid Therapy (88)

COMPARTMENTAL GLUCOCORTICOID ADMINISTRATION

Topical Glucocorticoids

Opthalmic Glucocorticoids

Inhaled Glucocorticoids

Nasal Glucocorticoids

Intraarticular Glucocorticoids

MONITORING OF PATIENTS ON GLUCOCORTICOID TREATMENT

CONCOMITANT USE OF GLUCOCORTICOIDS WITH OTHER DRUGS

Table 3: Interactions of Glucocorticoids with Other Drugs (20)

Table 4: Effects of Glucocorticoids on Blood Levels of Other Drugs (20)

Table 5: Effect of Drugs on Plasma Glucocorticoid Concentrations (20, 127, 128)

PREDICTING GLUCOCORTICOID-INDUCED HPA AXIS SUPPRESSION

Kind of Steroid Used and GC Potency

Systemic Versus Compartmental Therapy

Daily Therapy

Split Doses and Night Doses

Duration and Cumulative Dose of Glucocorticoid Treatment

Cushingoid Features

WEANING PATIENTS FROM GLUCOCORTICOID THERAPY

ACUTE ADRENAL CRISIS

GLUCOCORTICOID WITHDRAWAL SYNDROME (GWS)

BIOCHEMICAL DIAGNOSIS OF ADRENAL INSUFFICIENCY

FUTURE PERSPECTIVES ABOUT GLUCOCORTICOID THERAPY

REFERENCES

CLINICAL RECOGNITION

Table 1. Clinical and Genetic Features of Familial Syndromes Associated with ACC

Table 2. Signs and Symptoms of ACC and Recommended Testing for Confirmation of Hypersecretory Syndromes

PATHOPHYSIOLOGY

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS

HISTOPATHOLOGICAL DIAGNOSIS

PROGNOSIS

Table 3. Staging system for adrenocortical carcinomas proposed by the International Union against cancer (WHO 2004) and the European Network for the study of adrenal tumors (ENSAT).

THERAPY

Surgery

Mitotane

Cytotoxic Chemotherapy

Radiation Therapy

Evolving Therapies

FOLLOW-UP

GUIDELINES

REFERENCES

CLINICAL RECOGNITION

Table 1: Use of Glucocorticoid Therapy in Clinical Practice

PATHOPHYSIOLOGY

DIAGNOSIS and DIFFERENTIAL DIAGNOSIS

Table 2: Predictors of Glucocorticoid-Induced HPA Axis Suppression

Table 3: Examples of Different Glucocorticoid-Induced HPA Axis Suppression

Table 4: Glucocorticoid Equivalent Dose Compared to Cortisol

DIAGNOSTIC TESTS NEEDED TO DOCUMENT AI

Table 5: Diagnostic Tests Used to Diagnose Adrenal Insufficiency

Table 6: Diagnostic Tests Not Commonly Used to Diagnose and Differentiate Adrenal Insufficiency

THERAPY

Table 7: Tapering After a Long-Term Glucocorticoid Regimen

FOLLOW-UP

GUIDELINE

REFERENCES

CLINICAL RECOGNITION

PATHOPHYSIOLOGY

DIAGNOSIS and DIFFERENTIAL

Diagnosis

Differential Diagnosis

Table 1. Causes of Acute Renal Colic

Table 2. Differential Diagnosis of Acute Non-colicky Renal Pain