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Amiodarone Induced Thyrotoxicosis

CLINICAL RECOGNITION

 

Patients treated with amiodarone for a cardiac arrhythmia may develop amiodarone Induced thyrotoxicosis (AIT). The risk of AIT is increased in iodine-deficient regions. The incidence of AIT varies greatly (between 0.003% and 10%). AIT occurs in 3% of patients treated with amiodarone in North America, but is much more frequent (up to 10%) in countries with a low iodine dietary intake. In contrast to the other forms of hyperthyroidism, AIT is more frequent in males than in females (M/F = 3/1).

 

AIT manifests with clinical signs indistinguishable from spontaneous hyperthyroidism, however symptoms and signs of thyrotoxicosis are not apparent in all patients, and may be obscured by an underlying cardiac condition. The reappearance or exacerbation of an underlying cardiac disorder after amiodarone is started, in a patient previously stable, should prompt an investigation into thyroid function for suspected development of AIT. Sometimes worsening of a cardiac arrhythmia with recurrence of atrial fibrillation and palpitations is the only clinical evidence of AIT. The development of angina may also occur. Similarly, unexplained changes in warfarin sensitivity, requiring a reduction in the dosage of this drug, can be the consequence of increased thyroid hormone levels, since hyperthyroidism increases warfarin effects.

 

AIT may develop early during amiodarone treatment, after many months of treatment, and has even been reported occur several months after amiodarone withdrawal, since amiodarone and its metabolites have a long half-life due to accumulation in several tissues, especially fat.

PATHOPHYSIOLOGY

 

There are two different forms of AIT, and differential diagnosis between the two forms is critical, since treatments are different.

 

Type 1 AIT usually occurs in an abnormal thyroid gland (latent Grave’s disease, multinodular gland) and is the consequence of increased thyroid hormone biosynthesis due to iodine excess in patients with a preexisting thyroid disorder (Amiodarone contains 37% iodine by weight). Type 1 AIT is more common in iodine deficient regions. Type 2 AIT is a destructive process of the thyroid gland leading to the release of pre-formed hormone. This thyroiditis is an intrinsic toxic effect of amiodarone. Type 2 AIT usually persists for one to three months until thyroid hormone stores are depleted. In most countries Type 2 AIT is more common than Type 1 AIT. Differences between Type 1 and Type 2 AIT are described in table 1. Differentiating between AIT Type 1 and 2 is often very difficult on clinical grounds.

 

Table 1 Differences between Type 1 and 2 Amiodarone Induced Thyrotoxicosis

Type 1 Type 2
Underlying thyroid disease Yes (Multinodular goiter, Grave’s) No
Time after starting amiodarone Short (median 3 months) Long (median 30 months)
24-hour iodine uptake Low-Normal (may be high in iodine deficient regions) Low to Suppressed
Thyroid Ultrasound Diffuse or Nodular Goiter may be present Normal or small gland
Vascularity on Echo-color Doppler ultrasound Increased Absent
T4/T3 ratio Usually <4 Usually >4
TgAb / TPOAb/ TSI May be present Usually absent
Circulating interleukin-6 Normal to high Frequently markedly elevated

 

DIAGNOSIS and DIFFERENTIAL DIAGNOSTIC TESTS

 

To confirm the diagnosis of AIT it is necessary to demonstrate a suppressed serum TSH associated with an increase in serum FT3 and FT4 levels in a patient currently or previously treated with amiodarone. T3 levels may not be as elevated as expected as amiodarone inhibits the conversion of T4 to T3 and severe non-thyroidal illness may be present blocking the increase in T3. The presence of a preexisting thyroid disorder is suggestive for Type 1 AIT. Frequently in patients with Type 2 AIT an increased T4/T3 ratio is present as a feature of destructive thyroiditis. Thyroid antibodies may be present in Type 1 AIT depending upon the underlying thyroid disorder. High levels of thyroglobulin antibodies and TPO antibodies have also been reported in 8% of Type 2 AIT patients. Type 2 AIT develops as an inflammatory process in a normal thyroid and therefore the levels of IL-6 may be markedly elevated.

 

Color flow Doppler ultrasonography is useful to differentiate between Type 1 and Type 2 AIT. Intra-thyroidal vascular flow is increased in Type 1 AIT (pattern II-III) and reduced or absent in Type 2 (pattern 0).

In many patients with Type 1 AIT the 24-hr iodine uptake is low.  In some patients with Type 1 AIT, despite the very high iodine load, a normal or inappropriately elevated 24-hr iodine uptake may be observed, especially if the patients live in an iodine deficient area. Patients with Type 2 AIT typically have a radioactive iodine uptake < 1%.

 

In a small pilot study, 99mTc-sestaMIBI was successfully used in the differentiation between Type 1 and Type 2 AIT: uptake remained elevated in Type1 patients while uptake was absent in Type 2 patients.

 

While the distinction between Type 1 and Type 2 may sometimes be clear, in many patients neither the clinical findings nor the response to treatment clearly indicate whether the patient has Type 1 or Type 2 AIT. Some patients may have a mixed form of AIT.

 

TREATMENT

AIT may lead to increased morbidity and mortality, especially in older patients with impaired left ventricular function. Thus, in most patients, prompt restoration and stable maintenance of euthyroidism should be achieved as rapidly as possible.

Mild AIT may spontaneously resolve in about 20% of the cases. Type 1 AIT should be treated with high doses of thioamides (20-60 mg/day of methimazole; or 400-600 mg/day of propylthiouracil) to block the synthesis of thyroid hormones (Figure 1). The response to thionamides is often modest due to the high iodine levels in patients taking amiodarone. In selected patients, potassium perchlorate can also be used to increase sensitivity of the gland to thionamides by blocking iodine uptake in the thyroid. KClO4 should be used for no more than 30 days at a daily dose < 1 g/day, since this drug, especially in higher doses, is associated with aplastic anemia or agranulocytosis. Once thyroid hormone levels are back to normal, definitive treatment of the hyperthyroidism should be considered. If thyroid uptake is sufficient (>10%) radioactive iodine can be used. Thyroid surgery is a good alternative. If thyrotoxicosis worsens after initial control, a mixed form Type1-Type 2 should be considered, and treatment for Type 2 AIT should be started.

 

Type 2 AIT can be treated with prednisone, starting with an initial dose of 0.5-0.7 mg/kg body weight per day and the treatment is generally continued for three months. If a worsening of the toxicosis occurs during the taper, the prednisone dose should be increased. Thioamides are generally not useful in Type 2 AIT.

 

Because the distinction between AIT Type 1 and 2 is difficult and not always clear, and because some patients have mixed forms of AIT, these therapies for AIT Type 1 and 2 are occasionally combined.

 

For patients with persistent hyperthyroidism surgery is the optimal choice.  Treatment with iopanoic acid (if available), an iodinated cholecystographic agent, at a dose of 500 mg twice/day has been reported to quickly reduce FT3 levels, and can be used in preparation for the surgery. The treatment should be continued for about seven to 10 days after the surgery to prevent the T3 surge after the drug is withdrawn. Propylthiouracil can also be used to inhibit T4>T3 conversion. Beta blockers will be helpful in preparation for surgery.

 

Figure 1. Management of Patients with Amiodarone Induced Thyrotoxicosis

FOLLOW-UP

 

It is still debatable whether amiodarone should be discontinued once the diagnosis of AIT is made. Because of the long half-life, there is no immediate benefit in stopping the drug. However, some forms of Type 2 AIT may remit with amiodarone withdrawal. If feasible from the cardiological point of view, it is probably safer to withdraw amiodarone and use a different anti-arrhythmic drug, but no controlled trials have been published on this question. A good alternative to amiodarone in patients with atrial fibrillation and atrial flutter can be dronedarone, but this drug is contraindicated in patients with NYHA Class IV heart failure, or NYHA Class II–III heart failure with a recent decompensation. Some patients with Type 2 AIT may develop hypothyroidism due to thyroid gland destruction.

GUIDELINES

 

Bartalena L, Bogazzi F, Chiovato L, Hubalewska-Dydejczyk A, Links TP, Vanderpump M. 2018 European Thyroid Association (ETA) Guidelines for the Management of Amiodarone-Associated Thyroid Dysfunction. Eur Thyroid J. 2018 Mar;7(2):55-66

 

REFERENCES

 

Kopp P. Thyrotoxicosis of other Etiologies. 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.; 2000-2010 Dec 1.

 

Bogazzi F, Tomisti L, Bartalena L., Aghini-Lombardi F, Martino E. Amiodarone and the thyroid: a 2012 update. J Endocrinol. Invest. 2012; 35:340-48.

 

Bogazzi F, Bartalena L, Martino E. Approach to the patient with amiodarone-induced thyrotoxicosis. J Clin Endocrinol Metab 2010; 95:2529-35.

 

Cohen-Lehman J, Dahl P, Danzi S, Klein I. Effects of amiodarone therapy on thyroid function. Nat Rev Endocrinol 2010; 6:34-41.

 

Trohman RG, Sharma PS, McAninch EA, Bianco AC. Amiodarone and the thyroid physiology, pathophysiology, diagnosis and management. Trends Cardiovasc Med. 2018 Sep 20. pii: S1050-1738(18)30195-6.

 

Thyroid Storm

CLINICAL RECOGNITION


Thyroid (or thyrotoxic) storm is an acute, life-threatening syndrome due to an exacerbation of thyrotoxicosis. It is now an infrequent condition because of earlier diagnosis and treatment of thyrotoxicosis and better pre- and postoperative medical management. The incidence of thyroid storm currently may be as low as 0.2 cases/100,000 population. Thyroid storm may be precipitated by a number of factors including intercurrent illness, especially infections (Table 1). Pneumonia, upper respiratory tract infection, enteric infections, or any other infection can precipitate thyroid storm. Thyroid storm in the past most frequently occurred after surgery, but this is now unusual. Occasionally it occurs as a manifestation of untreated or partially treated thyrotoxicosis without another apparent precipitating factor. In the Japanese experience approximately 20% of patients developed thyroid storm before they received anti-thyroid drug treatment. Finally, if patients are not compliant with anti-thyroid medications thyroid storm may occur and this is a relatively common cause. Thyroid storm is typically associated with Graves' disease, but it may occur in patients with toxic nodular goiter or any other cause of thyrotoxicosis.

 

Table 1. Factors That May Precipitate Thyroid Storm
Infections
Acute Illness such as acute myocardial infarction, stroke, congestive heart failure, trauma, etc.
Non-thyroid surgery in a hyperthyroid patient
Thyroid surgery in a patient poorly prepared for surgery
Discontinuation of anti-thyroid medications
Radioiodine therapy
Recent use of iodinated contrast
Pregnancy particularly during labor and delivery

 

Classic features of thyroid storm include fever, marked tachycardia, heart failure, tremor, nausea and vomiting, diarrhea, dehydration, restlessness, extreme agitation, delirium or coma (Table 2). Fever is typical and may be higher than 105.8 F (41 C). Patients may present with a true psychosis or a marked deterioration of previously abnormal behavior. Rarely thyroid storm takes a strikingly different form, called apathetic storm, with extreme weakness, emotional apathy, confusion, and absent or low fever.

 

Signs and symptoms of decompensation in organ systems may be present. Delirium is one example. Congestive heart failure may also occur, with peripheral edema, congestive hepatomegaly, and respiratory distress. Marked sinus tachycardia or tachyarrhythmia, such as atrial fibrillation, are common. Liver damage and jaundice may result from congestive heart failure or the direct action of thyroid hormone on the liver. Fever and vomiting may produce dehydration and prerenal azotemia. Abdominal pain may be a prominent feature. The clinical picture may be masked by a secondary infection such as pneumonia, a viral infection, or infection of the upper respiratory tract.

 

Table 2. Clinical Manifestations of Thyroid Storm
History of thyroid disease
Goiter/thyroid eye disease
High fever
Marked tachycardia, occasionally atrial fibrillation
Heart Failure
Tremor
Sweating
Nausea and vomiting
Agitation/psychosis
Delirium/coma
Jaundice
Abdominal pain

 

Death from thyroid storm is not as common as in the past if it is promptly recognized and aggressively treated in an intensive care unit, but is still approximately 10-25%. In recent nationwide studies from Japan the mortality rate was >10%. Death may be from cardiac failure, shock, hyperthermia, multiple organ failure, or other complications. Additionally, even when patients survive, some have irreversible damage including brain damage, disuse atrophy, cerebrovascular disease, renal insufficiency, and psychosis.

 

PATHOPHYSIOLOGY

Thyroid storm classically began a few hours after thyroidectomy performed on a patient prepared for surgery by potassium iodide alone. Many such patients were not euthyroid and would not be considered appropriately prepared for surgery by current standards. Exacerbation of thyrotoxicosis is still seen in patients sent to surgery before adequate preparation, but it is unusual in the anti-thyroid drug-controlled patient. Thyroid storm occasionally occurs in patients operated on for some other illness while severely thyrotoxic. Severe exacerbation of thyrotoxicosis is rarely seen following 131-I therapy for hyperthyroidism; but some of these exacerbations may be defined as thyroid storm.

Thyroid storm appears most commonly following infection, which seems to induce an escape from control of thyrotoxicosis. Pneumonia, upper respiratory tract infections, enteric infections, or any other infection can cause this condition. Interestingly, serum free T4 concentrations were higher in patients with thyroid storm than in those with uncomplicated thyrotoxicosis, while serum total T4 levels did not differ in the two groups, suggesting that events like infections may decrease serum binding of T4 and cause a greater increase in free T4 responsible for storm occurrence. Another common cause of thyroid storm is a hyperthyroid patient suddenly stopping their anti-thyroid drugs.

 

DIAGNOSIS and DIFFERENTIAL

Diagnosis of thyroid storm is made on clinical grounds and involves the usual diagnostic measures for thyrotoxicosis. A history of hyperthyroidism or physical findings of an enlarged thyroid or hyperthyroid eye findings is helpful in suggesting the diagnosis. The central features are thyrotoxicosis, abnormal CNS function, fever, tachycardia (usually above 130bpm), GI tract symptoms, and evidence of impending or present CHF. There are no distinctive laboratory abnormalities. Free T4 and, if possible, free T3 should be measured. Note that T3 levels may be markedly reduced in relation to the severity of the illness, as part of the associated “non-thyroidal illness syndrome”. As expected, TSH levels are suppressed. Electrolytes, blood urea nitrogen (BUN), blood sugar, liver function tests, and plasma cortisol should be monitored. While the diagnosis of thyroid storm remains largely a matter of clinical judgment, there are two scales for assessing the severity of hyperthyroidism and determining the likelihood of thyroid storm (Figures 1 and 2). Recognize that these scoring systems are just guidelines and clinical judgement is still crucial. Data comparing these two diagnostic systems suggest an overall agreement, but a tendency toward underdiagnosis using the Japanese criteria. Unfortunately, there are no unique laboratory abnormalities that facilitate the diagnosis of thyroid storm.

Figure 1. Burch-Wartofsky Point Scale for the Diagnosis of Thyroid Storm

Figure 2. Japanese Thyroid Association Criteria for Thyroid Storm

THERAPY

Thyroid storm is a medical emergency that has to be recognized and treated immediately (Table 3). Admission to an intensive care unit is usually required. Besides treatment for thyroid storm it is essential to treat precipitating factors such as infections. As would be expected given the rare occurrence of thyroid storm there are very few randomized controlled treatment trials and therefore much of what is recommended is based on expert opinion.

 

Table 3. Treatment of Thyroid Storm
Supportive Measures
1. Rest
2. Mild sedation
3. Fluid and electrolyte replacement
4. Nutritional support and vitamins as needed
5. Oxygen therapy
6. Nonspecific therapy as indicated
7. Antibiotics
8. Cardio-support as indicated
9. Cooling, aided by cooling blankets and acetaminophen
Specific therapy
1. Propranolol (20 to 200 mg orally every 6 hours, or 1 to 3 mg intravenously
every 4 to 6 hours), Start with low doses
2. Antithyroid drugs (PTU 500–1000mg load, then 250mg every 4 hours or Methimazole 60-80mg/day), then taper as condition improves
3. Potassium iodide (one hour after first dose of antithyroid drugs):
250mg orally every 6 hours
4. Dexamethasone 2 mg every 6 hours or hydrocortisone 300mg intravenous load, then 100mg every 8 hours.
Second Line Therapy
1. Ipodate (Oragrafin) or other iodinated contrast agents
2. Plasmapheresis
3. Oral T4 and T3 binding resins- colestipol or cholestyramine
4. Dialysis

5. Lithium in patients who cannot take iodine

6. Thyroid surgery

 

It should be noted that if any possibility is present that orally given drugs will not be appropriately absorbed (e.g. due to stomach distention, vomiting, diarrhea or severe heart failure), the intravenous route should be used. If the thyrotoxic patient is untreated, an antithyroid drug should be given. PTU, 500–1000mg load, then 250mg every 4 hours, should be used if possible, rather than methimazole, since PTU also prevents peripheral conversion of T4 to T3, thus it may more rapidly reduce circulating T3 levels. Methimazole (60–80mg/day) can be given orally, or if necessary, the pure compound can be made up in a 10 mg/ml solution for parenteral administration. Methimazole is also absorbed when given rectally in a suppository. After initial stabilization, one should taper the dose and treat with Methimazole if PTU was started at the beginning as the safety profile of Methimazole is superior. If the thyroid storm is due to thyroiditis neither PTU not Methimazole will be effective and should not be used.

 

An hour after thiocarbamide has been given, iodide should be administered. A dosage of 250 mg every 6 hours is more than sufficient. The iodine is given after PTU or Methimazole because the iodine could stimulate thyroid hormone synthesis. Unless congestive heart failure contraindicates it, propranolol or other beta-blocking agents should be given at once, orally or parenterally, depending on the patient's clinical status. Beta-blocking agents control tachycardia, restlessness, and other symptoms. Additionally, propranolol inhibits type 1 deiodinase decreasing the conversion of T4 to T3. Probably lower doses should be administered initially, since administration of beta-blockers to patients with severe thyrotoxicosis has been associated with vascular collapse. Esmolol, a short-acting beta blocker, at a loading dose of 250 mcg/kg to 500 mcg/kg followed by 50 mcg/kg to 100 mcg/kg/minute can be used in an ICU setting.  For patients with reactive airway disease, a cardioselective beta blocker like atenolol or metoprolol can be employed.

 

Permanent correction of the thyrotoxicosis by either 131-I or thyroidectomy should be deferred until euthyroidism is restored. Other supporting measures should fully be exploited, including sedation, oxygen, treatment for tachycardia or congestive heart failure, rehydration, multivitamins, occasionally supportive transfusions, and cooling the patient to lower body temperature down. Antibiotics may be given on the presumption of infection while results of cultures are awaited.

 

The adrenal gland may be limited in its ability to increase steroid production during thyrotoxicosis. Therefore, hydrocortisone (100-300 mg/day) or dexamethasone (2mg every 6 hours) or its equivalent should be given. The dose can rapidly be reduced when the acute process subsides. Pharmacological doses of glucocorticoids (2 mg dexamethasone every 6 h) acutely depress serum T3 levels by reducing T4 to T3 conversion. This effect of glucocorticoids is beneficial in thyroid storm and supports their routine use in this clinical setting.

 

Usually rehydration, repletion of electrolytes, treatment of concomitant disease, such as infection, and specific agents (antithyroid drugs, iodine, propranolol, and corticosteroids) produce a marked improvement within 24 hours. A variety of additional approaches have been reported and may be used if the response to standard treatments is not sufficient. For example, oral gallbladder contrast agents such as ipodate and iopanoic acid in doses of 1-2 g, which inhibit peripheral T4 to T3 conversion, may have value. Plasmapheresis can remove circulating thyroid hormone and rapidly decrease thyroid hormone levels. Orally administered bile acid sequestrants (20-30g/day Colestipol-HCl or Cholestyramine) can trap thyroid hormone in the intestine and prevent recirculation. In most cases these therapies are not required but in the occasion patient that does not respond rapidly to initial therapy these modalities can be effective. Finally, in rare situations where medical therapy is ineffective or the patient develops side effects and contraindications to the available therapies thyroid surgery may be necessary.

 

FOLLOW-UP

Antithyroid treatment should be continued until euthyroidism is achieved, when a decision regarding definitive treatment of the hyperthyroidism with antithyroid drugs, surgery, or 131-I therapy can be made. Rarely urgent thyroidectomy is performed with antithyroid drugs, iodide, and beta blocker preparation.

 

Prevention of thyroid storm is key and involves recognizing and actively avoiding common precipitants, educating patients about avoiding abrupt discontinuation of anti-thyroid drugs, and ensuring that patients are euthyroid prior to elective surgery and labor and delivery.

 

GUIDELINES

Ross DS, Burch HB, Cooper DS, Greenlee MC, Laurberg P, Maia AL, Rivkees SA, Samuels M, Sosa JA, Stan MN, Walter MA. 2016 American Thyroid Association Guidelines for Diagnosis and Management of Hyperthyroidism and Other Causes of Thyrotoxicosis. Thyroid. 2016 Oct;26(10):1343-1421.

 

Satoh T, Isozaki O, Suzuki A, Wakino S, Iburi T, Tsuboi K, Kanamoto N, Otani H, Furukawa Y, Teramukai S, Akamizu T. 2016 Guidelines for the management of thyroid storm from The Japan Thyroid Association and Japan Endocrine Society (First edition). Endocr J. 2016 Dec 30;63(12):1025-1064

 

REFERENCES

Bartalena L. Graves’ Disease: Complications. 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 Feb 20.

 

Akamizu T1, Satoh T, Isozaki O, Suzuki A, Wakino S, Iburi T, Tsuboi K, Monden T, Kouki T, Otani H, Teramukai S, Uehara R, Nakamura Y, Nagai M, Mori MDiagnostic criteria, clinical features, and incidence of thyroid storm based on nationwide surveys.Thyroid.2012 Jul;22(7):661-79.

Swee du S, Chng CL, Lim A. Clinical characteristics and outcome of thyroid storm: a case series and review of neuropsychiatric derangements in thyrotoxicosis. Endocr Pract. 2015 Feb;21(2):182-9.

Angell TE, Lechner MG, Nguyen CT, Salvato VL, Nicoloff JT, LoPresti JS. Clinical features and hospital outcomes in thyroid storm: a retrospective cohort study. J. Clin. Endocrinol. Metab. 2015 Feb;100(2):451-9.

 

Chiha M, Samarasinghe S, Kabaker AS. Thyroid storm: an updated review. J Intensive Care Med. 2015 Mar;30(3):131-40

 

Akamizu T. Thyroid Storm: A Japanese Perspective. Thyroid. 2018 Jan;28(1):32-40

Age-Related Changes in the Male Reproductive System

ABSTRACT

 

In male mammals, changes at all levels of the hypothalamic-pituitary-testicular axis, including alterations in the GnRH pulse generator, gonadotropin secretion, and testicular steroidogenesis, in addition to alterations of feed-forward and feed-back relationships contribute to the age-related decline in circulating testosterone concentrations.  The rate of age-related decline in testosterone levels is affected by the presence of chronic illness, adiposity, medication, sampling time, and the methods of testosterone measurement.  Epidemiologic surveys reveal an association of low testosterone levels with changes in body composition, physical function and mobility, risk of diabetes, metabolic syndrome, coronary artery disease, and fracture risk. Age-related decline in testosterone should be distinguished from classical hypogonadism due to known diseases of the hypothalamus, pituitary and the testis. In young hypogonadal men who have a known disease of the hypothalamus, pituitary and testis, testosterone therapy is generally beneficial and has been associated with a low frequency of adverse events. However, neither the long-term benefits in improved health outcomes nor the long-term risks of testosterone therapy are known in older men with age-related decline in testosterone levels. Well-conducted studies of up to one-year duration have found improvements in sexual desire, erectile function, and overall sexual activity; mobility; and volumetric bone density, and correction of anemia with testosterone replacement of older men with unequivocally low testosterone levels. Although testicular morphology, semen production, and fertility are maintained up to a very old age in men, there is clear evidence of decreased fecundity with advancing age and an increased risk of specific genetic disorders related to paternal age among the offspring of older men. Thus, reproductive aging of men is emerging as an important public health problem whose serious societal consequences go far beyond the quality of life issues related to low testosterone levels. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

 

INTRODUCTION

 

Aging of male mammals is a very recent evolutionary event observed mostly in humans and animals in captivity.  Most animal species in the wild do not live beyond their reproductive years; during periods of food deprivation, many small animals may not even live beyond puberty. Even among humans, only the men and women of the past three generations have enjoyed a life expectancy of greater than fifty years. With increasing life expectancies, the reproductive problems of aging men and women have begun to receive the attention that they deserve. Aging of humans is associated with functional alterations at all levels of the reproductive axis that affect both the steroidogenic and gametogenic compartments. Even after forty years of investigation, the controversy surrounding the use of hormone replacement in postmenopausal women has grown only louder (1-4); in contrast, the issue of testosterone replacement in older men has been shrouded in acerbic debate from its very inception, even though not a single, adequately powered, long term, randomized trial of testosterone replacement has yet been conducted. There is agreement that in men with classical hypogonadism due to known diseases of the testis, pituitary, and the hypothalamus, testosterone replacement is relatively safe and has many beneficial effects in improving sexual function, maintaining secondary sex characteristics, energy, mood, and well-being, skeletal muscle mass and reducing fat mass, and is associated with low frequency of adverse events (5-8). However, the data from otherwise healthy men with classical hypogonadism should not be directly extrapolated to older men with age-related decline in serum testosterone concentrations (9,10). As discussed in this chapter, there is agreement that serum testosterone levels decline with age, a decline that is exacerbated by the accumulation of comorbidities; however, the long-term effects of testosterone supplementation on health-related outcomes in older men have not been fully examined. Long-term safety data on the effects of testosterone supplementation on the risk of prostate cancer and major adverse cardiovascular events are also lacking. The recent publication of several well-conducted placebo-controlled trials of testosterone in middle-aged and older men has greatly advanced our understanding of the effects of testosterone treatment on sexual function, mobility, vitality, lower urinary tract symptoms and atherogenesis progression (11-17). However, in the absence of long-term, adequately-powered randomized trials of the effects of testosterone on hard patient-important health outcomes – fractures, falls, physical disability, progression from prediabetes to diabetes, remission of depressive disorders, wellbeing, and progression to dementia - the risks and benefits of long-term testosterone replacement in older men remain incompletely understood.

 

CHANGES IN THE STEROIDOGENIC COMPARTMENT OF THE TESTIS

 

Many studies suggest that aging per seaffects the gonadal axis independently of the co-morbidities that accrue with aging, but there remains controversy about the relative contributions of the aging and the accumulation of co-morbidities to the age-related decline in testosterone levels. A few studies of older men have reported preservation of normal testosterone concentrations and its circadian rhythm in healthy older men (18,19). However, a number of cross-sectional studies have shown that even after accounting for the potential confounding factors such as time of sampling, concomitant illness and medications, and technical issues related to hormone assays, serum total testosterone levels are lower in older men in comparison to younger men (20-41). Several longitudinal studies (20-23)have confirmed a gradual but progressive decrease in serum testosterone concentrations from age 20 to 80. Adiposity, chronic illness, weight gain, lifestyle factors, medications, and genetic factors affect testosterone levels and the trajectory of the age-related decline in testosterone levels in men (18,21,24,42-45).  The rate of age-related decline is greater in older men with chronic illness and adiposity than in healthy, non-obese older men (24,42,43). In the European Male Aging Study, adiposity and comorbidities were more strongly associated with low testosterone levels than age (46); however, advanced age is often associated with weight gain and accumulation of comorbidities and extricating their individual contributions to age-related decline in testosterone levels is difficult.

 

In contrast to the sharp reduction in ovarian estrogen production at menopause, the age-related decline in men does not start at a discrete coordinate in old age; rather, total testosterone concentrations, after reaching a peak in the second and third decade, decline inexorably throughout a man’s life (Figure 1). Because of the absence of an identifiable inflection point at which testosterone levels begin to decline abruptly or more rapidly, many investigators have questioned the validity of the concept of “andropause”, which misleadingly implies an abrupt cessation of androgen production in men (28,47). The term ‘late-onset hypogonadism’ has been proposed to reflect the view that in some middle-aged and older men (> 65 years), the age-related decline in testosterone concentration is associated with a cluster of symptoms and signs in a syndromic constellation which resembles that observed in men with classical hypogonadism (36,48).

Figure 1. The distribution of total and free testosterone levels by decades of age in male participants of the Framingham Heart Study, the European Male Aging Study (EMAS) and the Study of Osteoporotic Fractures in Men (MrOS). Means and standard deviations are shown. To convert total testosterone from ng/dL to nmol/L, multiply concentrations in ng/dL with 0.0347. To convert free testosterone from pg/mL to pmol/L, multiply concentrations in pg/mL with 3.47. Reproduced with permission from Bhasin et al, J Clin Endocrinol Metab. 2011 Aug;96(8):2430-9.

Sex-hormone binding globulin concentrations are higher in older men than younger men (21,32,37). Thus, the age-related decline in free testosterone levels is of a greater magnitude than that in total testosterone levels. Similarly, there is a greater percent decline in bioavailable testosterone concentrations (the fraction of circulating testosterone that is not bound to SHBG) than in total testosterone concentrations.

 

 

An Expert Panel of the Endocrine Society defined androgen deficiency as a syndrome resulting from reduced production of testosterone and characterized by a set of signs and symptoms in association with unequivocally low testosterone levels (5). Many epidemiologic studies have defined androgen deficiency solely in terms of serum testosterone concentrations below the lower limit of the normal range for healthy, young men leading to exaggerated estimates of the prevalence of androgen deficiency in older men. Additionally, serum testosterone levels in most studies were measured using direct immunoassays, whose accuracy in the low range has been questioned. Not surprisingly, the estimates of the prevalence of androgen deficiency in older men have varied greatly among different studies. In the Baltimore Longitudinal Study of Aging (BLSA) (20), 30% of men over the age of 60 and 50% of men over the age of 70 had total testosterone concentration below the lower limit of normal range for healthy young men (325 ng/dL, 11.3 nmol/L). The prevalence was even higher when these investigators used a free testosterone index to define androgen deficiency (20). Several other studies have also reported a similarly high prevalence of low total and free testosterone levels in older men. In contrast, more recent studies, using liquid chromatography tandem mass found the prevalence of androgen deficiency to be significantly lower than that observed in the MMAS and BLSA (28,29,36-39).Although 10–15% of men aged ≥65 years have low total testosterone levels (Table 1) (36-39), the prevalence of late-onset hypogonadism defined by symptoms and a total testosterone level <8 nmol/l in the EMAS was 3.2% for menaged 60–69 years and 5.1% for those aged 70–79 years (36). The Healthy Man Study in Australia found no significant age-related decline in testosterone or dihydrotestosterone in men who reported being in good health (49). The authors of the Health Man Study have argued that ill health, rather than aging itself, is the major contributor to androgen deficiency in older men. A Finnish cross-sectional study also demonstrated very low prevalence of low serum

testosterone concentrations in older men who were healthy (28).

 

Table 1. Percent of Community-Dwelling Older Men with Unequivocally Low Testosterone Level in Population Studies.

Study

Principle Investigator

Number of Men > 65 Years of Age

% Men with Testosterone <250 ng/dL
Framingham Heart Study (FHS) Bhasin 1870 12.1%
Osteoporotic Fractures in Men Study (MrOs) Orwoll 2623 10%
European Male Aging Study (EMAS) Wu 1080 7.3%
Cardiovascular Health Study (CHS) Hirsch 639 14.3%

Data derived from Bhasin et al, JCEM 2011; Orwoll et al, JCEM 2009; Wu et al, NEJM 2010; Hirsch et al, JCEM 2009.

 

 

Circulating testosterone concentrations are a function of testosterone production and clearance rates; the age-related decline in serum testosterone concentrations is primarily a consequence of decreased production rates in older men (9,10,32-34,37). Plasma clearance rates of testosterone are, in fact, lower in older men than in younger men (43,44). The decline in testosterone production in older men is the result of abnormalities at all levels of the hypothalamic-pituitary-testicular axis (31-33,49-60).

 

GONADOTROPIN-RELEASING HORMONE SECRETION AND REGULATION IN OLDER MEN

 

Pulsatile GnRH secretion is attenuated in older men. In addition, there are disturbances of the feedback and feed-forward relationships between testosterone and LH secretion (52,60,61). Thus, the sensitivity of pituitary LH secretion to androgen-mediated feedbackinhibition is increased; in addition, the ability of LH to stimulate synchronously testicular testosterone secretion (feedforward) is attenuated (52,60,61). This insight has emerged largely from the research of Veldhuis who used novel algorithms to quantitate the orderliness of pulsatile hormone secretion, and the synchrony between secretion of related hormones (e.g., LH and testosterone, and LH and FSH) (52,60,61). This research has revealed that the orderliness of LH pulses and the synchrony between LH and testosterone pulses are decreased in older men (60,61); in addition, there is greater variability in LH pulse frequency, amplitude, and secretory mass in older men, in comparison to younger men (60,61).

 

GONADOTROPIN SECRETION AND REGULATION IN OLDER MEN

 

There is considerable heterogeneity in circulating LH and FSH concentrations in individual older men; both hypogonadotropic and hypergonadotropic hypogonadism have been reported (43,48). As a group, serum LH and FSH concentrations are higher in older men than in young men (21,22). Serum LH and FSH levels show an age-related increase in longitudinal studies. However, serum LH concentrations do not increase in proportion to the age-related decline in circulating testosterone levels, probably due to the impairment of GnRH secretion and alterations in gonadal steroid feedback and feedforward relationships (49-60); both of these mechanisms are operative in older men.

 

In the EMAS, secondary hypogonadism (low testosterone and low or normal LH)was more prevalent (nearly 12%) than primary hypogonadism (low testosterone and elevated LH, 2%) (46). Secondary hypogonadism was associated with obesity and comorbid conditions, while primary hypogonadism was associated predominately with age (46). Nearly 10% of men in EMAS had normal testosterone levels but elevated LH; these men with elevated LH tended to be older and in poor health and were at increased risk of developing low testosterone and other comorbid conditions (62).

 

The data on LH response to GnRH are somewhat contradictory. Urban et al (54)used an interstitial cell bioassay to measure serum concentrations of bioactive LH and found that although basal bioactive LH concentrations were similar in this sample of young and older men, older men demonstrated diminished LH response to GnRH administration. However, in a subsequent study, Zwart et al (55)found greater gonadotropin responsiveness to GnRH in older men than younger men; the maximal and incremental LH and FSH secretory masses in response to graded doses of GnRH were significantly higher in healthy, older men than in younger men. The estimated half-lives of LH, FSH, or alpha-subunit were not significantly different between young and older men.

 

The Brown Norway rat has been widely used as a model of reproductive aging. In this experimental model, the prepro-GnRH mRNA content and the number of neurons expressing prepro-GnRH mRNA are lower in older male rats in comparison to young rats (56,57). The GnRH content of several hypothalamic areas is also lower in intact older rats than younger rats (56). Older Brown Norway rats exhibit significant reductions in glutamate and -aminobutyric acid (GABA) levels in the hypothalamus compared to young rats (57). These observations suggest that the decreased hypothalamic excitatory amino acid expression and the reduced responsiveness of GnRH neurons to NMDA may contribute to the altered LH pulsatile secretion observed in old rats (57).

 

Infusions of testosterone and DHT are associated with greater reductions in mean serum LH and FSH levels and the frequency of LH pulses in older men in comparison to young men (58). Winters et al (53)reported that the degree of LH inhibition during testosterone replacement of older, hypogonadal men was significantly greater than in young, hypogonadal men suggesting that older men are more sensitive to the feedback inhibitory effects of testosterone on LH. Deslypere et al (58)also found decreased LH pulse frequency and a greater degree of LH inhibitory response to estradiol administration in older men than young controls. Age-related increase in FSH levels is not associated with a progressive or proportionate decrease in inhibin B levels (59). Thus, the mechanistic basis of FSH increase in not fully understood, although the lack of change in inhibin B levels suggests that Sertoli cell function is relatively preserved in older men.

 

TESTICULAR TESTOSTERONE PRODUCTION IN OLDER MEN

 

Testosterone secretion in healthy, young men is characterized by a diurnal rhythm with higher concentrations in the morning and lower levels in later afternoon. Many studies have revealed that the diurnal rhythm of testosterone secretion is dampened in older men (30,40). Testosterone response to LH and human chorionic gonadotropin is decreased in older men in comparison to younger men (31-33).

 

 

Many of the physiological changes that occur with advancing age, such as loss of bone and muscle mass, increased fat mass, impairment of physical, sexual and cognitive functions, loss of body hair, and decreased hemoglobin levels, are similar to those associated with androgen deficiency in young men. Aging is associated with loss of skeletal muscle mass (Figure 2), muscle strength and power, and progressive impairment of physical function (63-87). Epidemiological studies of older men have reported associations between low testosterone levels and health outcomes, although these associations are weak. For instance, in a number of epidemiologic studies, such as the St. Louis Inner City Study of Aging Men (66), the Olmsted County Epidemiological Study (65), and the New Mexico Elderly Health Study (68,69), low bioavailable testosterone levels (unbound and albumin-bound testosterone) were associated with low appendicular skeletal muscle mass. Low bioavailable testosterone levels also have been associated with decreased strength of upper as well as lower extremity muscles (66,67)and decreased performance in self-reported as well as performance-based measures of physical function (88-92). Low free testosterone levels have also been associated with the development of mobility limitation and the frailty syndrome (93-96).

 

Figure 2. A schematic diagram of the age-related changes in body composition in 7265 men.
Lines represent the longitudinal changes in body weight (black line), fat mass (red line) and fat-free mass (blue line) components from age 20 years. The estimated mass values at age 20 years were as follows: body mass, 72.72 kg; fat mass, 9.14 kg; fat-free mass, 64.09 kg. Figure adapted with permission from Jackson et al. Br J Nutr. 2012;107(7):1085-91.

The association of testosterone levels with sexual dysfunction has been inconsistent across studies because of the heterogeneity and variable quality of instruments used to assess sexual dysfunction, problems of testosterone assay quality, and failure to distinguish among various categories of sexual dysfunction (97-102). Androgen deficiency and erectile dysfunction are two independently distributed clinical disorders and because both disorders are prevalent in middle-aged and older men, they can often co-exist (101,102). Low testosterone levels were associated with low sexual desire In the MMAS (97). Among men enrolled in the testosterone trials, free and total testosterone levels were independently associated with sexual desire, erectile function, and sexual activity scores (103).

 

In the EMAS, total and free testosterone levels were associated with overall sexual functionin middle-aged and older men (36). This relationship was observed more robustly at testosterone concentrations <8 nmol/L, but not at higher testosterone concentrations (104). Men deemed to have low total and free testosterone levels in EMAS were more likely to report decreased morning erections, erectile dysfunction, and decreased frequency of sexual thoughts than those with normal testosterone levels (37). In another study of men over the age of 50 who had benign prostatic hyperplasia, sexual dysfunction, assessed by the Sexual Function Inventory, was reported only in men with serum total testosterone levels less than 225 ng/dL (99).

 

Aging of humans is attended by a decline in several aspects of cognitive function; of these multiple domains of cognition that decline with aging, declines in verbal memory, visual memory, spatial ability, and executive function are associated with the age-related decline in testosterone (98-102,104-113).

 

The relationship of testosterone levels with depression has been inconsistent across epidemiologic studies (114-118). Low testosterone levels in older men appear to be associated more with late-onset low grade persistent depressive disorder (dysthymia) than with major depression (117-119). In general, testosterone levels are lower in older men with dysthymic disorder than in those without any depressive symptoms (118).

 

Several epidemiologic studies of older men (120-124), including MrOS (120), Rancho Bernardo Study (121), Framingham Heart Study (122), and the Olmsted County Study (123)- have found bioavailable testosterone levels to be associated with bone mineral density, bone geometry, and bone quality (124); the associations are stronger with bioavailable testosterone and estradiol levels than with total testosterone levels. In the MrOS Study, the odds of osteoporosis in men with a total testosterone less than 200 ng/dL were 3.7-fold higher than in men with normal testosterone level (120); free testosterone was an independent predictor of prevalent osteoporotic bone fractures (125).

 

Several studies have evaluated the association of testosterone levels and mortality (126-129). Some, but not all, studies found higher all-cause mortality and cardiovascular mortality in men with low testosterone levels than in those with normal testosterone levels. In a meta-analysis of epidemiologic studies of community-dwelling men, low testosterone levels were associated with an increased risk of all-cause and CVD death (Figure 3) (130,131). However, the strength of the inferences of these meta-analyses was limited by considerable heterogeneity in study populations; it is possible that effects may have been driven by differences in the age distribution and the health status of the study populations (130-134).

 

Figure 3. The relationship of low testosterone level with all-cause mortality in a meta-analysis of epidemiologic studies of community-based men. Eleven studies which enrolled 16,184 subjects were included in this meta-analysis. There was considerable heterogeneity of the age distribution, health status, and other subject characteristics. Reproduced with permission from Araujo et al, J Clin Endocrinol Metab 2011;96:3007-19.

Testosterone levels are not correlated with aging-related symptoms assessed by the Aging Male Symptom (AMS) score or with lower urinary tract symptoms assessed by the IPSS/AUA prostate symptom questionnaire (132). Some cross-sectional studies found no difference in serum testosterone levels between men who had coronary artery disease and those who did not have coronary artery disease; other studies have reported testosterone levels to be lower in men with coronary artery disease than in men without coronary artery disease (133-138).

 

Epidemiologic studies, especially cross-sectional studies, can only demonstrate associations; a cause and effect relationship cannot be inferred from these studies. Furthermore, the associations between testosterone levels and health-related outcomes that have been found to be statistically significant are weak. The inferences are further confounded by the collinearity of aging-related co-morbid conditions, low testosterone levels, and age-related changes in body composition and inflammatory markers. Although epidemiologic studies have reported associations between the age-related changes in circulating testosterone levels and skeletal muscle mass, muscle strength and physical function; sexual and cognitive functions; areal and volumetric bone density and fracture risk; and mood, long-term randomized trials are needed to determine whether these relations are causal.

 

POTENTIAL BENEFICIAL EFFECTS OF TESTOSTERONE THERAPY IN OLDER MEN WITH LOW TESTOSTERONE LEVELS

 

It has been hypothesized that increasing serum testosterone concentrations in older men with low testosterone levels into a range that is mid-normal for healthy, young men would improve physical function and mobility, some domains of sexual and cognitive functions, energy and sense of wellbeing, and reduce the risk of falls and fractures, and improve overall quality of life. A number of randomized trials have demonstrated improvements in measures of sexual function, lean and fat mass, and areal and volumetric bone mineral density; however, there has been a paucity of long-term, placebo-controlled, randomized trials that are adequately powered to detect clinically meaningful changes in health outcomes such as fracture rates, physical disability, progression to dementia, remission of late onset low grade persistent depressive disorder (dysthymia), progression from prediabetes to diabetes, and overall quality of life. Furthermore, none of the previously published studies had sufficient power to address the long-term risks of prostate and cardiovascular disease.

 

The following section describes the effects of testosterone supplementation on multiple organ systems focusing on physical function, sexual function, vitality, bone health, mood, wellbeing, and depression, and cognitive function.

 

Effects of Testosterone Supplementation on Muscle Mass and Performance and Physical Function in Older Men with Low Testosterone Levels

 

 

Sarcopenia, the loss of muscle mass and function, is an important consequence of aging (64-68). The principal component of the decrease in fat-free mass is the loss of muscle mass; there is little change in non-muscle lean mass (70-76). Between 20 and 80 years of age, the skeletal muscle mass decreases by 35-40% in men (74), in part due to decreased muscle protein synthesis (81). Although there is a loss of both type I and type II fibers, there is a disproportionate decrease in the number of type II muscle fibers that are important for the generation of muscle power (82,83). In spite of the significant depletion of skeletal muscle mass, body weight does not decrease, and may even increase because of the corresponding accumulation of body fat (70-76)(Figure 2).

 

The loss of skeletal muscle mass that occurs with aging is associated with a reduction in muscle strength (84-87). There is a substantial decrease in muscle strength and power between 50 and 70 years of age, primarily due to muscle fiber loss and selective atrophy of type II fibers (82-87). The loss of muscle strength is even greater after the age of 70; 28% of men over the age of 74 could not lift objects weighing more than 4.5 kg (86). With increasing age, there is a progressive reduction in muscle power (139,140), the speed of strength generation, and fatigability, the ability to persist in a task.

 

Loss of muscle mass and strength leads to impairment of physical function, as indicated by the impaired ability to arise from a chair, climb stairs, generate gait speed, and maintain balance (139-142). The impairment of physical function contributes to loss of independence, and increased risk of physical disability, falls and fractures in older men. Therefore, function promoting therapies that can reverse or prevent aging-associated sarcopenia are desirable.

 

ANABOLIC EFFECTS OF TESTOSTERONE IN HUMANS: TESTOSTERONE TRIALS IN HEALTHY, HYPOGONDAL MEN, MEN WITH CHRONIC ILLNESS, AND OLDER MEN

 

The anabolic effects of testosterone on the muscle have been a source of intense controversy for over sixty years. The athletes and recreational body builders abuse large doses of androgenic steroids with the belief that these compounds increase muscle mass and strength. Until recently, the academic community was skeptical about such claims because of the problems of study design. However, a number of studies in healthy young men, healthy hypogonadal men, men with chronic illness, and in healthy older men have established that testosterone administration increases skeletal muscle mass, maximal voluntary strength, and leg power (142-149).

 

The anabolic effects of testosterone on fat-free mass, muscle size, and maximal voluntary strength are related to the administered testosterone dose and the circulating testosterone concentrations (150-152)(Figure 4). Testosterone effects on muscle performance are domain-specific: testosterone administration increases maximal voluntary strength and leg power, but does not affect fatigability or specific tension (151). The gains in maximal voluntary strength during testosterone administration are proportional to the increase in muscle mass; unlike resistance exercise training, testosterone does not improve the contractile properties of the human skeletal muscle (151).

Figure 4. Testosterone Dose Response Relationship in Young and Older Men. In this study, healthy, young men (18-34 years of age) and healthy older men (60-75 years of age) were treated with a long acting GnRH agonist plus graded doses of testosterone enanthate for 20 weeks. Shown are mean (±SEM) changes from baseline in fat free mass (upper left), skeletal muscle mass (upper right), fat mass (lower left), and leg press strength (lower right) in young (black bars) and older (lightly shaded bars) men. Adapted with permission from Bhasin et al. J Clin Endocrinol Metab. 2005 Feb;90(2):678-88.

Resistance exercise training augments the anabolic response to androgens; thus, men receiving testosterone and resistance exercise training together experience greater gains in fat-free mass and muscle strength than those receiving either intervention alone (153). The anabolic effects of testosterone are also augmented by concomitant recombinant growth hormone administration (154). Although it has been speculated in the sports medicine literature that increasing protein intake can enhance the muscle mass and strength gains in response to anabolic stimuli such as resistance exercise training or androgens, the evidence supporting such speculation is weak. In a recent controlled feeding study, increasing the daily protein intake to a level (1.3 g/kg/day) higher than the recommended daily allowance (0.8 g/kg/day) for six months did not increase lean body mass or maximal muscle strength more than that associated with the daily intake of the recommended daily allowance of 0.8 g/kg/day (155). The higher level of daily protein intake (1.3 g/kg/day) also did not augment the gains in lean body mass and muscle strength in response to testosterone administration above that observed in participants eating the recommended dietary allowance for protein (155,156).

 

Testosterone replacement of young, hypogonadal men increases muscle protein synthesis (146,157,158); the effects of testosterone replacement on muscle protein degradation need further investigation.

 

In a systematic review of testosterone trials in healthy, hypogonadal men, testosterone therapy increased fat-free mass and body weight (Figure 5) (142-149). Testosterone replacement therapy also improves maximal voluntary strength (145,148), and decreases whole body fat mass (144,147-149). The administration of supraphysiologic doses of testosterone in eugonadal men increases fat-free mass, muscle size, and maximal voluntary strength (150-153).

 

Systematic reviews (143,159,160)of randomized, placebo-controlled trials in HIV-infected men with weight loss (160-165)have revealed that testosterone therapy for 3 to 6 months was associated with greater gains in lean body mass than placebo administration (difference in lean body mass change between placebo and testosterone arms 1.22 kg, 95% CI 0.23-2.22 for the random effect model). In two (160,164)out of three trials that measured muscle strength (160,164,165), testosterone administration was associated with significantly greater improvements in maximal voluntary strength than placebo. Testosterone therapy had a moderate effect on depression indices (-0.6, 95% CI -1.0, -0.2) (166)and fatigue (167), but did not improve overall quality of life (166,167). Changes in CD4+ T lymphocyte counts, HIV copy number, PSA, plasma HDL cholesterol, and adverse event rates were not significantly different between the placebo and testosterone-treatment groups (160-167).Overall, short-term (3-6 months) testosterone use in HIV-infected men with low testosterone levels and weight loss can induce modest gains in body weight and lean body mass with minimal changes in quality of life and mood. This inference is weakened by inconsistency of results across trials, and heterogeneity in inclusion and exclusion criteria, disease status, testosterone formulations and doses, treatment duration, and methods of body composition analysis (143). Data on testosterone effects on physical function, risk of disability, or long-term safety in HIV-infected men are limited.

 

Testosterone administration increases fat-free mass and decreases fat mass in older men with low testosterone levels. Meta-analyses (143,167)of randomized trials (168-172)that included middle-aged and older men with low or low normal testosterone levels, and that used testosterone or its esters in replacement doses for >90 days, have confirmed that testosterone administration is associated with a significantly greater increase in fat-free mass, hand grip strength, and a greater reduction in whole body fat mass than placebo (Figure 5). The average gains in fat-free mass generally were greater in trials that used injectable testosterone esters than in those which used transdermal testosterone gel, presumably because of the higher doses of testosterone delivered by the injectable formulations than by transdermal gel formulations. The change in body weight did not differ significantly between the testosterone and placebo groups.

 

Figure 5. The effects of testosterone therapy on body composition, muscle strength, and sexual function in intervention trials. The point estimates and the associated 95% confidence intervals are shown. Panel A shows the effects of testosterone therapy on, grip strength, fat mass and lean body mass in a meta-analysis of randomized trials (data derived from Bhasin et al. Nat Clin Pract Endocrinol Metab. 2006;2(3):146-59; figure reproduced with permission from Spitzer et al. Nat Rev Endocrinol. 2013;9(7):414-24). Panel B shows the effects of testosterone therapy on sexual function in a meta-analysis of randomized trials (figure adapted with permission from Ponce et al. J Clin Endocrinol Metab. 2018;103(5):1745-54).

Testosterone administration improves stair climbing speed and power, and self-reported physical function, as assessed by the Medical Outcomes Study Short Form 36 (MOS SF36) questionnaire. Testosterone’ Effects on Atherosclerosis Progression in Aging Men Trial (The TEAAM Trial), arandomized trial conducted in healthy community-dwelling older men without functional limitations and low to low-normal testosterone levels, showed that testosterone replacement for 3-years was associated with modest improvements in leg-press and chest-press power and the stair-climb power (173).Changes in gait speed generally have been modest and inconsistent across randomized trials (14,169,172,174). Testosterone administration is associated with small improvements in aerobic capacity and attenuation of the age-related decline in VO2peak(Figure 6)(175,176).

 

One reason for the variable improvements in physical function in testosterone trials is that the measures of physical function used in previous studies had low ceilings. Another confounder of the effects of anabolic interventions on muscle function is the learning effect. For instance, subjects who are unfamiliar with weight lifting exercises often demonstrate improvements in measures of muscle performance because of increased familiarity with the exercise equipment and technique. Because of the considerable test-to-test variability in tests of physical function, it is possible that previous studies did not have adequate power to detect meaningful differences in measures of physical function between the placebo and testosterone-treated groups. It is also possible that neuromuscular adaptations needed to translate strength gains into functional improvements require a lot longer than the 3 to 6-month duration of most of the previous trials. The measures of physical function that are more robustly related to lower extremity muscle strength, such as stair climbing speed and power, have shown more consistent improvements in testosterone trials than walking speed (11,12,177).

Figure 6. Effects of testosterone administration on measures of muscle performance and physical function in randomized testosterone trials in older men. Panel A shows the mean (SD) change from baseline to maximal voluntary strength in the leg press and chest press exercises and on loaded stair climbing power at either the end of the intervention period or at the last measurement performed in who dropped out before study completion in the testosterone in older men with mobility limitation (The TOM Trial). The minimal clinically important difference (MCID) for each outcome was determined using an anchor-based method within the trial. The proportion of men (percent) whose change from baseline either equaled or exceeded the MCID is shown below the figure along with the P-value for the comparison of placebo and testosterone groups (figure adapted with permission from Spitzer et al. Nat Rev Endocrinol. 2013;9(7):414-24). Panel B shows the long-term effects of testosterone administration on aerobic capacity in older men participating in the TEAAM trial. Data points represent mean changes from baseline and error bars are 95% CI in VO2peak (L/min) and in peak work rate. P values indicate the overall effect of the testosterone intervention over time (figure reproduced with permission from Traustadóttir et al, J Clin Endocrinol Metab. 2018;103(8):2861-2869).

Only a few testosterone trials have been conducted in older men with functional limitations (11,15,17,177-179). In a trial of pre-frail or frail men (17), administration of 50 mg testosterone gel daily for 6 months induced greater improvements in lean mass, knee extension peak torque and sexual symptoms than did placebo gel (17). Performance-based measures of physical function did not differ significantly between groups, but they improved in the subgroup of frail elderly men (17). In Testosterone in Older Men (TOM) Trial, older men with mobility limitation were randomly assigned to either placebo or 10 g testosterone gel daily for 6 months(11,177). The testosterone dose was adjusted to achieve testosterone levels between 17.4 nmol/l and 34.7 nmol/L (500 to 1000 ng/dL). The improvements in leg-press strength, chest-press strength and power, and loaded stair-climbing speed and power were significantly greater in men assigned to testosterone arm than in those receiving placebo (Figure 6). A greater proportion of men in the testosterone arm improved more than the minimal clinically important difference for leg-press and chest-press strength and stair-climbing speed than that in the placebo arm. Because of a higher frequency of cardiovascular-related events in the testosterone arm compared with the placebo arm, the trial’s data and safety monitoring board stopped further administration of study medication (11,177).The findings of the TOM trial and other epidemiologic studies have heightened the concern that frail elderly men with a high burden of chronic co-morbidities may be at an increased risk of adverse events (11), providing the impetus to develop, strategies to achieve increased selectivity and a more favourable risk to benefit ratio (11).

 

The Testosterone Trials were a coordinated set of seven randomized double-blind, placebo-controlled trials designed to determine the benefits of testosterone therapy in older men 65 years and older with low testosterone levels and clinical symptoms of androgen deficiency on a variety of androgen-dependent outcomes (179). To participate in these trials, the men had to be eligible for at least one of the three main trials (the Sexual Function Trial, the Physical Function Trial, or the Vitality Trial). The Testosterone Trials are the largest controlled trials of testosterone to date. These men were assigned to testosterone or placebo gel for 1 year and the dose was adjusted to maintain testosterone concentrations within the normal range for healthy young men. The Physical Function Trial of the TTrials recruited older men with self-reported difficulty walking or climbing stairs and walking speed less than 1.2 m/second and an average of two morning fasting testosterone levels less than 275 ng/dL. There was no significant difference in gains in 6-minute walking distance between men assigned to the testosterone or placebo arms. Statistically significant differences in the change from baseline in 6-minute walking distance were detected when all men enrolled in the TTrials were included; the magnitude of the effect was small. The changes in self-reported physical function, assessed using the physical component of MOS SF36 (PF10), were significantly greater in the testosterone-treated men than in placebo-treated men (180). The men in the testosterone arm were more likely to report that their walking ability was significantly improved from baseline, suggesting that these small effects may be clinically meaningful. The number of falls was similar in the testosterone and placebo arms.

 

Innovative strategies to translate gains in muscle mass and strength induced by testosterone into functional improvements are needed (7). Resistance exercise training has been demonstrated to augment the anabolic effects of androgens on muscle mass and performance and physical function (181). Thus, adjunctive exercise training might be required to induce the neuromuscular and behavioural adaptations that are necessary to translate the gains in muscle mass and strength into clinically-meaningful functional improvements (7). In addition, there is some evidence that the anabolic response of skeletal muscle to dietary protein attenuates with age (182,183)calling into question whether the current recommended dietary allowance (RDA) for protein (0.8 g/kg/day) is adequate to preserve lean body mass and physical function in older adults. In a recent clinical trial, functionally limited older men with usual protein intake less than or equal to RDA for protein were randomized for 6 months to controlled diets with 0.8 g/kg/day of protein plus placebo, 1.3 g/kg/day of protein plus placebo, 0.8g/kg/day of protein plus testosterone and 1.3g/kg/day of protein plus testosterone (155). This trial demonstrated that higher protein intake exceeding the RDA did not increase lean body mass, muscle performance or physical function nor augmented the anabolic response to testosterone. Thus, these findings suggest that the current RDA for protein is adequate to maintain lean body mass and higher protein intake above the RDA does not promote additional gains in muscle mass or physical function with or without testosterone supplementation.

 

MECHANISMS OF ANDROGEN ACTION ON MUSCLE

 

Testosterone-induced increase in muscle mass is associated with hypertrophy of both type I and II muscle fibers (184). The absolute number and the relative proportion of type I and type II fibers do not change during testosterone administration. Testosterone-induced muscle fiber hypertrophy is associated with dose-dependent increases in myonuclear number and satellite cell number (185), suggesting that testosterone administration increases the number of muscle progenitor cells.

 

Testosterone administration has been shown to increase fractional muscle protein synthesis and improve the reutilization of amino acids (157,158). The effects of testosterone on muscle protein degradation have not been well studied. However, the muscle protein synthesis hypothesis does not explain the reciprocal decrease in fat mass or the increases in myonuclear and satellite cell number that occur during testosterone administration (185). Testosterone promotes the differentiation of mesenchymal multipotent muscle progenitor cells into the myogenic lineage and inhibits the differentiation of these precursor cells into the adipogenic lineage (186,187). Thus, testosterone promotes the formation of myosin heavy chain II positive myotubes in multipotent cells and up-regulates markers of myogenic differentiation, such as MyoD and myosin heavy chain (186,187). Testosterone and DHT inhibit adipogenic differentiation and downregulate markers of adipogenic differentiation, such as PPAR-¡and C/EBPµ  (188).

 

Testosterone’s effects on myogenic differentiation are mediated largely through its binding to the classical androgen receptor, which induces a conformational change in the androgen receptor protein, promoting its association with its co-activator, beta-catenin, causing the complex to translocate into the nucleus (187,189). The androgen receptor – beta-catenin complex associates with TCF-4 and activates a number of Wnt target genes (187,189), including follistatin. Follistatin cross-communicates the signal from the AR-beta- catenin pathway to the TGF-beta signaling pathway, blocking signaling through the TGF-beta / Smad 2/3 (188,190). Follistatin plays an essential role in mediating the effects of testosterone on myogenic differentiation (190,191). In a remarkable discovery, Jasuja et al (191)found that the administration of recombinant follistatin selectively increased muscle mass and decreased fat mass but had no effect on prostate growth. Recombinant follistatin and testosterone each regulated the expression of a large number of common genes in the skeletal muscle, but they differed substantially in the expression profile of genes activated in the prostate (191). Among the genes activated differentially by testosterone but not by follistatin in the prostate, Jasuja et al (191)identified polyamine pathway as an important signaling pathway. The polyamine pathway has been known to be involved in regulating prostate growth. Administration of testosterone in combination with an inhibitor of ornithine decarboxylase-1, a key enzyme in the polyamine pathway, to castrated male mice restored levator ani muscle mass but not prostate mass, indicating that ODC1 plays an important role in mediating the effects of testosterone on the prostate (Figure 7) (191). Therefore, combined administration of testosterone plus ODC1 inhibitor provides a novel approach for achieving selectivity of testosterone’s anabolic effects on the muscle while sparing the prostate (191).

Figure 7. Testosterone Plus Ornithine Decarboxylase 1 Inhibitor as a Selective Prostate Sparing Anabolic Therapy. Intact and castrated adult male mice were treated for 2-weeks with vehicle or testosterone with and without α-difluoromethylornithine (DFMO), a specific Odc1 inhibitor, as follows: Intact, castrated (Cx), castrated + 15µg/day T (Cx+T), castrated +15µg/day T+ 15µg/day DFMO (Cx+T+DFMO). Levator ani weights (right panel) in mice treated with testosterone plus DFMO were similar to those in intact controls and testosterone-treated castrated mice. Prostate weights in castrated mice were lower than in intact controls and were restored by testosterone administration to levels seen in intact mice (left panel). Mice treated with testosterone plus DFMO had significantly lower prostate weights than intact controls or castrated mice treated with testosterone alone, but not significantly different from those in castrated mice treated with vehicle alone. Thus, testosterone plus ODC1 inhibitor could serve as prostate-sparing selective anabolic therapy. Reproduced with permission from Jasuja et al. Aging Cell. 2014 Apr;13(2):303-10.

THE ROLE OF STEROID 5-ALPHA REDUCTASE AND DHT IN MEDIATING ANDROGEN EFFECTS IN MUSCLE

Although the enzyme steroid 5-alpha-reductase is expressed at low concentrations within the muscle (192,193), we do not know whether conversion of testosterone to dihydrotestosterone is required for mediating the androgen effects on the muscle. Men with benign prostatic hypertrophy who are treated with a 5-alpha reductase inhibitor do not experience muscle loss (194). Similarly, individuals with congenital 5-alpha-reductase deficiency have normal muscle development at puberty (194). These data suggest that 5-alpha reduction of testosterone to DHT is not obligatory for mediating its effects on the muscle. However, all the kindred with steroid 5-alpha reductase deficiency that have been published to-date have had mutations of type 2 isoform of the enzyme. Similarly, finasteride is a weak inhibitor of only the type 2 isoform of the enzyme. The circulating concentrations of DHT in male patients with congenital mutation of type 2 steroid 5-alpha reductase enzyme or in men treated with finasteride are lower than eugonadal men; however, these patients still produce significant amounts of DHT and their circulating DHT concentrations are often in the lower end of the male range. Long-term administration of dutasteride, a dual and potent inhibitor of both 5-alpha reductase isoforms, has not been associated with significant reductions in bone mineral density (194); the data on the effects of dutasteride on muscle mass are not available. This issue is important because if 5-alpha reduction of testosterone to DHT were not obligatory for mediating its anabolic effects on the muscle, then it might be beneficial to administer testosterone with an inhibitor of steroid 5-alpha reductase or to develop selective androgen receptor modulators that do not undergo 5-alpha reduction.

 

To determine whether testosterone’s effects on muscle mass and strength, sexual function, hematocrit, prostate, sebum production, and lipids are attenuated when its conversion to DHT is blocked, we administered to healthy men, 18-50 years, a long-acting GnRH-agonist to suppress endogenous testosterone. We randomized them to placebo or dutasteride (dual inhibitor of steroid 5-alpha reductase type 1 and 2) 2.5-mg daily, plus 50, 125, 300, or 600-mg testosterone enanthate weekly for 20-weeks (195). Changes in lean and fat mass, leg-press and chest-press strength, were related to testosterone dose but did not differ between placebo and dutasteride groups (195). The relationship between testosterone concentrations and the change in lean body mass, maximum voluntary muscle strength, hematocrit, and sebum production were similar between dutasteride and placebo arms (Figure 8) (195). Changes in sexual-function scores, bone markers, prostate volume, and PSAdid not differ between groups (195). These data indicate that testosterone’s conversion to DHT is not essential for mediating its effects on muscle mass and strength, sexual function, hematocrit, or sebum in men over the range of testosterone concentrations achieved in this trial (195). These data are consistent with studies that have reported that administration of steroid 5α-reductase inhibitors has little or no effect on muscle or bone mass (196-198).

 

Figure 8. The Role of 5-alpha-Dihydrotestosterone in Men. In this randomized trial, healthy men, 18-50 years, received a long-acting GnRH-agonist to suppress endogenous testosterone. They were then randomized to either placebo or dutasteride (dual inhibitor of steroid 5-alpha reductase types 1 and 2) 2.5-mg daily, plus 50, 125, 300, or 600-mg testosterone enanthate weekly for 20-weeks (535). Changes in fat-free mass (upper panel) and leg-press strength (lower panel), were related to testosterone dose but did not differ between placebo and dutasteride groups (535). The relationship between change in total testosterone (TT) levels and change in fat-free mass and leg press strength (right panels) did not differ between men assigned to placebo or duatsteride arms. Reproduced with permission from Bhasin et al, JAMA. 2012 Mar 7;307(9):931-9.

THE ROLE OF CYP19A1 (AROMATASE) IN MEDIATING TESTOSTERONE’S EFFECTS ON MUSCLE

 

Studies of aromatase knockout mice have revealed higher fat mass and lower muscle mass in mice that are null for the P450-linked CYP19 aromatase gene (199). Similarly, humans with CYP19A1 mutations have decreased muscle mass and increased fat mass, and they exhibit insulin resistance (200). Data from these gene-targeting experiments suggest that aromatization of testosterone to estradiol might also be important in mediating androgen effects on body composition. Finkelstein et al (201)have recently examined the relative roles of testosterone and estradiol in regulation of muscle and fat mass, and sexual function. These investigators found that testosterone’s effects on lean mass, muscle size, and strength were not significantly attenuated when its conversion to estradiol was blocked by administration of an aromatase inhibitor (201).

 

Regulation of Fat Mass, Fat Distribution, and Metabolism by Testosterone

 

Testosterone is an important regulator of fat mass and distribution. Lowering testosterone concentrations by administration of a GnRH agonist increases fat mass, and testosterone administration decreases whole body fat mass (147,202-204). The loss of fat mass during testosterone administration occurs both in the appendices as well as the trunk and is distributed evenly between the superficial subcutaneous and deep intra-abdominal and intermuscular compartments (150,203). The effects of testosterone on whole body fat mass are related to the administered testosterone dose and the circulating testosterone concentrations (150,203).

 

MECHANISMS OF TESTOSTERONE’S EFFECTS ON FAT MASS AND METABOLISM

 

The effects of testosterone on fat mass are mediated through its conversion to estradiol by the aromatase enzyme encoded by CYP19A1 (201).  Men with inactivating mutations of CYP19A1 are characterized by increased fat mass, metabolic syndrome, hepatic steatosis, and insulin resistance (205-207). Estradiol replacement of male aromatase knockout mice reverses the adiposity and metabolic abnormalities associated with estrogen deficiency (208).

 

Testosterone regulates adipose tissue mass and metabolism through multiple mechanistic pathways. Androgens inhibit adipogenic differentiation of multipotent mesenchymal progenitor cells; these effects are blocked by androgen receptor blocker, bicalutamide (187,188,209). Testosterone regulates fat oxidation but does not appear to affect triglyceride secretion at least over short durations (210).

 

Testosterone, after its aromatization to estradiol, acts through the estrogen receptors in specific brain regions to regulate eating behavior, energy expenditure, and adipose tissue metabolism. The deletion of estrogen receptor α (ER-α) in specific brain regions is associated with adiposity, hyperphagia, and hypometabolism (211); estradiol acting through ER-α regulates eating behavior and energy expenditure differentially through actions on different hypothalamic neurons (211). Activation of estrogen receptor β (ER-β) by selective agonists inhibits weight gain, adiposity, increases energy expenditure and thermogenesis, and reverses hepatic steatosis in mice through direct effects on xenobiotic and bile acid receptors in the liver (212).

 

Testosterone and Sexual Function in Older Men

 

REGULATION OF SEXUAL FUNCTION BY TESTOSTERONE

 

Sexual function in men is a complex process that includes central mechanisms for regulation of sexual desire and arousability, and local mechanisms for penile tumescence, orgasm, and ejaculation (213). Primary effects of testosterone are on sexual interest and motivation (213-218). Testosterone replacement of young, androgen deficient men improves a wide range of sexual behaviors including frequency of sexual activity, sexual daydreams, sexual thoughts, feelings of sexual desire, spontaneous erections, and attentiveness to erotic stimuli (213-221). Kwan et al (217)demonstrated that androgen-deficient men have decreased frequency of sexual thoughts and lower overall sexual activity scores; however, these men can achieve erections in response to visual erotic stimuli. Hypogonadal men have lower frequency and duration of the episodes of nocturnal penile tumescence; testosterone replacement increases both the frequency and duration of sleep-entrained, penile erections (219-221). Although both orgasm and ejaculation are believed to be androgen-independent, hypogonadal men have decreased ejaculate volume and their orgasm may be delayed.

 

Although hypogonadal men can achieve erections, it is possible that achievement of optimal penile rigidity might require physiologic testosterone concentrations. Testosterone regulates nitric oxide synthase activity in the cavernosal smooth muscle (222). Testosterone administration in orchiectomized rats increases penile blood flow and has trophic effects on cavernosal smooth muscle (223-225).

 

In male rodents, all measures of mating behavior are normalized by relatively low testosterone levels that are insufficient to maintain prostate and seminal vesicle weight (226,227). Similarly, in men, sexual function is maintained at relatively low normal levels of serum testosterone (201,218,228). Testosterone’s effects on libido may be mediated through its conversion to estradiol (201).

 

RELATIONSHIP OF ANDROGEN DEFICIENCY AND SEXUAL DYSFUNCTION IN MIDDLE-AGED AND OLDER MEN

 

Total and free serum testosterone levels are positively associated with sexual desire, erectile function and sexual activity in older men with unequivocally low testosterone levels and symptoms of sexual dysfunction (103). These findings suggest that low testosterone levels may contribute to impaired sexual functioning in older men, and these men may benefit from testosterone therapy.

 

Erectile dysfunction and androgen deficiency are two common but independently distributed, clinical disorders that sometimes co-exist in the same patient (101,102,213,229). Hypogonadism is a clinical syndrome that results from androgen deficiency (5); in contrast, erectile dysfunction is usually a manifestation of a systemic vasculopathy, often of atherosclerotic origin. Thus, androgen deficiency and erectile dysfunction have distinct pathophysiology. Eight to ten percent of middle-aged men presenting with erectile dysfunction have low testosterone levels (102,229-231).

 

CLINICAL TRIALS OF THE EFFECTS OF TESTOSTERONE THERAPY ON SEXUAL FUNCTION OF OLDER MEN WITH LOW CIRCULATING TESTOSTERONE CONCENTRATIONS

 

In mostly open-label trials, testosterone treatment has been shown to improve sexual function in young men with classical hypogonadism due to disorders of the hypothalamus, pituitary or testes (147,232). However, previous trials evaluating the benefits of testosterone therapy in men 60 years and older with age-related decline in testosterone levels on sexual functioning have yielded inconsistent results (233), with some studies showing improvement (234,235), while others have suggested no clear benefit (12). The inconsistencies in these previous studies are due to several factors, including small sample sizes, inclusion of men who were not clearly hypogonadal or did not have sexual symptoms, inclusion of men with heterogeneous sexual disorders, variable treatment durations, and the use of outcomes assessment tools that had not been rigorously validated.

 

Only a small number of placebo-controlled trials of testosterone have been conducted in men with sexual symptoms and low testosterone levels (13,15,149). In these trials, testosterone replacement was associated with a small but significant increase in sexual desire, erectile function, and sexual satisfaction.  A meta-analysis of these placebo-controlled trials found that testosterone replacement of hypogonadal men was associated with a small but significant increase in sexual desire [standardized mean difference (SMD): 0.17; 95% CI, 0.01, 0.34], erectile function (SMD: 0.16; 95% CI, 0.06, 0.27), and sexual satisfaction (SMD: 0.16; 95% CI, 0.01, 0.31) (236).

 

The Sexual Function Trial of the TTrials determined the efficacy of testosterone treatment for 1-year on sexual function in symptomatic, but relatively healthy older men ≥65 years with low testosterone levels (15). Testosterone administration for 1-year raised testosterone concentrations into a range that is mid-normal for healthy young men and was associated with significant improvements in sexual activity, desire, and erectile function; the treatment effects tended to wane over time, and the effect on erectile function was smaller than that reported with phosphodiesterase 5 inhibitors. The magnitude of increase in testosterone levels was related to the improvements in sexual activity and desire, but not erectile function (237). There was no clear testosterone threshold level of effect. The effects of testosterone replacement on erectile function were substantially smaller than those reported in older men taking phosphodiesterase type 5 inhibitors (238).

 

Testosterone does not improve any domain of sexual function in middle-aged and older men who have normal testosterone levels and do not have any sexual symptoms (12). Testosterone replacement therapy does not improve ejaculatory function in men with an ejaculatory disorder (239).

 

It had been speculated that testosterone administration might improve erectile response of men with ED to selective phosphodiesterase inhibitors (240-242). To determine whether the addition of testosterone to a phosphodiesterase-5-inhibitor improves erectile response, we conducted a randomized, placebo-controlled trial (243), in men, 40-to-70 years, with erectile dysfunction and low total testosterone< 11.5 nmol/L (330ng/dL) and/or free testosterone <173.5 pmol/L (50pg/mL). All participants were initially started on sildenafil alone and the sildenafil dose was optimized based on their response during a 3 to 7-week run-in period (243). The participants were then randomized to 10-g testosterone or placebo gel for 14-weeks in combination with the optimized sildenafil dose (243). The administration of sildenafil alone was associated with substantial increases in erectile function domain (EFD) score and total and satisfactory sexual encounters (243). However, the change in EFD score in men assigned to testosterone plus sildenafil did not differ significantly from that in men assigned to placebo plus sildenafil (243). Changes in total and successful sexual encounters, quality-of-life, and marital-intimacy did not differ between testosterone and placebo groups. Even among the subsets of men with baseline testosterone <250ng/dL or those without diabetes, there were no significant differences in EFD scores between the two arms (243). Another placebo-controlled trial of men with erectile dysfunction who were non-responders to tadalafil also did not show a greater improvement in erectile function in men assigned to the testosterone arm than in those assigned to the placebo arm (242). Thus, in randomized trials, the addition of testosterone to PDE5Is has not been shown to improve erectile function in men with erectile dysfunction (242,243).

 

SYNOPSIS OF THE EFFECTS OF TESTOSTERONE ON SEXUAL FUNCTION

 

Androgen deficiency is an important cause of low sexual desire disorder (213). Therefore, serum testosterone concentrations should be measured in the diagnostic evaluation of hypoactive sexual desire disorder, recognizing that low sexual desire is often multifactorial; systemic illness, relationship and differentiation (the ability of individuals in a relationship to maintain their distinct identities) issues, depression, and many medications can be important antecedents or contributors to low sexual desire and sexual dysfunction. In older hypogonadal men with low sexual desire, testosterone treatment improves sexual desire, erectile function, and overall sexual activity.

 

Testosterone Effects on Bone Mineral Metabolism

THE EFFECTS OF ANDROGEN DEFICIENCY ON BONE MASS

 

Testosterone deficiency is associated with a progressive loss of bone mass (244-247). In one study performed in sexual offenders (244), surgical orchiectomy was associated with a progressive decrease in bone mineral density of a magnitude similar to that seen in women after menopause. Similarly, androgen deficiency induced by the administration of a GnRH agonist, surgical orchiectomy, or an androgen antagonist for the treatment of prostate cancer leads to loss of bone mass (245-247). In male rats, surgical orchiectomy or androgen blockade by administration of an androgen receptor antagonist is associated with loss of bone mass (248).

 

Androgen deficiency that develops before the completion of pubertal development is associated with reduced cortical and trabecular bone mass (249,250). During the pubertal years, significant bone accretion occurs under the influence of sex steroids; therefore, individuals with sex-steroid deficiency before or during peri-pubertal years may end up with suboptimal peak bone mass. Similarly, men with acquired androgen deficiency have lower bone mineral density than age-matched controls (143).

 

CLINICAL TRIALS ON THE EFFECTS OF TESTOSTERONE THERAPY ON BONE IN YOUNG, HYPOGONADAL MEN

 

Testosterone therapy of healthy, young, hypogonadal men is associated with significant increases in vertebral bone mineral density (144,251-255). However, bone mineral density is typically not normalized after 1-2 years of testosterone replacement therapy (144). The reasons for the failure of testosterone replacement therapy to normalize bone mineral density in androgen-deficient men are not entirely clear. Some hypogonadal participants patients included in these testosterone trials had panhypopituitarism and also suffered from growth hormone deficiency. It is possible that concomitant GH replacement might be necessary for restoration of normal bone mineral density. Excessive glucocorticoid replacement might also contribute to bone loss in these patients. In addition, some participants had experienced testosterone deficiency before the onset and completion of pubertal development. Because maximal bone mass is achieved in part through bone accretion during the peripubertal period under the influence of sex-steroid hormones, the individuals who develop androgen deficiency during the critical pubertal developmental window of bone accretion, may end up with decreased peak bone mass, and testosterone administration may not be able to restore bone mass to levels seen in eugonadal age-matched controls. Many testosterone replacement trials were less than 3 years in duration, and it is possible that a longer period of testosterone administration might be necessary to achieve maximal improvements in bone mineral density. Indeed, Behre et al (251)reported that bone mineral density in some hypogonadal men continued to increase even after many years of testosterone treatment using a scrotal transdermal patch and reached the levels expected for age-matched eugonadal controls.

 

CROSS-SECTIONAL STUDIES OF THE RELATIONSHIP BETWEEN SEX-HORMONE CONCENTRATIONS AND OSTEOPOROSIS IN OLDER MEN

 

The age-related decline in sex hormones is associated with age-related changes in bone mineral density and increased risk of osteoporotic fractures (120-125,256,257). Older men with hip fractures have lower testosterone levels than age-matched controls (258). In general, epidemiologic studies have reported bioavailable testosterone and estradiol levels to be more strongly associated with bone mineral density of the spine, hip, and distal radius than total testosterone levels (121,123,124,257).

 

CLINICAL TRIALS OF THE EFFECTS OF TESTOSTERONE THERAPY ON BONE OF OLDER MEN WITH LOW CIRCULATING TESTOSTERONE CONCENTRATIONS

 

Earlier studies of testosterone replacement of relatively healthy older men have examined the effects of testosterone on bone mineral density but have reported inconsistent results (172,174,259,260). One study found greater increases in vertebral bone mineral density in the testosterone arm of the trial than in the placebo arm, while another study did not find any significant differences between the change in vertebral or femoral bone mineral density between testosterone and placebo groups (260). Another study reported greater gains in bone mineral density of the femoral neck but not of other regions in men randomized to receive testosterone compared to those who received placebo. A meta-analysis of randomized trials found a significantly greater increase in lumbar bone mineral density but not in femoral bone mineral density in the testosterone arms of trials that used intramuscular testosterone than in placebo arms (Figure 9) (261); transdermal testosterone had no significant effect.

Figure 9. The effects of testosterone therapy on bone health in intervention trials. Panel A shows the effects of testosterone therapy on lumbar and femoral bone mineral density in a meta-analysis of randomized trials (data derived from a meta-analysis by Tracz et al, J Clin Endocrinol Metab. 2006;91(6):2011-6.; figure adapted with Spitzer et al. Nat Rev Endocrinol. 2013;9(7):414-24). Panels B and C show the effects of testosterone replacement for 12 Months on volumetric bone mineral density and estimated bone strength of trabecular, peripheral, and whole bone of the spine and hip, as assessed by quantitative computed tomography (figure reproduced with permission from Snyder et al. JAMA Intern Med. 2017;177(4):471-479).

The Bone Trial of the TTrials determined the effects of testosterone replacement for 1-year in men 65 years or older with low testosterone levels on volumetric bone mineral density and bone strength using quantitative computed tomography (262). This trial found significant increases in volumetric bone mineral density and estimated bone strength in the testosterone arms compared to placebo; specifically, these increases were most prominent in the spine than hip and more in trabecular than peripheral bone (Figure 9). The treatment effects on volumetric bone density and bone strength observed in the TTrials compare favorably with those reported in trials of bisphosphonates and some selective estrogen receptor modulators. Future studies are needed to determine whether these improvements from testosterone treatment are associated with reduced fracture risk in older men with low testosterone levels.

 

MECHANISMS OF ANDROGEN ACTION ON THE BONE

 

Testosterone increases bone mass by several mechanisms (263). Short-term studies of androgen replacement have shown inconsistent increases in markers of bone formation, but a more consistent reduction in markers of bone resorption (255,263-265). These observations suggest that testosterone increases bone mineral density in part through its aromatization to estrogen, which inhibits bone resorption. Estrogen deficiency contributes to increased bone resorption and remodeling by multiple mechanisms. Estrogens regulate the activation frequency of bone functional basic multicellular units, the duration of the resorption phase and the formation phase, and osteoclast recruitment (266). The protective effects of estrogen on bone in both male and female mice during growth and maturation are mediated largely through estrogen receptor-alpha (267-273). Dias and colleagues found that treatment with testosterone for 12-months improved lumbar spine bone mineral density compared to placebo but not in men treated with anastrozole, suggesting that aromatization of testosterone to estrogen may be required for maintaining bone mineral density (274). Similarly, another recent study found significant reduction in spine bone mineral density in men treated with testosterone and anastrozole for 16-weeks that was independent of testosterone dose (275).In addition, treatment with lower testosterone doses were associated with greater increases in bone turnover markers; an effect that was significantly greater in combination with anastrozole. There is increasing evidence that testosterone also directly stimulates osteoblastic bone formation. Androgen receptors have been demonstrated on osteoblasts and on mesenchymal stem cells (276). Testosterone stimulates cortical bone formation (277).

Testosterone also stimulates the production of several growth factors within the bone, including IGF-1; these growth factors may contribute to bone formation (278). Testosterone increases muscle mass, which may indirectly increase bone mass by increased loading. Testosterone might inhibit apoptosis of osteoblasts through non-genotropic mechanisms (279,280). In addition to its effects on bone mineral density, testosterone might reduce fall propensity because of its effects on muscle strength and reaction time.

 

In men androgens and estrogens both play independent roles in regulating bone resorption (266). Estradiol levels above 10 pg/ml and testosterone levels above 200 ng/dl are generally sufficient to prevent increases in bone resorption and decreases in BMD in men (275).

 

SYNOPSIS OF THE EFFECTS OF TESTOSTERONE ON BONE

 

Testosterone replacement has been shown to increase vertebral bone mineral density in young and older men with unequivocally low testosterone levels (5). Testosterone increases bone mass by multiple mechanisms. Testosterone’s aromatization to estrogen plays an important role in regulating bone health in men. Testosterone’s effects on fracture risk have not been studied.

 

Testosterone Effects on Cognitive Function

 

CROSS-SECTIONAL AND LONGITUDINAL STUDIES CORRELATING SEX-HORMONE LEVELS AND COGNITIVE FUNCTION

 

Testosterone is aromatized to estrogen in the brain, and some effects of testosterone on cognition might be mediated through its conversion to estradiol. Androgen receptors are expressed in the brain (281), and androgen effects on brain organization during development (282,283)are mediated through androgen receptor. Androgens increase neurite arborization, facilitating intercellular communication (282-285). Testosterone also affects serotonin, dopamine, acetylcholine (284), and calcium signaling (285).

 

An age-related decline in serum testosterone levels has been associated with impairment in cognitive function (286). Androgens effects on cognitive function are domain-specific. For instance, observations that men outperform women in a variety of visuo-spatial skills suggest that androgens enhance visuo-spatial skills (287). In !Kung San hunter-gatherers of Southern Africa, testosterone, but not estradiol, levels correlated with better spatial ability and with worse verbal fluency (288). Women with congenital adrenal hyperplasia with high androgen levels score higher on tests of spatial cognition than their age- and gender-matched siblings (289). 46, XY rats with androgen insensitivity perform worse on tests of spatial cognition than their age-matched controls (290). Other studies have reported a complex relationship between androgen levels and spatial ability (112,291-293). Circulating levels of dihydrotestosterone, a metabolite of testosterone that is not converted to estrogen, positively correlated with verbal fluency (288). Barrett-Conner et al (111)found positive associations between total and bioavailable testosterone levels, and global cognitive functioning and mental control, but not with visuospatial skills. In the Baltimore Longitudinal Study of Aging (294), higher free testosterone index was associated with better scores on visual and verbal memory, visuospatial functioning, and visuomotor scanning. Men with low testosterone levels had lower scores on visual memory and visuospatial performance (294). In contrast, other studies have shown no association of serum testosterone levels with domains of visual and verbal memory, and executive function in older men (295,296). Neither total testosterone level nor the free testosterone index was correlated with verbal knowledge, mental status, or depressive symptoms (294). Recently, in the Concord Health and Aging in Men Project, the authors found that changes in serum testosterone levels over time, rather than baseline testosterone levels, were predictive of cognitive decline (286).

 

INTERVENTION TRIALS OF THE EFFECTS OF TESTOSTERONE SUPPLEMENTATION ON COGNITIVE FUNCTION

 

Several small clinical trials in elderly hypogonadal men have provided conflicting results (107,110,297-301); not surprisingly, a systematic review of clinical trials revealed no significant effects of testosterone on cognition (5). Janowsky et al (107)tested verbal and visual memory, spatial cognition, motor speed and cognitive flexibility in a group of healthy older men who received 3 months of testosterone supplementation. Testosterone replacement was associated with a significant improvement in spatial cognition only. Serum testosterone levels were not significantly correlated with spatial performance, but estradiol levels showed a significant inverse relationship with spatial performance suggesting that estradiol might inhibit spatial ability. Vaughan et al (297)found no effect of testosterone administration on cognition, while Cherrier et al (298-300)reported an effect on visuo-spatial cognition. Testosterone also enhanced verbal fluency. Hypogonadal men performed worse on tests of verbal fluency than eugonadal men, and showed improvement after testosterone replacement (110,301,302). In transsexual males (303), administration of anti-androgen and estrogen, prior to surgery for gender reassignment, decreased anger and aggression proneness, sexual arousability, and spatial skills, and increased verbal fluency ability. Conversely, testosterone administration to females decreased verbal fluency and increased spatial skills. Testosterone administration may also improve verbal memory in women (304).

 

In a double-blind randomized placebo-controlled trial, Huang et alinvestigated the effect of testosterone administration for 3-years on multiple domains of cognitive function in a large cohort (n=280) of men 60 years and older with low or low-normal testosterone levels (305). In this trial of older, cognitively healthy men, testosterone administration was not associated with significant improvement in any domain of cognitive function (Figure 10). These findings are similar to another recent placebo-controlled clinical trial conducted in older men 65-years and older (n=493) with low testosterone levels, which showed that treatment with testosterone for 1-year was not associated with improved cognitive function or memory (Figure 10) (306). Sensitivity analysis that were limited to men with minimal cognitive impairment also did not find significant differences in measures of cognition between the testosterone and placebo groups (306).

 

Figure 10. Effects of testosterone therapy on cognition domains in older men. Left panels show the long-term effects of testosterone therapy in visual and verbal memory, spacial ability and executive function in the TEAAM trial (36 months of treatment). Data displayed as baseline and post-randomization cognitive function test scores by group and study visit. Error bars are 95% CIs for mean scores and p-values are for the estimated difference between treatment effects, controlling for baseline values, age, and education (figure adapted from Huang et al. J Clin Endocrinol Metab. 2018;103(4):1678-1685.) Right panels show adjusted mean change from baseline to 6 months and 12 months for men with age-associated memory impairment by treatment group in cognition domains in the Cognitive Function Trial of the TTrials (12 months duration; figure adapted from Resnick et al. JAMA. 2017;317(7):717-727).

SYNOPSIS OF THE EFFECTS OF TESTOSTERONE ON COGNITION

 

The literature on testosterone and cognition is highly equivocal; more recent larger and longer-term studies have shown no clear benefit in older men with low testosterone levels. The inconsistency in findings cannot yet be interpreted as conclusive evidence that there is no effect. Limitations of previous studies include limited sample sizes with heterogeneous, poorly defined samples; short treatment durations; the use of a variety of neuropsychological tests, including some that lack psychometric validation; the use of differing protocols in clinical trials; and inclusion of men with no clear cognitive deficit. The effects of testosterone therapy on clinically important outcomes in men with cognitive impairment have not been studied. Furthermore, the efficacy of testosterone replacement in men with cognitive impairment, such as in patients with Alzheimer’s disease, needs further investigation in larger randomized controlled trials.

 

 

Circulating testosterone concentrations have not been consistently associated with major depressive disorder in men (117,118,307-310). Rather, circulating testosterone levels appear to be associated with a late-life low grade persistent depressive disorder (dysthymia) (117,118,307-310). Intervention trials have failed to demonstrate statistically significant or clinically meaningful improvements in patients with major depressive disorder (311). Placebo-controlled trials of testosterone in men with refractory depression have not consistently shown a beneficial effect of testosterone (311-314). A meta-analysis of randomized clinical trials did not reveal a clinically meaningful effect of testosterone on depression (315). Two small trials in men with dysthymia have reported greater improvements in depressive symptoms in testosterone-treated men than in placebo-treated men (316,317).

 

Meta-analyses of randomized trials have reported modest improvements in depressive symptoms in testosterone-treated men than in placebo-treated men, but there is no convincing evidence that testosterone treatment can induce remission in men with major depressive disorder. Adequately-powered long term randomized trials are needed to determine whether testosterone replacement therapy can induce remission in older hypogonadal men with late-onset, low grade persistent depressive disorder (dysthymia).

 

There is anecdotal evidence that androgens improved energy and reduced sense of fatigue (318). Testosterone administration increases hemoglobin and red cell mass, stimulates 2, 3 BPG concentrations thereby shifting the oxygen – hemoglobin dissociation curve favorably to improve greater oxygen delivery, and induces muscle capillarity (319-321). Additionally, testosterone stimulates mitochondrial biogenesis and mitochondrial quality (322). All of these adaptations would be expected to improve net oxygen delivery to the muscle, improve aerobic performance and reduce fatigability. The effects of testosterone on fatigue and vitality have been studied in some randomized trials. Endogenous levels of total and free testosterone are not significantly associated with vitality in older hypogonadal men with sexual dysfunction, diminished vitality and/or mobility limitation (103). In the Vitality Trial of the TTrials, testosterone treatment for 1-year did not improve vitality in older men with low vitality measured using the Functional Assessment of Chronic Illness Therapy (FACIT)-scale but men receiving testosterone did report small but statistically significant improvements in mood.  These findings are consistent with other randomized controlled studies (12,155,177), showing no clear benefit on fatigue and health-related quality of life with testosterone therapy.

 

Supraphysiologic doses of androgenic steroids such as those abused by athletes and recreational body builders have been associated with aggressive responses to provocative situations (323), increased scores on Young’s manic scale, and with affective and psychotic disorders in some individuals (324); these adverse effects have not been reported with physiologic testosterone replacement.

 

By improving some aspects of physical and sexual function, testosterone supplementation might be expected to improve health-related quality of life. However, only a few small trials have evaluated the effects of testosterone on health-related quality of life. A systematic review of a small number of randomized trials has not revealed a significant improvement in composite health-related quality of life scores, but testosterone therapy improves scores on the physical function domain of SF-36 (5,147).

 

CONSIDERATIONS IN TESTOSTERONE THERAPY OF OLDER MEN WITH LOW TESTOSTERONE LEVELS

 

Recent large randomized trials, especially the TTrials have substantially expanded our understanding of the efficacy and short-term safety of testosterone in older men. However, the long-term safety and benefits of long-term testosterone therapy on health-related outcomes in older men with symptomatic conditions associated with low testosterone levels still remain incompletely understood. None of the trials has been long enough or large enough to determine the effects of testosterone treatment on major adverse cardiovascular events and prostate cancer risk. Furthermore, the long-term efficacy of testosterone treatment in improving hard outcomes – physical disability, fractures, falls, progression to dementia, progression from prediabetes to diabetes, remission of late-life low grade persistent depressive disorder (dysthymia) and health-related quality of life remains to be established. Adherence with testosterone treatment is poor and in one survey, nearly 50% of men prescribed testosterone, discontinued treatment within 3 months.

 

Recognizing the lack of evidence of the safety and long-term efficacy of testosterone therapy in older men with symptomatic androgen deficiency, the expert panel of the Endocrine Society recommended against testosterone therapy of all men 65 years or older with low testosterone levels (5). Instead, the panel suggested that “in men >65 years who have symptoms or conditions suggestive of testosterone deficiency (such as low libido or unexplained anemia) and consistently and unequivocally low morning testosterone, clinicians offer testosterone therapy on an individualized basis after explicit discussion of the potential risks and benefits” (5). The panel’s recommendations were guided mainly by the sober realization that high quality evidence of the cardiovascular and prostate safety will not be available for a very long time.

 

Population level screening of all older men for androgen deficiency is not justified because of the lack of agreement on a case definition, the paucity of data on the performance characteristics of the screening instruments (e.g., the ADAM questionnaire (325), the Aging Male Symptoms questionnaire (326), and the MMAS questionnaire (327)and the lack of clarity on the public health impact of the androgen deficiency syndrome in the general population.

 

Prior to prescribing testosterone therapy, a careful general health evaluation is necessary to identify any potential conditions that might increase the risk of testosterone therapy. An explicit discussion of the uncertainties about the benefits and risks of testosterone therapy should precede prescription of testosterone therapy. Men receiving testosterone therapy should be monitored using a standardized monitoring plan to facilitate early detection of adverse events and to minimize the risk of unnecessary prostate biopsies (Table 2), as recommended by the Endocrine Society expert panel (Table 3).

 

Table 2. Potential Adverse Effects of Testosterone Replacement in Older Men
Adverse Events for Which There is Evidence of Association with Testosterone Administration

1.              Erythrocytosis

2.              Acne and oily skin

3.              Detection of subclinical prostate cancer

4.              Growth of metastatic prostate cancer

5.              Reduced sperm production and fertility

Potential Adverse Events for Which There is Weak Evidence of Association with Testosterone Administration

1.     Gynecomastia

2.     Male pattern balding (familial)

3.     Growth of breast cancer

4.     Induction of worsening of obstructive sleep apnea

Formulation Specific Adverse Effects

1.     Oral Tablets (not recommended)

¨                 Effects on liver enzymes and HDL cholesterol (methyltestosterone)

1.     Pellet Implants

¨                 Infection, extrusion of pellet

2.     Intramuscular Injections

¨                 Fluctuations in mood or libido

¨                 Pain at injection site

¨                 Coughing episodes immediately after injection

3.     Transdermal Patches

¨                 Skin reaction at the patch application site

4.     Transdermal Gel

¨                 Potential risk of transference to partner

¨                 Skin irritation and odor at application site

¨                 Stickiness, slow drying, dripping

5.     Buccal Testosterone Tablets

¨                 Alterations in taste

¨                 Irritation of gums

 

Adapted with permission from the Endocrine Society Guideline for Testosterone Therapy in Men With Hypogonadism (Bhasin et al J Clin Endocrinol Metab 2018;103(5):1715-1744).

 

Testosterone therapy can be instituted using any of the available approved formulations based on considerations of pharmacokinetics, patient convenience and preference, cost, and formulation-specific adverse effects.  The testosterone dose and regimen should be adjusted based on measurement of serum testosterone levels after initiation of therapy. The aim should be to raise testosterone levels into the mid-normal range for healthy young men (5).

 

Table 3. Recommendations for Monitoring of Men Receiving Testosterone Therapy
A. Explain the potential benefits and risks of monitoring for prostate cancer and engage the patient in shared decision making regarding the prostate monitoring plan.
B. Evaluate the patient at 3–12 months after treatment initiation and then annually to assess whether symptoms have responded to treatment and whether the patient is suffering from any adverse effects
C. Monitor testosterone concentrations 3–6 months after initiation of therapy:

·       Therapy should aim to raise testosterone into the mid-normal range.

·       Injectable testosterone enanthate or cypionate: measure testosterone midway between injections. If mid-interval T is >600 ng/dL (24.5 nmol/L) or <350 ng/dL (14.1 nmol/L), adjust dose or frequency.

·       Transdermal gels: assess testosterone 2–8 h following the gel application, after the patient has been on treatment for at least 1 week; adjust dose to achieve testosterone in the mid-normal range.

·       Transdermal patches: assess testosterone 3–12 h after application; adjust dose to achieve concentration in the mid-normal range.

·       Buccal T bioadhesive tablet: assess concentrations immediately before or after application of fresh system.

·       Testosterone pellets: measure concentrations at the end of the dosing interval. Adjust the number of pellets and/or the dosing interval to maintain serum T concentrations in the mid-normal range.

·       Oral T undecanoate: monitor serum T concentrations 3–5h after ingestion with a fat-containing meal.

·       Injectable testosterone undecanoate: measure serum T levels at the end of the dosing interval just prior to the next injection and aim to achieve nadir levels in low-mid range.

D. Check hematocrit at baseline, 3–6 months after starting treatment, and then annually. If hematocrit is >54%, stop therapy until hematocrit decreases to a safe level; evaluate the patient for hypoxia and sleep apnea; reinitiate therapy with a reduced dose.
E. Measure BMD of lumbar spine and/or femoral neck after 1–2 year of testosterone therapy in hypogonadal men with osteoporosis, consistent with regional standard of care.
F. For men 55–69 years of age and for men 40–69 years of age who are at increased risk for prostate cancer who choose prostate monitoring, perform digital rectal examination and check PSA level before initiating treatment; check PSA and perform digital rectal examination 3–12 months after initiating testosterone treatment, and then in accordance with guidelines for prostate cancer screening depending on the age and race of the patient.
G. Obtain urological consultation if there is:

·       An increase in serum PSA concentration.1.4 ng/mL within 12 months of initiating testosterone treatment

·       A confirmed PSA > 4 ng/mL at any time

·       Detection of a prostatic abnormality on digital rectal examination

·       Substantial worsening of lower urinary tract symptoms

Adapted with permission from the Endocrine Society Guideline for Testosterone Therapy in Men With Hypogonadism (Bhasin et al J Clin Endocrinol Metab 2018;103(5):1715-1744).

 

RISKS OF TESTOSTERONE ADMINISTRATION IN OLDER MEN

 

Short-term testosterone administration in healthy, young, androgen-deficient men with classical hypogonadism is associated with a low frequency of relatively mild adverse effects such as acne, oiliness of skin, and breast tenderness. However, the long-term risks of testosterone supplementation in older men are largely unknown. There are several unique considerations in older men that may increase their risks of testosterone administration. Serum total and free testosterone concentrations are higher in older men than young men at any dose of testosterone therapy, presumably due to decreased testosterone clearance (50). Older men exhibit greater increments in hemoglobin and hematocrit in response to testosterone administration than young men (328). Altered responsiveness of older men to testosterone administration might make them susceptible to a higher frequency of adverse events, such as erythrocytosis, or to unique adverse events not observed in young hypogonadal men. The baseline prevalence of disorders such as prostate cancer, benign prostatic hypertrophy, and cardiovascular disease that might be exacerbated by testosterone administration is high in older men; therefore, small changes in risk in either direction could have enormous public health impact. Furthermore, the clustering of co-morbid conditions in the frail elderly might render these men more susceptible to the adverse effects of testosterone therapy than healthy young hypogonadal men.

 

The contraindications for testosterone administration include history of prostate or breast cancer (5). Benign prostatic hypertrophy by itself is not a contraindication, unless it is associated with severe symptoms, as indicated by IPSS symptom score of greater than 21. Testosterone should not be given without prior evaluation and treatment to men with baseline hematocrit greater than 50%, severe untreated sleep apnea, or congestive heart failure with Class III or IV symptoms (5). Testosterone suppresses spermatogenesis and should not be prescribed to men who are considering having a child in the near future.

 

The risks of testosterone administration include acne, oiliness of skin, erythrocytosis, induction or exacerbation of sleep apnea, leg edema, transient breast tenderness or enlargement and reversible suppression of baseline spermatogenesis (5) (Table 2). Abnormalities of liver enzymes, hepatic neoplasms, and peliosis hepatis that have been reported previously with orally administered, 17-alpha alkylated androgens, have not been observed with replacement doses of parenterally administered testosterone formulations. The two major areas of concern and uncertainty are the effects of long-term testosterone administration on prostate cancer and cardiovascular events.

 

The Effects of Testosterone on the Risk of Cardiovascular Disease

 

The long-term consequences of testosterone supplementation on the risk of heart disease remain unknown and have been the subject of contentious debate (133,329-332). Some known effects of testosterone such as increase in hematocrit, suppression of plasma HDL cholesterol, salt and water retention, might be expected to increase cardiovascular risk. Some other effects such as testosterone’s vasodilator effect on coronary arteries resulting in increased coronary blood flow, reduction of whole body and abdominal fat mass, and improved brachial reactivity might be perceived as beneficial. Testosterone’s effects on coagulation are complex; testosterone administration is associated with stimulation of both anti-coagulant and pro-coagulant proteins.

 

ANDROGEN EFFECTS ON PLASMA LIPIDS

 

Cross-sectional studies of middle-aged men found a positive relationship between serum testosterone levels and plasma HDL-cholesterol concentrations (331,333-336). Lower testosterone levels in men are associated with higher levels of dense LDL particles (333), triglycerides (336,337)and prothrombotic factors (338).

 

The effects of androgen supplementation on plasma lipids depend on the dose, the route of administration (oral or parenteral), the type of androgen (aromatizable or not) and the subject population (whether young or old, and hypogonadal or not). Supraphysiological doses of testosterone and non-aromatizable androgens frequently employed by bodybuilders undoubtedly decrease plasma HDL-cholesterol levels (339-342). However, administration of replacement doses of testosterone in older men has been associated with only a modest or no decrease in plasma HDL-cholesterol (5,11,12,14,168,170-172,174,343-345), and without significant effect on cholesterol efflux capacity from macrophages (346), suggesting preserved HDL function.

 

ANDROGENS AND OTHER CARDIOVASCULAR RISK FACTORS

 

Cross-sectional studies have found a positive association between circulating testosterone concentrations and tissue plasminogen activator activity (347), and a negative relationship between testosterone and plasminogen activator inhibitor-1 activity, fibrinogen, and some other prothrombotic factors (347), suggesting an antithrombotic effect of testosterone. However, testosterone increases hematocrit (348). Additionally, testosterone administration increases thromboxane A2 receptor density on human platelets, increasing platelet aggregability ex vivo(349,350). Observational studies have not found a consistent relationship between testosterone treatment and the risk of venous thromboembolism (351-355), although one study reported a small increase in VTE risk in the first few months after starting testosterone treatment (351).

 

Cross-sectional studies have reported conflicting findings on the association of endogenous testosterone levels and inflammatory markers (356-361). Intervention trials of testosterone generally have not found a significant effect of testosterone on inflammatory markers (345,362). Even supraphysiological doses of testosterone have been found not to affect C-reactive protein (363). Similarly, a prospective cohort study did not find meaningful changes in inflammatory markers in men with prostate cancer receiving androgen deprivation therapy (364).

 

ANDROGENS AND CORONARY ARTERY DISEASE

 

Whether variation of testosterone within the normal range is associated with risk of coronary artery disease remains controversial. Of the 30 cross-sectional studies reviewed by Alexandersen (133), 18 reported lower testosterone levels in men with coronary heart disease, 11 found similar testosterone levels in controls and men with coronary artery disease and 1 found higher levels of DHEAS. Prospective studies have failed to reveal an association of total testosterone levels and coronary artery disease (134-138,365-367). The common carotid artery intimal media thickness, a marker of generalized atherosclerosis, has been reported to be negatively associated with circulating testosterone levels (138).

 

One interventional study (368), reported that testosterone undecanoate given orally improved angina pectoris in men with coronary heart disease. Testosterone infusion acutely improves coronary blood flow in a canine model and in men with coronary artery disease (369-375). Short-term administration of testosterone induces a beneficial effect on exercise-induced myocardial ischemia in men with coronary artery disease (374). This effect may be related to a direct coronary-relaxing effect. Testosterone replacement has been shown to increase the time to 1-mm ST-segment depression (372). However, in another study, there were no differences between the placebo or testosterone groups in peak heart rate, systolic blood pressure, maximal rate pressure product, perfusion imaging scores, or the onset of ST-segment depression (374). Yue et al (375)reported that testosterone induces endothelium-independent relaxation of rabbit coronary arteries via potassium conductance. Testosterone is a potent vasodilator; it induces nitric oxide synthesis in human aortic endothelial cells in vitro (376).Testosterone has been shown to be an inhibitor of L-type Ca2+channel.In human cells transfected with α1Csubunit of the human cardiovascular L-type Ca2+channel, testosterone inhibits these calcium channels with a potency that is similar to that of dihydropyridine calcium channel blockers (377).

 

EFFECTS OF TESTOSTERONE SUPPLEMENTATION ON ATHEROSCLEROSIS PROGRESSION

 

In some animal models, orchiectomy accelerates and testosterone administration retards atherogenesis progression (331,378). The protective effect of testosterone on aortic atherogenesis is mediated through its conversion to estradiol by the CYP19A1 in the blood vessel wall.

 

Two large placebo-controlled trials have evaluated the effects of testosterone treatment on atherogenesis progression in middle-aged and older men. The Testosterone’s Effects on Atherosclerosis Progression in Aging Men (TEAAM) Trial determined the effects of testosterone therapy on progression of subclinical atherosclerosis in the common carotid artery using sonographic measurement of common carotid artery intima-media thickness (CCA-IMT) and the coronary artery calcium scores measured using MDCT. The participants in the TEAAM Trial were 308 men, 60 years and older, with total testosterone between 100 and 400 ng/dL or free testosterone below 50 pg/mL (12). Men were randomized to receive either 75 mg of transdermal testosterone gel or placebo gel daily and received for 3 years.  Neither the progression of CCA-IMT nor coronary artery calcium scores differed between the men randomized to the testosterone and placebo groups (Figure 11) (12).

Figure 11. Effects of testosterone administration on atherosclerosis progression. Panels A and B show data from the TEAAM trial (Basaria et al. JAMA. 2015;314(6):570-81; figure reproduced with permission from JAMA) Panel C shows data from the Cardiovascular Trial of the TTrials (data from Budoff et al. JAMA. 2017;317(7):708-716; figure adapted from Gagliano-Jucá & Basaria, Asian J Androl. 2018;20(2):131-137).

In the cardiovascular trial of the TTrials, 138 men with serum total testosterone below 275 ng/dL received either testosterone gel or placebo gel for one year and were evaluated by coronary computed tomographic angiography for progression of non-calcified and calcified coronary artery plaque volume, as well as coronary artery calcium score (379). Consistent with the findings of the TEAAM Trial, the changes in coronary artery calcium scores did not differ between the testosterone and placebo groups over one year of intervention. However, the increase in non-calcified plaque volume (primary endpoint) was significantly greater in men assigned to the testosterone arm than in those assigned to placebo arm (Figure 11) (379); there were baseline differences in non-calcified plaque volume between the two groups. The clinical implications of these findings to cardiovascular risk remain to be established.

 

TESTOSTERONE AND CARDIAC ARRHYTHMIAS

 

Testosterone has important effects in cardiac electrophysiology (380); it increases potassium currents derived from the human ether-a-go-go related gene (hERG) (381), and inhibits the depolarizing delayed calcium current (ICaL) (382), with its effects on ICaLbeing more meaningful than on hERG (383). These effects lead to shortening of ventricular cardiomyocyte repolarization time, which can be seen in the electrocardiogram as shortening of the heart-rate corrected QT interval (QTc). Indeed, cross-sectional studies have observed a negative association between serum testosterone levels and QTc duration (384). Additionally, in randomized trials of testosterone replacement to men with low testosterone levels, testosterone treatment shortened QTc duration in community-dwelling older men (385)and in men with chronic heart failure (386). Similarly, in a prospective cohort study, androgen deprivation therapy in men with prostate cancer was associated with QTc prolongation compared with men with prostate cancer not receiving the therapy (387). As QTc prolongation is associated with an increased risk of ventricular tachyarrhythmias (torsades de pointes) and sudden cardiac death (388-390), low testosterone might predispose men to these arrhythmias. Indeed, androgen deprivation therapy is associated with a higher risk of arrhythmia, cardiac conduction disturbances and sudden death (391,392). A small case series study and analysis of the European pharmacovigilance database concluded that “conditions or drugs leading to male hypogonadism were associated with torsades de pointes”, and “correction of hypogonadism with testosterone replacement therapy can treat or prevent torsades de pointes” (393).

 

Cross-sectional studies have also linked low androgen levels in men to an increased risk of atrial fibrillation (394-396), and normalization of testosterone levels with testosterone replacement is associated with a decreased incidence of atrial fibrillation compared with untreated hypogonadal men or with men on testosterone replacement with non-normalized testosterone levels (397). These findings need corroboration in randomized trials.

 

EFFECT OF TESTOSTERONE ON MAJOR ADVERSE CARDIOVASCULAR EVENTS (MACE)

 

To-date, no randomized trials have been large enough or of sufficiently long duration to determine the effects of testosterone treatment on MACE (332). Therefore, the published data have been derived necessarily from the analyses of the reported adverse events in randomized clinical trials. The number of cardiovascular-related events reported in randomized testosterone trials has been strikingly low—even lower than that expected for the age and comorbid conditions of the participants (7,398,399). A randomized trial of testosterone in older men (The TOM Trial) with mobility limitation was stopped early due to a higher frequency of cardiovascular-related events in men assigned to testosterone than in those assigned to placebo (11), heightening concern about the cardiovascular safety of testosterone in frail older men. In contrast to many other testosterone trials in older men, which recruited relatively healthy older men, the participants in the TOM trial had a high prevalence of chronic conditions, such as heart disease, diabetes mellitus, obesity, hypertension, and hyperlipidaemia (11). Men, 75 years of age or older, and men with higher on-treatment testosterone levels seemed to be at the greatest risk of cardiovascular-related events.In secondary analyses, these events were found to be associated with changes in serum free testosterone and estradiol levels (400).The dose of testosterone used in the TOM trial was higher than that used in some previous trials, but not dissimilar from or lower than that used in some other trials. The cardiovascular events were small in number and of variable clinical significance. The TOM trial was not designed for cardiovascular events; therefore, the cardiovascular events were not a pre-specified endpoint,and were not collected in a standardized manner, nor adjudicated prospectively. Additionally, many of the cardiovascular events were not MACE.

 

The higher cardiovascular adverse event incidence in testosterone-treated older men observed in the TOM trial was not reproduced in two larger trials of longer duration published more recently; in the TEAAM trial,the incidence of major adverse cardiac events throughout the 3 years of intervention was similar between groups (12).Similarly, in the TTrials, the number of MACE (myocardial infarction, stroke or death related to cardiovascular disease) during the one year of treatment was similar in the two groups, with seven men in each group experiencing an event (15). The number of MACE in the TEAAM and Ttrials were too few to permit strong inferences on the effects of testosterone treatment on MACE.

 

The Hormonal Regulators of Muscle and Metabolism in Aging (HORMA) trial reported a significantly greater increase in blood pressure in men treated with testosterone than in those treated with placebo (401). Testosterone administration causes salt and water retention (402), which can induce edema and worsen pre-existing heart failure. Thus, large prospective randomized trials of long duration are needed to determine the effects of testosterone therapy on cardiovascular health.

 

Several meta-analyses of randomized testosterone trials have been published (329,398,399,403,404); however, these meta-analyses are limited by the small size of most trials, heterogeneity of study populations, poor quality of adverse-event reporting, and short treatment duration in many trials. None of the testosterone trials to date was sufficiently powered to adequately assess safety outcomes. The rigor of adverse-event reporting varied greatly among studies. The MACE were not ascertained rigorously nor adjudicated in most trials except in the TTrials. A meta-analysis of randomized testosterone trials by Xu et al. included 2,994 men from 27 eligible trials of 12 weeks or longer duration. Randomization to testosterone was associated with an increased the risk of a cardiovascular-related event (odds ratio (OR) 1.54, 95% confidence interval (CI) 1.09 to 2.18) (399). A remarkable finding of this meta-analysis was that the effect of testosteronetherapy varied with the source of the trial’s funding (399). The risk of a cardiovascular-related event on testosteronetherapy was greater (OR 2.06, 95% CI 1.34 to 3.17) in trials that were not funded by the pharmaceutical industry; in contrast, the trials funded by the pharmaceutical industry did not reveal a significant increase in cardiovascular events. Some meta-analyses of randomized testosterone trials published after the report by Xu and colleagues have not reproduced their findings (Figure 12) (403-406).

 

Retrospective analyses of electronic medical records data have yielded conflicted results. Two recent studies performed retrospective analyses of the association of testosterone administration with either mortality or cardiovascular events. Vigen et al (407)conducted a retrospective cohort study to determine the association between testosterone therapy and all-cause mortality, myocardial infarction, and stroke in middle-aged and older men with low testosterone levels who underwent a coronary angiography within the Veteran’s Administration system. In the adjusted analyses, there was an increased risk of the composite endpoint for men receiving testosterone therapy, after adjusting for the presence of coronary artery disease, but unadjusted analyses did not reveal this risk. In contrast, Shores et al (408)evaluated another subset of men within the VA system with low testosterone levels and no history of prostate cancer, who received T therapy during routine clinical care. After adjusting for age, BMI, baseline testosterone, and other co-morbidities, testosterone-treated men had lower risk of death than untreated men (408).

Figure 12. A meta-analysis of cardiovascular events in randomized testosterone trials. In this meta-analysis of cardiovascular-related events in randomized testosterone trials included from 91 eligible trials in the analysis of major adverse cardiovascular events (MACE). Compared with placebo, exogenous testosterone treatment did not show any significant increase in risk of MACE occurrence (odds ratio [OR] 0.97; 95% CI, 0.64 to 1.46). Figure reproduced with permission from Corona et al. J Sex Med. 2018 Jun;15(6):820-838.

Among studies that have examined the association of testosterone administration and cardiovascular events, Finkle et al (409)reported on a retrospective cohort study of men before and after receiving testosterone replacement and found an increased risk of non-fatal myocardial infarctions in the 3 months following a testosterone prescription than in the year prior to receiving testosterone prescription for patients 65 years of age or older and in patients younger than 65 with a history of heart disease.  Baillargeon et al (410), however, found no increase in the risk of myocardial infarction in men treated with testosterone in a sample of Medicare beneficiaries on testosterone therapy matched to beneficiaries not on therapy. In this study, testosterone use was found to be modestly protective against myocardial infarctions among men at higher MI risk.

 

These epidemiologic studies suffer from many limitations that are inherent in epidemiologic studies and in retrospective analysis of electronic medical records data. These studies included heterogeneous populations, and differed in the duration of intervention and study design. They used variable definitions and ascertainment of cardiovascular outcomes. Treatments indications, treatment regimens, on-treatment testosterone levels and exposure differed among studies. These studies also suffered from a potential for residual confounding in that the patients assigned to testosterone therapy differed from comparators in baseline cardiovascular risk factors. Because of these inherent limitations and inconsistency of findings, these epidemiologic studies do not permit strong inferences about the relation between testosterone therapy and mortality and cardiovascular outcomes.

 

SYNOPSIS OF THE EFFECTS OF TESTOSTERONE ON CARDIOVASCULAR RISK

 

The long-term effects of testosterone replacement therapy on MACE remain unknown. Some cohort and cross-sectional studies collectively suggest a neutral or favorable effect of testosterone on coronary heart disease in men, although the evidence is far from conclusive. It is possible that frail elderly men with high burden of chronic diseases and cardiovascular risk factors may be at increased risk of cardiovascular-related adverse events (7). Long-term randomized trials of the effects of testosterone replacement on MACE are needed and are particularly important because even small changes in incidence rates could have significant public health impact.

 

Fortunately, a large randomized, placebo-controlled trial of the effects of testosterone replacement therapy on major adverse cardiovascular events in men 45 to 85 years of age with low testosterone levels and one or more symptoms of testosterone deficiency, who are at increased risk for cardiovascular events is currently underway (The TRAVERSE Trial). The intervention duration is up to 5 years in this trial of over 6,000 men. The efficacy outcomes include adjudicated clinical fractures, remission of low-grade persistent depressive disorder (dysthymia), progression from pre-diabetes to diabetes, correction of anemia, and overall sexual activity, sexual desire, and erectile function. This historical randomized, placebo-controlled trial offers an historical opportunity to advance our understanding of the cardiovascular safety and long-term efficacy of testosterone replacement in middle-aged and older hypogonadal men.

 

Testosterone, Diabetes, and Metabolic Syndrome

 

Spontaneous(144)and experimentally induced (202)androgen deficiency is associated with increased fat mass, and testosterone replacement decreased fat mass in older men with low testosterone levels (5). In epidemiologic studies, low testosterone levels are associated with higher levels of abdominal adiposity (411,412). Testosterone administration promotes the mobilization of triglycerides from the abdominal adipose tissue in middle-aged men (413).Surgical castration in rats impairs insulin sensitivity; physiologic testosterone replacement reverses this metabolic derangement (414). However, high doses of testosterone impair insulin sensitivity in castrated rats (414), suggesting a biphasic relationship in which both low and high testosterone levels impair insulin resistance. Androgens increase insulin-independent glucose uptake (415)and modulate LPL activity in a region-specific manner (416).

 

Testosterone levels are lower in men with type 2 diabetes mellitus compared with controls (417-422). Low total testosterone levels have been associated with lower insulin sensitivity (417,423)and increased risk of type 2 diabetes mellitus and metabolic syndrome in community dwelling men both cross-sectionally and longitudinally (420-422,424-432). However, the association of free testosterone and type 2 diabetes mellitus has been inconsistent; some studies have reported a weak relationship (421,422,424)while others have failed to find any relationship (420,426). Circulating sex hormone binding globulin (SHBG) and some SHBG polymorphisms also have been associated negatively with the risk of type 2 diabetes (420-422,424-428,433-436). For instance, individuals with the rs6257and rs179994 variant alleles of the SHBG single nucleotide polymorphism (SNP) have lower plasma SHBG levels and a higher risk of type 2 diabetes (433-436). Similarly, individuals with the rs6259 variant have higher SHBG levels and lower type 2 diabetes risk (436).As total testosterone and SHBG levels are highly correlated(437), we performed longitudinal analyses of men participating in the Massachusetts Male Aging Study (438), a population-based study of men aged 40-70 years (Figure 13) to evaluate whether SHBG is an independent predictor of T2DM. After adjustment for age, body mass index, hypertension, smoking, alcohol intake and physical activity, the hazard ratio for incident type 2 diabetes was 2.0 for each one SD decrease in SHBG and 1.29 for each one SD decrease in total testosterone (438). Free testosterone was not significantly associated with type 2 diabetes. The strong association of T2DM risk with SHBG persisted even after additional adjustment for free testosterone. Thus, SHBG, but not free testosterone, is an independent predictor of incident type 2 diabetes. Although it is possible that SHBG is a marker of insulin resistance, and low SHBG levels reflect the effects of hyperglycemia or insulin resistance, the association of SHBG polymorphisms with type 2 diabetes suggests an important mechanistic role of SHBG in the pathogenesis of type 2 diabetes.

 

Figure 13. Circulating Concentrations of SHBG, but not total or free testosterone, were associated prospectively with risk of incident diabetes in the Massachusetts Male Aging Study (MMAS). In a prospective analysis of data from the Massachusetts male Aging Study, total testosterone (left panel) and free testosterone (middle panel) were not associated significantly with risk of incident diabetes. Only SHBG concentrations were associated with incident diabetes in longitudinal analysis. Reproduced with permission from Lakshman et al J Gerontol A Biol Sci Med Sci. 2010;65(5):503-9.

Interventional studies have yielded inconsistent results. Acute and severe androgen deficiency induced by administration of a GnRH agonist or antagonist worsens measures of insulin sensitivity; acute withdrawal of testosterone therapy in men with idiopathic hypogonadotropic hypogonadism (439)and administration of androgen deprivation therapy in men with prostate cancer is associated with the worsening of insulin sensitivity (364,440,441). Men with prostate cancer who are receiving androgen deprivation therapy are at increased risk of developing type 2 diabetes and metabolic syndrome (391,440,442). In the TIMES2 study (443), men with type 2 diabetes mellitus and/or metabolic syndrome were randomized to either 2% testosterone gel or placebo gel for 6 months. Randomization to testosterone arm was associated with greater improvements in sexual function and plasma lipid levels than placebo. However, the changes in HbA1clevels did not differ between groups. Homeostasis model assessment of insulin resistance (HOMA-IR), a marker of insulin resistance, improved modestly in men who were assigned to testosterone compared with placebo (443). More recently, Dhindsaet al reported improvement of insulin sensitivity with testosterone replacement for 24 weeks in hypogonadotropic hypogonadal men with type 2 diabetes (423). Overall, studies have failed to show improvements in diabetes outcomes or consistent changes in measures of insulin sensitivity (7,345,363,443-447)even though interventional trials have found a consistent reduction in whole body fat as well as abdominal fat (7,112,291). Indeed, in the TEAAM trial, 3 years of testosterone supplementation decreased fat mass in community-dwelling older men with low or low-normal serum testosterone concentrations, but did not improve insulin sensitivity (Figure 14) (448).

 

Figure 14. Long-term effects of testosterone therapy on insulin sensitivity in older men. Change in insulin sensitivity over time measured by the octreotide insulin suppression test and estimated as the mean concentration of glucose at equilibrium (SSPG). Figure adapted from Huang et al. J Clin Endocrinol Metab. 2018;103(4):1678-1685.

Testosterone and Prostate Cancer Risk

 

There is no evidence that testosterone causes prostate cancer (449). A retrospective analysis of the Registry of Hypogonadism in Men (RHYME) (450)and several meta-analyses of randomized controlled trials (398,451,452)did not find an increased risk of prostate cancer in men receiving testosterone. Also, there is no consistent relationship between endogenous serum testosterone levels and the risk of prostate cancer (5,7,112,291,452-455). A meta-analyses of prospective cohort studies did not find a significant association between endogenous total testosterone levels and prostate cancer (452). Conversely, an analysis of 20 prospective studies found that menin the lowest tenth of free testosterone concentration had a lower risk of prostate cancer (OR=0.77, 95%CI= 0.69 to 0.86; p<0.001) compared with men with higher concentrations (455);however, this finding might be due to detection bias, as we further discuss in this section. Prostate cancer is a common, androgen–dependent tumor, and androgen administration may promote the growth of a pre-existing metastatic prostate cancer (454,456). Therefore, testosterone administration has been historically contraindicated in men with history of prostate cancer (5,453). The prevalence of subclinical, microscopic foci of prostate cancer in older men is high (457-464). There is concern that testosterone administration might make these subclinical foci of cancer grow and become clinically overt. In addition, older men with low testosterone levels may have prostate cancer (465,466). Morgentaler et al (465,466)reported a high prevalence of biopsy-detectable prostate cancer in men with low total or free testosterone levels despite normal PSA levels and normal digital rectal examinations. However, this study did not have a control group, and we do not know whether sextant biopsies of age-matched controls with normal testosterone levels would yield a similarly high incidence of biopsy-detectable cancer. Therefore, this study should not be interpreted to conclude that there is a higher prevalence of prostate cancer in older men with low testosterone levels, or that low testosterone levels are an indication for performing prostate biopsy.

 

EFFECTS OF TESTOSTERONE THERAPY ON PROSTATE EVENTS

 

None of the testosterone trials in middle-aged or older men to date had sufficient power to detect meaningful differences in prostate event rates between testosterone and placebo-treated men. A systematic review of randomized testosterone trials in middle-aged and older men found higher rates of prostate events in testosterone-treated men than in placebo-treated men (Figure 15) (329). Men treated with testosterone in these trials were at significantly higher risk for undergoing prostate biopsy than placebo-treated men (329). Because of the high prevalence of subclinical prostate cancer in older men, the higher number of prostate biopsies in testosterone-treated men is likely to yield higher detection rates of prostate cancer in comparison with placebo-treated men. Thus, testosterone therapy of middle-aged and older men is associated with a higher risk of prostate biopsy and a bias towards detection of a higher number of prostate events (7,329).

Figure 15. Adverse events associated with testosterone therapy in randomized trials. The relative risk and 95% CI for development of erythrocytosis (RR= 8.14; 95%CI= 1.87 to 35.40) and lower urinary tract symptoms (LUTS; RR= 0.38; 95%CI= -0.67 to 1.43) in randomized testosterone trials derived from meta-analyses published by Ponce et al., 2018 are shown. The figure was adapted with permission from Ponce et al. J Clin Endocrinol Metab. 2018;103(5):1745-54.

Administration of exogenous testosterone or suppression of circulating levels of testosterone by administration of a GnRH antagonist is not associated with proportionate changes in intra-prostatic testosterone or DHT concentrations. For instance, in a randomized controlled trial, Marks et al (467)measured intraprostatic testosterone and DHT levels in older men treated with placebo or testosterone. Surprisingly, intraprostatic DHT concentrations were not significantly higher in testosterone-treated men than in placebo-treated men (467). Similarly, the expression levels of androgen-dependent genes in the prostate were not significantly altered by testosterone administration (467). In separate studies, lowering of circulating testosterone levels by administration of a GnRH antagonist was not associated with changes in intraprostatic androgen concentrations (468,469).

 

EFFECTS OF TESTOSTERONE REPLACEMENT IN SERUM PSA LEVELS

 

Serum PSA levels are lower in androgen–deficient men and are restored to normal following testosterone replacement (5,470-478). Lowering of serum testosterone concentrations by withdrawal of androgen therapy in young, hypogonadal men is associated with a decrease in serum PSA levels. Similarly, treatment of men with benign prostatic hyperplasia with a 5-alpha reductase inhibitor, finasteride, is associated with a significant lowering of serum and prostatic PSA levels (478,479). Conversely, testosterone supplementation increases PSA levels (471-478).  However, serum PSA levels do not increase progressively in healthy hypogonadal men with replacement doses of testosterone. Placebo–controlled trials of testosterone administration in older men have reported either minimal increase or no significant change in serum PSA levels in testosterone–treated men (169,170). The increase in PSA levels during testosterone replacement might trigger evaluation and biopsy in some patients (5,453).

 

More intensive PSA screening and follow-up of men receiving testosterone replacement might lead to an increased number of prostate biopsies and the detection of subclinical prostate cancers that would have otherwise remained undetected (5,453). Serum PSA levels tend to fluctuate when measured repeatedly in the same individual over time (480-482). When serum PSA levels in androgen deficient men on testosterone replacement therapy show a change from a previously measured value, the clinician has to decide whether the change warrants detailed evaluation of the patient for prostate cancer, or whether it is simply due to test–to–test variability in PSA measurement. Therefore, it is important to set criteria for monitoring PSA changes during testosterone supplementation. Criteria that use very low thresholds for performing prostate biopsy relative to test-retest variability will likely result in an excessive number of biopsies with their associated costs, psychological trauma, and morbidity. On the other hand, criteria that use unreasonably high thresholds for performing prostate biopsies may fail to detect clinical prostate cancers at an early stage.

 

There is considerable test-retest variability in PSA measurements (480-482). Some of this variability is due to the inherent assay variability, and a significant portion of this variability is due to unknown factors. Fluctuations are larger in men with high mean PSA levels. Variability can be even greater if measurements are performed in different laboratories that use dissimilar assay methodology (480-482).

 

From a clinical perspective, an important issue is what increment in PSA level should warrant a prostate biopsy in older men receiving testosterone replacement. To address this issue, we conducted a systematic review of published studies of testosterone replacement in hypogonadal men (453). This review indicated that the weighted effect size of the change in PSA after testosterone replacement in young, hypogonadal men is 0.68 standard deviation units (95% confidence interval 0.55 to 0.82). This means that the effect of testosterone replacement therapy is to increase PSA levels by an average 0.68 standard deviations over baseline. Because the average standard deviation was 0.47 in this systematic analysis, the standard deviation score of 0.68 translates into an average increase in serum PSA levels of about 0.30 ng/ml in young hypogonadal men (453). There is considerable variability in the magnitude of change in PSA after testosterone supplementation among these studies, in part due to heterogeneity of study populations, inclusion of older men in some studies but not others, and differences in PSA assays. In addition, many patients who were enrolled in these studies were likely receiving testosterone replacement therapy previously; we do not know whether the washout period was sufficient to return PSA levels to baseline. Therefore, it is possible that because of inadequate washout, the increments in serum PSA levels after testosterone administration might have been under-estimated.

 

We performed a separate systematic review of data from placebo-controlled trials of testosterone supplementation in older men with low or low normal testosterone concentrations (453).The weighted effect size in six studies of older men was 1.48 standard deviation units, with a 95% confidence interval of 1.21 to 1.75. Thus, on average, older men experience a greater increase in serum PSA concentrations than younger men. The average effect of testosterone replacement in older men is to increase PSA levels by almost 1.5 standard deviations over baseline. There is, however, significant variability in the results among these six studies (p<0.0001), and the average standard deviation was skewed by one study, which had a very high standard deviation (453). After excluding this study, the average change in serum PSA levels after testosterone replacement in studies of older men was 0.43 ng/mL.

 

The data from the Proscar Long-Term Efficacy and Safety Study (PLESS) demonstrated that the 90% confidence interval for the change in PSA values measured 3 to 6 months apart is 1.4 ng/mL (478). Therefore, a change in PSA of >1.4 ng/ml between any two values measured 3 to 6 months apart in the same patient is unusual (5,453).  In the TTrials, 2.4% of men receiving testosterone had increases above 1.4 ng/mL at 3 months, and 4.7% at 12 months (15).

 

Carter et al, based on the analysis of PSA data from the Baltimore Longitudinal Study of Aging, reported that PSA velocity, defined as the annual rate of change of PSA, is different in men who develop prostate cancer than in those who do not (483-485). Thus, PSA velocity greater than 0.7 ng/ml/year was unusual in men without prostate cancer whose baseline PSA was between 4 and 10 ng/ml (483-485). However, most men being considered for testosterone replacement will have baseline PSA less than 4 ng/ml. In a subsequent analysis, the same group reported that the PSA velocity in men with baseline PSA between 2 and 4 ng/ml was 0.2 ng/ml/year (485). Because test-to-retest variability in PSA measurement is far greater than this threshold, it is likely that the use of this threshold of 0.2 ng/ml/year to select men for prostate biopsy would lead to many unnecessary biopsies.

 

In eugonadal, young men, administration of supraphysiological doses of testosterone does not further increase serum PSA levels (150,153,486). These data are consistent with dose response studies in young men that demonstrate that maximal serum concentrations of PSA are achieved at testosterone levels that are at the lower end of the normal male range; higher testosterone concentrations are not associated with higher PSA levels (150,153).

 

In summary, these data suggest that the administration of replacement doses of testosterone to androgen-deficient men can be expected to produce a modest increment in serum PSA levels. Increments in PSA levels after testosterone supplementation in androgen-deficient men are generally less than 0.5 ng/mL, and increments in excess of 1.0 ng/mL over a 3-6-month period are unusual. Nevertheless, administration of testosterone to men with baseline PSA levels between 2.6 and 4.0 ng/mL will cause PSA levels to exceed 4.0 ng/mL in some men. Increments in PSA levels above 4 ng/mL will trigger a urological consultation and many of these men will be asked to undergo prostate biopsies. However, considering the controversy over prostate cancer screening and monitoring, clinicians should engage their patient in the decision-making process before starting treatment, informing them of the risks and benefits of prostate cancer screening and monitoring.

 

MONITORING PSA LEVELS IN OLDER MEN RECEIVING TESTOSTERONE REPLACEMENT (TABLES 3 AND 4)

 

Older men considering testosterone supplementation should undergo evaluation of risk factors for prostate cancer; the Endocrine Society guideline suggest a baseline PSA measurement and a digital prostate examination (5). Prostate cancer screening has some risks; therefore, initiation of prostate monitoring should be a shared decision, made only after a discussion of the risks and benefits of prostate cancer monitoring. Men with history of prostate cancer, should not be given androgen supplementation and those with palpable abnormalities of the prostate or PSA levels greater than 3 ng/ml should undergo urological evaluation. After initiation of testosterone replacement therapy, PSA levels should be repeated at 3 months and annually thereafter (5). Although measurements of free PSA and PSA density have been proposed to enhance the specificity of PSA measurement, long term data, especially from studies of testosterone replacement in older men, are lacking. Considering the interassay variability and the longitudinal change in PSA previously discussed, an Endocrine Society Expert Panel recently suggested that men receiving testosterone replacement should be referred to urological consultation if: 1) PSA increases more than 1.4 ng/mL in the first 12 months of treatment; 2) a PSA above 4 ng/mL is confirmed; or 3) a prostatic abnormality is detected on digital rectal examination (5). After 12 months of treatment, prostate monitoring should follow standard guidelines for prostate cancer screening taking into account the age and race of the patient (5).

 

Table 4. Indications for Urological Consultation in Men Receiving Testosterone Replacement
1.     An increase in serum or plasma PSA concentration >1.4 ng/mL within any 12-month period after initiating testosterone treatment
2.     A PSA >4.0 ng/mL
3.     Detection of a prostatic abnormality on digital rectal examination
4.     An AUA/IPSS prostate symptom score of >19

Adapted with permission from the Endocrine Society Guideline for Testosterone Therapy in Men With Hypogonadism in: Bhasin et al J Clin Endocrinol Metab 2018;103(5):1715-1744.

 

TESTOSTERONE AND BENIGN PROSTATIC HYPERTROPHY

 

Testosterone replacement can be administered safely to men with benign prostatic hypertrophy who have mild to moderate symptom scores. The severity of symptoms associated with benign prostatic hypertrophy can be assessed by using either the International Prostate Symptom Score (IPSS) or the American Urological Association (AUA) Symptom questionnaires. Androgen deficiency is associated with decreased prostate volume and androgen replacement increases prostate volume compared to age–matched controls (467,470,474,475). Meta-analyses of testosterone trials have not found statistically significant difference in lower urinary tract symptoms scores in hypogonadal men receiving testosterone replacement compared to placebo (236,487). However, in patients with pre–existing, severe symptoms of benign prostatic hypertrophy, even small increases in prostate volume during testosterone administration may exacerbate obstructive symptoms. In these men, testosterone should either not be administered or administered with careful monitoring of obstructive symptoms.

 

Erythrocytosis

 

Testosterone replacement is associated with increased red cell mass and hemoglobin levels (Figure 15) (236,488-494). Therefore, testosterone replacement should not be administered to men with baseline hematocrit of 52% or greater without appropriate evaluation and treatment of erythrocytosis (5)(Table 3). Administration of testosterone to androgen–deficient young men is typically associated with a small increase in hemoglobin levels. Clinically significant erythrocytosis is uncommon in young hypogonadal men during testosterone replacement therapy, but can occur in men with sleep apnea, significant smoking history, or chronic obstructive lung disease. Testosterone administration in older men is associated with more variable and somewhat greater increments in hemoglobin than observed in young, hypogonadal men (328). The magnitude of hemoglobin increase during testosterone therapy appears related to the testosterone dose, the increase in testosterone concentrations during testosterone therapy, and age (328). Testosterone replacement by means of a transdermal system has been reported to produce a lesser increase in hemoglobin levels than that associated with intramuscular testosterone enanthate and cypionate presumably because of the substantially higher testosterone dose and average circulating testosterone levels achieved with testosterone esters (495).

 

Testosterone increases hemoglobin and hematocrit by multiple mechanisms (319-321,496). Suppression of testosterone secretion in men receiving androgen deprivation therapy reduces hematocrit and hemoglobin levels by slowing erythropoiesis independently from changes in erythropoietin (497). Testosterone administration stimulates erythropoiesis, suppresses hepcidin transcription by blocking bone morphogenetic protein signaling, and increases iron availability for erythropoiesis (319-321,496). Additionally, testosterone stimulates erythropoiesis by a direct effect on bone marrow progenitors and appears to alter the set-point of the relationship between erythropoietin and hemoglobin (319). Testosterone supplementation can correct anemia in older men with unexplained anemia of aging and anemia of inflammation (319,321,493).

 

MONITORING HEMATOCRIT DURING TESTOSTERONE REPLACEMENT THERAPY (TABLE 3)

 

Hemoglobin levels should be measured at baseline and 3 months after institution of testosterone replacement or after increase in dosage, and every 12 months thereafter. It is not clear what absolute hematocrit level or magnitude of change in hematocrit warrants discontinuation of testosterone administration. Plasma viscosity increases disproportionately as hematocrit rises above 50%. Hematocrit levels above 54% are also associated with increased risk of stroke. Therefore, testosterone dose should be withheld if hematocrit rises above 54%; once hematocrit falls to a safe level, testosterone therapy may be re-initiated at a reduced dose or with a different formulation (5).

 

Sleep Apnea

 

Circulating testosterone concentrations are related to sleep rhythm and are generally higher during sleep than during waking hours (498-501). Testosterone secretory peaks coincide with the onset of rapid-eye movement sleep. Aging is associated with decreased sleepefficiency, reduced numbers of REM sleepepisodes, and altered REM sleeplatency, which may contribute to lower circulating testosterone concentrations (499-503).  The degree of sleep-disordered breathing increases with age, and is associated with reduced overnight plasma bioavailable testosterone. Thus, changes in sleep efficiency and architecture are associated with alterations in testosterone levels in older men (499-503).Sleep apnea and disordered sleep are often associated with low testosterone levels (504), particularly in patients with more severe cases of OSA (i.e. severe hypoxemia) (505). Some potential mechanisms by which OSA may decrease endogenous testosterone levels include disruption of pulsatile luteinizing hormone secretion from restricted sleep and/or recurrent nocturnal hypoxia (506,507), which is further exacerbated by obesity. OSA treatment with continuous positive airway pressure has been demonstrated to increase serum testosterone levels (508).

Testosterone can induce or exacerbate sleep apnea in some individuals, particularly those with obesity or chronic obstructive lung disease (498-503,509). This appears to be due to direct effects of testosterone on laryngeal muscles. However, the occurrence of sleep apnea, de novo, in healthy older men treated with physiologic testosterone replacement, is rare; it should be considered in men who develop erythrocytosis with low dosages of testosterone therapy.

 

Testosterone administration depresses hypercapnoeic ventilator drive and induces apnea in primate infants (503). Short-term administration of high doses of testosterone shortenssleepduration and worsens sleep apnea in older men (510). The frequency of sleep apnea in randomized testosterone trials in older men has been very low (5,398)and no randomized trial has reported an increased incidence of OSA or OSA worsening in men randomized to the testosterone arm compared to the placebo arm.

 

Testosterone should probably not be given to men with severe untreated OSA without evaluation and treatment of sleep apnea. Several screening instruments can be used to detect sleep apnea. A history of loud snoring, and daytime somnolence, in an obese individual with hypertension increases the likelihood of sleep apnea.

 

Breast Enlargement and Tenderness

 

Testosterone administration can induce breast enlargement due to testosterone conversion to estradiol, although these effects are very uncommon complications of testosterone replacement therapy. Even with administration of supraphysiological doses of testosterone enanthate, less than 4% of men in a contraceptive trial developed detectable breast enlargement (488). Breast cancer is listed as a contraindication for testosterone replacement therapy primarily because of concern that increased estrogen levels during testosterone treatment might exacerbate breast cancer growth. There are, however, few case reports of breast cancer occurring as a complication of testosterone treatment. Men with Klinefelter’s syndrome have a higher risk of breast cancer than the general population (511).

 

CHANGES IN THE SPERMATOGENIC COMPARTMENT OF THE TESTIS

 

Women are more fertile below the age of 40, and fertility ceases at the inception of menopause, around age 50. Increasing age in women confers greater risk for infertility, spontaneous abortion, and genetic and chromosomal defects among offspring. In contrast, there is no critical age at which sperm production or function, and fertility cease in men (512-519).  Although serum testosterone concentrations decrease below the normal range in a significant minority of older men, men over the age of 60 years commonly father children; the oldest father on record was 94-years old (512,514). Even though many older men are fertile, the overall fertility and fecundity decline with aging. The interpretability of data on the effects of aging on male fertility is limited by the small size of the studies and the low overall event rates.

 

Paternal age has been associated with a significant increase in the risk of germ line mutations in FGFR2, FGFR3, and RET genes and inherited autosomal dominant diseases, such as Apert's syndrome, achondroplasia, and Costello Syndrome, respectively, in the offspring of older men (519-528). These monogenic disorders have been referred to as paternal age effect (PAE) disorders. Approximately one third of babies with diseases due to new autosomal dominant mutations are fathered by men aged 40 years or older (529).

 

Some other disorders such as schizophrenia, autism and bipolar disorder have also been linked to paternal age (Figure 16) (520,521,527,528). The rate of de novo mutations increases with paternal age (527), which may contribute to the increase risk of neurodevelopmental diseases such as schizophrenia and autism (527). The accumulation of these de novo germ line mutations with increasing paternal age has been explained by the “selfish spermatogonial selection" hypothesis (523,524).  According to this hypothesis, the somatic mutations in male germ cells that enhance the proliferation of germ cells could lead to within-testis expansion of mutant clonal lines (525,526), thus favoring the propagation of germ cells carrying these pathogenic mutations, and increasing the risk of mutations in the offspring of older fathers (525,526). Interestingly, the risk of autism has also been associated with the age of the father as well as the grandparent (528). These concerns have prompted the American Society of Reproductive Medicine to state in their guidelines that semen donors should be younger than 40 years of age so that potential hazards related to aging are diminished (515).

Figure 16. Impact of paternal age on incidence of schizophrenia and early-onset bipolar disorder. Increasing paternal age at conception increases the relative risk of having an offspring with schizophrenia (panel A; figure adapted from Malaspina et al. Arch Gen Psychiatry. 2001 Apr;58(4):361-7.) and the odds ratio of having a child with early-onset bipolar disorder (compared to fathers aged 20 to 24 years; panel B; data derived from Frans et al. Arch Gen Psychiatry. 2008 Sep;65(9):1034-40)

 

Some cardiac defects have also been attributed to aberrant genetic input from older men.  For instance, a case-control study of 4,110 individuals with congenital heart defects born between 1952 and 1973 in British Columbia, found a general pattern of increasing risk with increasing paternal age among cases relative to controls for ventricular septal defects, atrial septal defects and patent ductus arteriosus (522,529). The risk of schizophrenia has also been reported to increase with paternal age (523)and possible loci affecting this risk have been identified (530). In addition, a modest proportion of preeclampsia, normally associated with increased maternal risk factors including age, might be attributable to an increase in paternal age although no gene loci have been identified (531). These observations need further corroboration.

 

Although there is a positive association between paternal age and incidence of aneuploidy, it has been difficult to dissociate the effect of paternal age from the confounding influence of the advanced maternal age. After accounting for various confounders, there does not appear to be a major independent effect of increased paternal age on the incidence of autosomal aneuploidies (513,514,520,521,532,533). The existence of a paternal age effect on Down syndrome is controversial. Earlier studies from the 1960s and 1970s found no correlation between Down syndrome and paternal age (e.g., (534)). However, a study in New York from 1983 to 1997 found significantly greater numbers of mothers and fathers 35 years of age and older, respectively, among parents of patients with Down’s syndrome (535). Among the cases of Down syndrome evaluated, paternal age had a significant effect only in mothers 35 years of age or older, and was the greatest in couples greater than 40 years of age where the risk was 6 times the rate of couples younger than 35 years of age (535).

 

Changes in Fertility of Older Men

 

A review of studies examining fertility at different ages demonstrated significant age-related differences in fertility rates in men; men older than 50 have lower pregnancy rates, increased time to pregnancy, and subfecundity compared to younger men (513,514,520,536,537). Some changes in fertility rates might be related to age-related decrease in sexual activity.  A literature review found no significant change in sperm concentration with aging when comparing men under the age of 30 to those greater than 50 years (518). However, in general, semen volume, sperm motility, and the number of morphologically normal sperm decrease with advancing age (Table 5; (34,513-519, 527, 532, 533, 536, 538). A number of these studies, however, did not control for important confounding variables. Of the 21 studies in which sperm densities were compared among men of different age groups (518), only four studies adjusted for the duration of abstinence, well known to affect sperm concentration. In addition, there is significant heterogeneity in the populations studied; most of the studies examined data from semen of sperm donors while others examined men from infertility clinics. Sperm donors might represent a healthier group of men than the general population; conversely men in infertility clinics might be more likely to have abnormalities of sperm number or function. Even studies that have controlled for abstinence as well as alcohol and tobacco use have shown an age-related decrease in semen volume. In one study of men whose partners had bilateral tubal obstruction or absence of both tubes and who were treated by conventional IVF, the odds ratio of failure to conceive was higher for men 40 years of age or older (539).

 

Table 5: Changes in Semen Quality and Fertility in Men with Age in a Literature Review by Kidd et al., 2001 (518).
Parameter Age comparison Change
Semen volume 30 versus³50 years 3-22% decrease
Sperm concentration Varying None
Abnormal sperm morphology £30 versus ³50 years 4-18% increase
Time to pregnancy <30-35 versus >30-50 years 5-20% increase
Pregnancy rates <30 versus > 50 years 23-38% decrease
Subfecundity Varying 11-250% increase

 

Changes in the Germ Cell Compartment

 

In a comparison of younger men (21-25 years) with older men (>50) referred for andrological evaluation, the ejaculate volume, progressive sperm motility, and sperm morphology were lower in older men than younger men after adjustment for duration of sexual abstinence (540). The older men also had a higher frequency of sperm tail defects, suggesting epididymal dysfunction (541). In addition, the fructose content was significantly lower in older men suggesting a defect in the seminal vesicle contribution to semen. There were no significant differences in sperm concentration and testicular size between the young and older men in this study.

 

Necropsies in adult men of different ages have revealed that the testicular volume was lower only in men in the 8th decade of life (542). A recent study examined testicular germ cells obtained by orchiectomy from 36 older men with advanced prostate cancer and by testicular biopsy from 21 younger men with obstructive azoospermia, as controls (543). The ratios of primary spermatocytes, round spermatids, and elongated spermatids to Sertoli cells were significantly decreased in the testes of older men, but the ratio of spermatogonia to Sertoli cell number remained unchanged (543,544). Older men are characterized by lower rates of germ cell apoptosis and cell proliferation compared with younger men, suggesting that germ cell proliferation and apoptosis diminish with aging (544).

 

Other studies evaluating the fidelity of the germ cell compartment are cross-sectional and depend on analyses of sperm number and semen quality; large-scale chromosomal analyses in healthy community dwelling men are scarce as most data are derived from fertility clinics.  A review of studies examining semen quality at different ages demonstrated significant age-related decrease in semen volume and sperm morphology.  The change in sperm morphology has been hypothesized to be due to an increase in aneuploidy with age.  Härkönen et al (533)found that sperm morphology was directly associated with the number of chromosomes in sperm and that men with higher aneuploidy rates for chromosomes 13, 18, 21, X and Y had lower sperm motility and sperm concentrations. Despite the changes in sperm morphology and motility from older men, in vitrofertilizing capacity of the sperm is well preserved (34,539). In some older men, degenerating germ cells can be observed suggesting loss of germ cells with age.

 

There are several difficulties in interpreting these data on age-related changes in sperm density and function. The normal range for sperm concentration in men is wide where sperm concentration above 15 million/ml (total sperm per ejaculate > 39 million) is considered normal. Thus, even though average sperm concentrations might decline with aging, they might still be in the normal range (34,537,543). Furthermore, normal sperm counts might not always correlate with normal sperm function.

 

Studies in flies demonstrate more germ cells during larval than adult stages suggesting age-related quiescence of the germ line (545). Significant age-related decreases in germ cells and spermatogenesis also have been reported in rodents and primates (546-550). The Brown Norway rat has been studied as a model of aging of the human male reproductive system because in this rodent model, serum testosterone levels decrease with aging, as they do in humans (547-549). Along with changes in hypothalamic-pituitary hormones, alterations in sperm counts, sperm maturation, Sertoli cell number, and progeny outcomes have been observed in this rodent model (Table 6; (531,547-556)). Analysis of ribosomal DNA from germ cells of the male brown Norway rat has revealed hypermethylation of ribosomal DNA (550,557). Alterations in ribosomes have been theorized to promote aging of cells by multiplying errors in protein synthesis which initially might elude gross morphological analysis but eventually might lead to germ cell degeneration (557). Further assessment of spermatogonial stem cell populations is needed.  In many animal models of life span extension, there is a trade-off between longer life and fecundity, although there are some exceptions (558).

 

Changes in Supportive Cells and Accessory Glands

 

Since Sertoli and Leydig cells are crucial to spermatogenesis, changes in these cells could affect sperm number and function. Age-related changes in the supporting structures for sperm maturation have been described in the Brown Norway rat.  These changes include reductions in the numbers of Leydig and Sertoli cells, seminiferous tubules, and in epididymal cell number and function (548-550). Changes in the supporting cells and structures for sperm maturation have been invoked to explain the age-related decrease in sperm number and fecundity in rats. In stallions, the numbers of Sertoli cells decreases with aging but individual Sertoli cells display a remarkable capacity to accommodate greater numbers of developing germ cells (559).

 

Table 6.  Changes in the Reproductive Axis in the Brown-Norway Rat.
Parameter Change Reference
GnRH ¯ 547, 548
FSH 547, 548
LH ® 547, 548
Testosterone ¯ 547-549, 551
Germ Cells ¯ 552
Sertoli Cells ¯ 548, 554
Spermatogenesis ¯ 548, 554
Seminiferous Tubules ¯ 548, 554
Seminiferous Tubule Function altered 551, 554
Epididymal function ¯ 555
Sperm morphology altered 555
Sperm motility ¯ 555
Sperm Count ¯ 549

 

In men, Sertoli cell number has been reported to be lower in men aged 50 to 85 years than in men aged 20 to 48 years (560). The apoptotic rate of primary spermatocytes in aged men was also significantly elevated compared with that of younger men, resulting in a decrease of the number of primary spermatocytes per Sertoli cell (544), leading the authors to suggest that there might be a failure of the Sertoli cells to support spermatogenesis in older men.

Sertoli cells produce inhibin, which regulates gonadotropin expression from the pituitary. Inhibin B has been identified as the physiologically important form of inhibin in men and as a valuable serum marker of Sertoli cell function and spermatogenesis. Higher gonadotropins and lower inhibin levels in older men suggest a decline in Sertoli cell function (560); however, changes in circulating inhibin B levels with advancing age have been inconsistent (59,560-562). Overall, these data suggest a possible decline in Sertoli cell number and function in older men with little effect on spermatogenesis.

Aging is accompanied by a progressive, albeit variable, decline of Leydig cell function with a decrease of mean serum free (or bioavailable) testosterone levels in the population between age 25 and 75 years(563). Total Leydig cell volume and the absolute number of Leydig cells decline with advancing age, although total testis weight does not change substantially with age (563-567). In one study, age accounted for more than a third of the variation in Leydig cell number, and explained more than half the variation in daily sperm production (566). This might in part be explained by a fusion of Leydig cells resulting in fewer but multinucleated Leydig cells with age (567). The functionality of the multinucleated cells is not known.

 

CONCLUSION

 

In male mammals, changes at all levels of the hypothalamic-pituitary-testicular axis, including alterations in the GnRH pulse generator, gonadotropin secretion, and testicular steroidogenesis, in addition to alterations of feed-forward and feed-back relationships contribute to the age-related decline in circulating testosterone concentrations.  The rate of age-related decline in testosterone levels is affected by the presence of chronic illness, adiposity, medication, sampling time, and the methods of testosterone measurement.  Epidemiologic surveys reveal an association of low testosterone levels with changes in body composition, physical function and mobility, risk of diabetes, metabolic syndrome, coronary artery disease, and fracture risk.

 

Age-related decline in testosterone should be distinguished from classical hypogonadism due to known diseases of the hypothalamus, pituitary and the testis. In young hypogonadal men who have a known disease of the hypothalamus, pituitary and testis, testosterone therapy is generally beneficial and has been associated with a low frequency of adverse events. However, neither the long-term benefits in improved health outcomes nor the long-term risks of testosterone therapy are known in older men with age-related decline in testosterone levels. Well-conducted studies of up to one-year duration have found improvements in sexual desire, erectile function, and overall sexual activity; mobility; and volumetric bone density, and correction of anemia with testosterone replacement of older men with unequivocally low testosterone levels.

 

Although testicular morphology, semen production, and fertility are maintained up to a very old age in men, there is clear evidence of decreased fecundity with advancing age and an increased risk of specific genetic disorders related to paternal age among the offspring of older men. Thus, reproductive aging of men is emerging as an important public health problem whose serious societal consequences go far beyond the quality of life issues related to low testosterone levels.

 

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Cholesterol Lowering Drugs

ABSTRACT

 

There are currently six different classes of drugs available for lowering cholesterol levels. There are currently seven HMG-CoA reductase inhibitors (statins) approved for lowering cholesterol levels and they are the first line drugs for treating lipid disorders and can lower LDL cholesterol levels by as much as 60%. Statins also are effective in reducing triglyceride levels in patients with hypertriglyceridemia. Statins lower LDL levels by inhibiting HMG-CoA reductase activity leading to decreases in hepatic cholesterol content resulting in an up-regulation of hepatic LDL receptors, which increases the clearance of LDL. The major side effects are muscle complications and an increased risk of diabetes. The different statins have varying drug interactions. Ezetimibe lowers LDL cholesterol levels by approximately 20% by inhibiting cholesterol absorption by the intestines leading to the decreased delivery of cholesterol to the liver, a decrease in hepatic cholesterol content, and an up-regulation of hepatic LDL receptors. Ezetimibe is very useful as add on therapy when statin therapy is not sufficient or in statin intolerant patients. Ezetimibe has few side effects. Bile acid sequestrants lower LDL cholesterol by10-30% by decreasing the absorption of bile acids in the intestine which decreases the bile acid pool consequently stimulating the synthesis of bile acids from cholesterol leading to a decrease in hepatic cholesterol content and an up-regulation of hepatic LDL receptors. Bile acid sequestrants can be difficult to use as they decrease the absorption of multiple drugs, may increase triglyceride levels, and cause constipation and other GI side effects. They do improve glycemic control in patients with diabetes, which is an additional benefit. PCSK9 monoclonal antibodies lower LDL cholesterol by 50-60% by binding PCSK9, which decreases the degradation of LDL receptors. PCSK9 inhibitors also decrease Lp(a) levels. PCSK9 inhibitors are very useful when maximally tolerated statin therapy does not reduce LDL sufficiently and in statin intolerant patients. PCSK9 inhibitors have very few side effects. Mipomersen and lomitapide are approved for lowering LDL levels in patients with Homozygous Familiar Hypercholesterolemia. Mipomersen is a second-generation apolipoprotein anti-sense oligonucleotide that decreases apolipoprotein B synthesis resulting in a reduction in the formation and synthesis of VLDL. Lomitapide inhibits microsomal triglyceride transfer protein decreasing the formation of chylomicrons in the intestine and VLDL in the liver. Both mipomersen and lomitapide have the potential to cause liver toxicity and therefore they were approved with a risk evaluation and mitigation strategy (REMS) to reduce risk.

 

INTRODUCTION

 

This chapter will discuss the currently available drugs for lowering total cholesterol levels, especially LDL cholesterol: statins, ezetimibe, bile acid sequestrants, PCSK9 inhibitors, lomitapide, and mipomersen. We will not discuss the effect of lifestyle changes or food additives, such as phytosterols, on LDL cholesterol as this is addressed in detail in the chapter entitled “Lifestyle Changes: Effect of Diet, Exercise, Functional Food, and Obesity Treatment, on Lipids and Lipoproteins” (1). Additionally, we will not discuss guidelines for determining who to treat, how aggressively to treat, or targets of treatment as these issues are discussed in detail in the chapter entitled “Risk Assessment and Guidelines for the Management of High Blood Cholesterol” (2).

 

STATINS

 

Introduction

 

In the 1970s Dr. Akira Endo, working at Sankyo, discovered that compounds isolated from fungi inhibited the activity of HMG-CoA reductase, a key enzyme in the synthesis of cholesterol (3). Further studies at Merck led to the development of the first HMG-CoA reductase inhibitor, lovastatin, approved in 1987 for the treatment of hypercholesterolemia (4). There are currently seven HMG-CoA reductase inhibitors (statins) approved in the United States for lowering cholesterol levels. Three statins are derived from fungi (lovastatin, simvastatin, and pravastatin) and four statins are synthesized (atorvastatin, rosuvastatin, fluvastatin, and pitavastatin). Most of these statins are now generic drugs and therefore they are relatively inexpensive. Which particular statin one elects to use may depend on the degree of cholesterol lowering needed and the potential of drug-drug interactions. Statins are the first line drugs for treating lipid disorders and therefore one of the most widely utilized class of drugs. Statins have revolutionized the field of preventive cardiology and make an important contribution to the reduction in atherosclerotic cardiovascular events.

 

Effect on Statins on Lipid and Lipoprotein Levels

 

The major effect of statins is lowering LDL cholesterol levels. The effect of the various statins at different doses on LDL cholesterol levels is shown in Table 1. As can be seen in Table 1 different statins have varying abilities to lower LDL cholesterol with maximal reductions of approximately 60% seen with rosuvastatin 40mg. Doubling the dose of a statin results in an approximate 6% further decrease in LDL cholesterol levels. The percent reduction in LDL cholesterol levels is similar in patients with high and low starting LDL cholesterol levels but the absolute decrease is greater if the starting LDL cholesterol is high. Because of this profound ability of statins to lower LDL cholesterol levels, treatment with these drugs as monotherapy is often sufficient to lower LDL cholesterol below target levels.

Table 1: Approximate Effect of Different Doses of Statins on LDL Cholesterol Levels
% LDL Reduction Simvastatin

(Zocor)

Atorvastatin

(Lipitor)

Lovastatin

(Mevacor)

Pravastatin

(Pravachol)

Fluvastatin

(Lescol)

Rosuvastatin

(Crestor)

Pitavastatin

(Livalo)

27 10mg - 20mg 20mg 40mg - -
34 20mg 10mg 40mg 40mg 80mg - 1mg
41 40mg 20mg 80mg 80mg - - 2mg
48 80mg 40mg - - - 10mg 4mg
54 - 80mg - - - 20mg -
60 - - - - - 40mg -

Data modified from package inserts

 

As would be predicted from the effect of statins on LDL cholesterol levels, statins are also very effective in lowering non-HDL cholesterol levels (LDL cholesterol is the major contributor to non-HDL cholesterol levels) (5, 6). In addition statins also lower plasma triglyceride levels(7, 8). The ability of statins to lower triglyceride levels correlates with the reduction in LDL cholesterol (8). Statins that are most efficacious in lowering LDL cholesterol are also most efficacious in lowering plasma triglyceride and VLDL cholesterol levels. Notably the percent reduction in plasma triglyceride levels is dependent on the baseline triglyceride levels (8). For example, in patients with normal triglyceride levels (<150mg/dl), simvastatin 80mg per day lowered plasma triglyceride levels by 11%. In contrast, if plasma triglyceride levels were elevated (> 250mg/dl), simvastatin 80mg per day lowered triglyceride levels by 40% (8). In patients with both elevated LDL cholesterol and triglyceride levels statin therapy can be very effective in improving the lipid profile and are therefore the first line class of drugs to treat patients with mixed hyperlipidemia unless the triglyceride levels are markedly elevated (>500-1000mg/dl). As expected, given the ability of statins to lower LDL cholesterol and triglyceride/VLDL  levels, statin therapy is very effective in lowering apolipoprotein B levels (5, 6).

 

Of note despite the ability of statins to lower LDL cholesterol, non-HDL cholesterol, and apolipoprotein B levels, statins do not lower Lp(a) levels (9). Finally statins modestly increase HDL cholesterol levels (7, 10, 11). In most studies HDL cholesterol levels increase between 5-10% with statin therapy. Interestingly, while low dose atorvastatin increases HDL levels similar to other statins at high doses the effect of atorvastatin is blunted with either very modest increases or no change observed (10).

 

Non-Lipid Effects of Statins

 

In addition to effects on lipid metabolism statins also have pleiotropic effects that may not be directly related to alterations in lipid metabolism (12). For example, statins are anti-inflammatory and consistently decrease CRP levels (13). Other pleiotropic effects of statins include anti-proliferative effects, antioxidant properties, anti-thrombosis, improving endothelial dysfunction, and attenuating vascular remodeling (12). Whether these pleiotropic effects contribute to the beneficial effects of statins in preventing cardiovascular disease is uncertain and much of the beneficial effect of statins on cardiovascular disease can be attributed to reductions in lipid levels.

 

Mechanism Accounting for the Statin Induced Lipid Effects

 

Statins are competitive inhibitors of HMG-CoA reductase, which leads to a decrease in cholesterol synthesis in the liver. This inhibition of hepatic cholesterol synthesis results in a decrease in cholesterol in the endoplasmic reticulum resulting in the movement of sterol regulatory element binding proteins (SREBPs) from the endoplasmic reticulum to the golgi where they are cleaved by proteases into active transcription factors(14). The SREBPs then translocate to the nucleus where they increase the expression of a number of genes including HMG-CoA reductase and, most importantly, the LDL receptor (14). The increased expression of HMG-CoA reductase restores hepatic cholesterol synthesis towards normal while the increased expression of the LDL receptor results in an increase in the number of LDL receptors on the plasma membrane of hepatocytes leading to the accelerated clearance of apolipoprotein B and E containing lipoproteins (LDL and VLDL) (Figure 1) (14). The increased clearance of LDL and VLDL accounts for the reduction in plasma LDL cholesterol and triglyceride levels. In patients with a total absence of LDL receptors (Homozygous Familiar Hypercholesterolemia) statin therapy is not very effective in lowering LDL cholesterol levels.

Figure 1: Mechanism for the Decrease in LDL Levels

In addition to lowering LDL and VLDL levels by accelerating the clearance of lipoproteins some studies have also shown that statins reduce the production and secretion of VLDL particles by the liver (15). This could contribute to the decrease in triglyceride levels. The mechanism by which statins increase HDL cholesterol levels is not clear.

 

Pharmacokinetics and Drug Interactions

 

Statins have different pharmacokinetic properties which can explain clinically important differences in safety and drug interactions (16-19). Most statins are lipophilic except for pravastatin and rosuvastatin, which are hydrophilic. Lipophilic statins can enter cells more easily but the clinical significance of this difference is not clear. Most of the clearance of statins is via the liver and GI tract (16-18). Renal clearance of statins in general is low with atorvastatin having a very low renal clearance making this particular drug the statin of choice in patients with significant renal disease. The half-life of statins varies greatly with lovastatin, pravastatin, simvastatin, and fluvastatin having a short half-life (1-3 hours) while atorvastatin, rosuvastatin, and pitavastatin having a long half-life (16-19). In patients intolerant of statins, the use of a long acting statin every other day or 2 times per week has been employed. Short acting statins are most effective when administered in the evening when HMG-CoA reductase activity is maximal while the efficacy of long acting statins is equivalent whether given in the AM or PM (20). In patients who prefer to take their statin in the morning one should use a long acting statin.

 

A key difference between statins is their pathway of metabolism. Simvastatin, lovastatin, and atorvastatin are metabolized by the CYP3A4 enzymes and drugs that affect the CYP3A4 pathway can alter the metabolism of these statins (16-19, 21). Fluvastatin is metabolized mainly by CYP2C9 with a small contribution by CYP2C8 (16-18, 21). Pitavastatin and rosuvastatin are minimally metabolized by the CYP2C9 pathway (16-18, 21). Pravastatin is not metabolized at all via the CYP enzyme system (16-18).

 

Drugs that inhibit CYP3A4 can impede the metabolism of simvastatin, lovastatin, and to a smaller extent atorvastatin resulting in high serum levels of these drugs (16-19, 21). These higher levels are associated with adverse effects particularly muscle toxicity. Drugs that inhibit CYP3A4 include intraconazole, ketoconazole, erythromycin, clarithromycin, HIV protease inhibitors (amprenavir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, and saquinavir), amiodarone, diltiazem, verapamil, and cyclosporine (16-19, 21). It should be noted that grapefruit juice contains compounds that inhibit CYP3A4 and the consumption of grapefruit juice can significantly increase statin blood levels (22). The inhibition of CYP3A4 by grapefruit juice is dose dependent and increases with the concentration and volume of grapefruit juice ingested. One glass of grapefruit juice everyday can influence the metabolism of statins that are metabolized by the CYP3A4 pathway (22). If a patient requires treatment with a drug that inhibits CYP3A4 the clinician has a number of options to avoid potential drug interactions. One could use a statin that is not metabolized via the CYP3A4 system such as pravastatin or rosuvastatin, one could use an alternative drug to the CYP3A4 inhibitor (for example instead of using erythromycin use azithromycin), or one could temporary suspend for a short period of time the use of the statin that is metabolized by the CYP3A4 pathway (this is particularly useful when a short course of treatment with an antifungal or antibiotic is required). Drugs that inhibit CYP2C9 do not seem to increase the toxicity of fluvastatin, pitavastatin, or rosuvastatin probably because metabolism via the CYP2C9 pathway is not a dominant pathway.

 

Most statins are transported into the liver and other tissues by organic anion transporting polypeptides, particularly OATP1B1 (16-18, 21). Drugs, such as clarithromycin, ritonavir, indinavir, saquinavir, and cyclosporine that inhibit OATP1B1 can increase serum statin levels thereby increasing the risk of statin muscle toxicity (16-18, 21). Fluvastatin is the statin that is least affected by OATP1B1 inhibitors. In fact fluvastatin 40mg per day has been studied in patients receiving renal transplants concomitantly treated with cyclosporine and over a five year study period the risk of myopathy or rhabdomyolysis was not increased in the fluvastatin treated patients compared to those treated with placebo (23).

 

Gemfibrozil inhibits the glucuronidation of statins, which accounts for a significant portion of the metabolism of most statins (21). This can lead to the reduced clearance of statins and elevated blood levels increasing the risk of muscle toxicity (21). The only statin whose metabolism is not altered by gemfibrozil is fluvastatin (21). Notably, fenofibrate, another fibrate that has very similar effects on lipid and lipoprotein levels as gemfibrozil, does not inhibit statin glucuronidation (21). Therefore, in patients on statin therapy who also need treatment with a fibrate one should use fenofibrate and not gemfibrozil in combination with statin therapy. Studies have shown that fenofibrate combined with statins does not increase toxicity (24).

 

There are other drug interactions with statins whose mechanisms are unknown. For example, the lopinavir/ritonavir combination used to treat HIV increases rosuvastatin levels by 2-5 fold and atazanavir/ritonavir increases rosuvastatin levels by 2-6 fold (25-29). Similarly, the tipranavir/ritonavir combination increases rosuvastatin levels 2 fold and atorvastatin levels 8 fold (28). When HIV patients are on these drugs other statins should be used to lower LDL cholesterol levels.

 

Thus, despite the excellent safety record of statins, careful attention must be paid to the potential drug-drug interactions. For additional information see Kellick et al (19, 21).

 

Effect of Statin Therapy on Clinical Outcomes

 

A large number of studies using a variety of statins in diverse patient populations have shown that statin therapy reduces atherosclerotic cardiovascular disease. The Cholesterol Treatment Trialists have published meta-analyses derived from individual subject data. Their first publication included data from 14 trials with over 90,000 subjects (30). There was a 12% reduction in all-cause mortality in the statin treated subjects, which was mainly due to a 19% reduction in coronary heart disease deaths. Non-vascular causes of death were similar in the statin and placebo groups indicating that statin therapy and lowering LDL cholesterol did not increase the risk of death from other causes such as cancer, respiratory disease, etc. Of particular note there was a 23% decrease in major coronary events per 1 mmol/L (39mg/dl) reduction in LDL cholesterol. Decreases in other vascular outcomes including non-fatal MI, coronary heart disease death, vascular surgery and stroke were also reduced by 20-25% per 1 mmol/L (39mg/dl) reduction in LDL cholesterol. Additionally, analysis of these studies demonstrated that the greater the reduction in absolute LDL cholesterol levels the greater the decrease in cardiovascular events.  For example, while a 40mg/dl decrease in LDL cholesterol will reduce coronary events by approximately 20%, an 80mg/dl decrease in LDL cholesterol will reduce events by approximately 40%. These results support aggressive lipid lowering with statin therapy.

 

Of note the decrease in the number of events begins to be seen in the first year of therapy indicating that the ability of statins to reduce events occurs relatively quickly and increases over time. The ability of statins to reduce cardiovascular events was seen in a wide diversity of patients including those with and without a history of prior cardiovascular disease, patients over age 65 and younger than age 65, males and females, and patients with and without a history of diabetes or hypertension. Additionally, the beneficial effects of statins were seen regardless of the baseline lipid levels. Subjects with elevated or low LDL cholesterol, HDL cholesterol, or triglyceride levels all had similar decreases in the relative risk of cardiovascular events.

 

A subsequent publication by the Cholesterol Treatment Trialists has focused on five studies with over 39,000 subjects that have compared usual vs. intensive statin therapy (31). It was noted that there was a 0.51mmol/L (20mg/dl) further reduction in LDL cholesterol in the intensively treated subjects. This further decrease in LDL cholesterol resulted in a15% reduction in cardiovascular events. The strong numerical relationship between decreases in LDL cholesterol levels and the reduction in cardiovascular events provides evidence indicating that much of the beneficial effect of statins is accounted for by lipid lowering.

 

In addition, the authors added 7 additional trials to their original comparison of statin treatment vs. placebo for a total of 21 trials with over 129,000 subjects. In this larger cohort a 1mmol/L (39mg/dl) decrease in LDL was associated with a 21% reduction in major cardiovascular events. As seen previously the benefits of statin therapy were seen in a wide variety of subjects including patients older than age 75, obese patients, cigarette smokers, patients with decreased renal function, and patients with low and high HDL cholesterol levels. Additionally, a reduction of cardiovascular events with statin therapy was seen regardless of baseline LDL cholesterol levels.

 

A more recent meta-analysis by the Cholesterol Treatment Trialists examined the effect of statins in 27 trials that included 46,675 women and 127,474 men (32). They found that statin therapy was similarly effective in reducing cardiovascular events in both men and women. Thus, there is an overwhelming database of randomized clinical outcome trials showing the benefits of statin therapy in reducing cardiovascular disease, which, coupled with their excellent safety profile, has resulted in statins becoming a very widely used class of drugs and first line therapy for the prevention of cardiovascular disease.

 

Effect of Statins Therapy on Clinical Outcomes in Specific Patient Groups

 

PRIMARY PREVENTION

 

While there is no doubt that individuals with pre-existing cardiovascular disease require statin therapy, the use of statins for primary prevention was initially debated. There are now a large number of statin primary prevention studies. The Cholesterol Treatment Trialists reported that statin therapy was very effective in reducing cardiovascular events in subjects without a history of vascular disease and the magnitude of risk reduction was similar to subjects with a history of cardiovascular events (31). Additionally, vascular deaths were reduced by statin treatment even in subjects without a history of vascular disease. As expected, non-vascular deaths were not altered in these subjects without a history of pre-existing vascular disease. Additionally, the Cholesterol Treatment Trialists compared the benefits of statin therapy based on baseline risk of developing cardiovascular disease (<5%, ≥5% to <10%, ≥10% to <20%, ≥20% to <30%, ≥30%) (33). The proportional reduction in major vascular events was at least as big in the two lowest risk categories as in the higher risk categories indicating that subjects at low risk benefit from statin therapy. Similar to the Cholesterol Treatment Trialists analysis, a Cochrane review published in 2013 on the effect of statins in primary prevention patients reached the following conclusion: “Reductions in all-cause mortality, major vascular events, and revascularisations were found with no excess adverse events among people without evidence of CVD treated with statins” (34). Recently, an additional study (HOPE-3 trial) has been completed that focused on intermediate risk patients without cardiovascular disease. In this trial 12,705 men and women who had at least one risk factor for cardiovascular disease were randomized to 10mg rosuvastatin vs. placebo (35). Rosuvastatin treatment resulted in a 27% reduction in LDL cholesterol levels and a 24% decrease in cardiovascular events providing additional evidence that statin treatment can reduce events in primary prevention patients. It is therefore clear that statins are effective in safely reducing events in primary prevention patients.

 

The key issue is “which primary prevention patients should be treated” and this is still controversial.  It should be noted that the higher the baseline risk the greater the absolute reduction in events with statin therapy. For example, in a high-risk patient with a 20% risk of developing a vascular event, a 25% risk reduction will result in a 15% risk of an event (absolute decrease of 5%). In contrast in a low risk patient with a 4% risk of developing a vascular event, a 25% risk reduction will result in a 3% risk (absolute decrease of only 1%). Thus, the absolute benefit of statin therapy over the short term will depend on the risk of developing cardiovascular disease.

 

Additionally, based on the Cholesterol Treatment Trialists results the reduction in cardiovascular events is dependent on the absolute decrease in LDL cholesterol levels. Thus, the effect of statin treatment will be influenced by baseline LDL levels. For example, a 50% decrease in LDL is 80mg/dl if the starting LDL is 160mg/dl and only 40mg/dl if the starting LDL is 80mg/dl. Based on studies showing that a decrease in LDL of 1 mmol/L (40mg/dl) reduces cardiovascular events by ~20% the relative benefit of statin therapy will be greater in the patient with the starting LDL of 160mg/dl (40% decrease in events) than in the patient with the starting LDL of 80mg/dl (20% decrease in events). Thus, decisions on treatment need to factor in both relative risk and baseline LDL levels.

 

Finally, it should be recognized that clinical trials represent short term reductions in LDL cholesterol levels (typically 2-5 years) in a disorder that begins early in life and progresses over decades. Life-long decreases in LDL cholesterol levels due to genetic polymorphisms are associated with a much greater reduction in cardiovascular events than would be expected based on the clinical trial results (36). These results suggest that earlier and longer lasting therapy that decreases LDL cholesterol levels will result in a greater reduction in cardiovascular events (37).

 

ELDERLY

 

The Cholesterol Treatment Trialists reported that cardiovascular events were reduced by 22% in subjects age 65 years or younger, 22% in subjects 66 to 75 years old, and 16% for those older than 76 years for every 1mmol/L (39mg/dl) decrease in LDL cholesterol (31). The Cholesterol Treatment Trialists results included both primary and secondary prevention trials and there has been questions of whether the benefit of statins applied to primary prevention in the elderly patients. In a meta-analysis of eight randomized controlled trials focusing on primary prevention of cardiovascular disease in subjects ≥65 years of age (n= 25,952), it was reported that statins significantly reduced the risks of composite major adverse cardiovascular events (RR 0.82, 95% CI 0.74-0.92 (38). A meta-analysis of the Jupiter trial and HOPE trial, two primary prevention trials, examined the effect of statin therapy on patients over 70 years of age (n=8,781) (39). A 26% reduction in nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death was observed (HR, 0.74; 95% CI, 0.61–0.91; P=0.0048).

 

Thus, current data indicate that statin induced reductions in LDL cholesterol will decrease cardiovascular events in the elderly. The STAtins in Reducing Events in the Elderly (STAREE) study is currently recruiting 10,000 healthy men and women over 70 years of age in Australia who will be randomized to atorvastatin 40mg per day or placebo (NCT: 02099123). The results of this study will provide further information on the role of statin therapy in the elderly. Unfortunately, studies on the very old (>80-85 year) are not available. For additional information on lipid lowering in the elderly see the chapter entitled “Management of Dyslipidemia in the Elderly” (40).

 

WOMEN

 

As noted above a recent meta-analysis by the Cholesterol Treatment Trialists examined the effect of statins in 27 trials that included 46,675 women and 127,474 men (32). They found that statin therapy was similarly effective in reducing cardiovascular events in both men and women.

 

ASIANS

 

Pharmacokinetic data have shown that the serum levels of statins are higher in Asians than in Caucasians (41). Moreover, Asians achieve similar LDL lowering at lower statin doses than Caucasians (41). Therefore, the statin dose used should be lower in Asians. For example, the starting dose of rosuvastatin is 5mg in Asians as compared to 10mg in Caucasians. Additionally, the maximum recommended dose of statin is lower in Japan vs. the United States (Table 2). In contrast, studies suggest that South Asian patients may be treated with atorvastatin and simvastatin at doses typically applied to white patients (42).

 

Table 2: Maximum Statin Dose in Japan and United States
Statin Japan United States
Atorvastatin 40 80
Fluvastatin 60 80
Pravastatin 20 80
Rosuvastatin 20 40
Simvastatin 20 40

DIABETES

 

Statin trials, including both primary and secondary prevention trials, have consistently shown the beneficial effect of statins on cardiovascular disease in patients with diabetes (43). The Cholesterol Treatment Trialists analyzed data from 18,686 subjects with diabetes (mostly type 2 diabetes) from 14 randomized trials (44). In the statin treated group there was a 9% decrease in all-cause mortality, a 13% decrease in vascular mortality, and a 21% decrease in major vascular events per 1mmol/L (39mg/dl) reduction in LDL cholesterol. The beneficial effect of statin therapy was seen in both primary and secondary prevention patients. The effect of statin treatment on cardiovascular events in patients with diabetes was similar to that seen in non-diabetic subjects. It should be noted that while the data for patients with type 2 diabetes is robust, the number of patients with type 1 diabetes in these trials is relatively small and the results less definitive. Also, of note is that information on young patients with diabetes (< age 40) is very limited. Thus, these studies indicate that statins are beneficial in reducing cardiovascular disease in patients with diabetes. For addition details on the treatment of dyslipidemia in patients with diabetes see the chapter entitled “Diabetes and Dyslipidemia” (43).

 

RENAL DISEASE

 

The Cholesterol Treatment Trialists examined the effect of renal function on statin effectiveness. They reported that the relative risk reduction for cardiovascular events was similar if the eGFR was < 60ml/min as compared to > 90 or 60-90 (31). In a follow-up analysis it was reported that the relative risk reduction per 1mMol/l (~39mg/dl) decrease in LDL cholesterol levels with statin therapy was 0·78 for an eGFR ≥60 mL/min, 0·76 for an eGFR 45 to <60 mL/min, 0·85 for an eGFR 30 to <45 mL/min, and 0·85 for an eGFR <30 mL/min in patients not on dialysis (45). In patients on dialysis the relative risk reduction was 0·94 (99% CI 0·79-1·11). Similarly, a meta-analysis of 57 studies with >143,000 participants with renal disease not on dialysis reported a 31 % reduction in major cardiovascular events in statin treated subjects compared to placebo groups (46). Thus, in patients with renal disease not on dialysis, treatment with statins is beneficial and should be utilized in this population at high risk for vascular disease.

 

In contrast to the above results, studies examining the role of statins in dialysis patients have not found a benefit from statin therapy. The Deutsche Diabetes Dialyse Studie (4D) randomized 1255 type 2 diabetic subjects on hemodialysis to either 20 mg atorvastatin or placebo (47). The LDL-cholesterol reduction was similar to that seen in non-dialysis patients but there was no significant reduction in cardiovascular death, nonfatal myocardial infarction, or stroke in the atorvastatin treated compared to the placebo group. Similarly, A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis (AURORA) randomized 2776 subjects on hemodialysis to rosuvastatin 10 mg or placebo (48). Again, the LDL-cholesterol lowering in dialysis patients was similar to that seen in other studies but there was no significant effect on the primary endpoint of cardiovascular death, nonfatal myocardial infarction, or stroke. A meta-analysis of 25 studies involving 8289 dialysis patients found no benefit of statin therapy on major cardiovascular events, cardiovascular mortality, all-cause mortality, or myocardial infarction, despite efficacious lipid lowering. The reason for the failure of statins in patients on maintenance dialysis is unclear but could be due to a number of factors including the possibility that the marked severity of atherosclerosis in end stage renal disease may limit reversal, that different mechanisms of atherosclerosis progression occurs in dialysis patients (for example an increased role for inflammation, oxidation, or thrombosis), or that cardiovascular events in this patient population may not be due to atherosclerosis. We would recommend continuing statin therapy in patients on dialysis who have been previously treated with statins but not initiating therapy in the rare statin naïve patient beginning dialysis.

 

Statins are primarily metabolized in the liver and therefore the need to adjust the statin dose is not usually needed in patients with renal disease until the eGFR is < 30ml/min. The effect of renal dysfunction on statin clearance varies from statin to statin (49). For some statins such as atorvastatin, there is no need to adjust the dose in renal disease because there is limited renal clearance (49). However, for other statins it is recommended to adjust the dose in patients when the eGFR is < 30ml/min. In patients with an eGFR < 30ml/min the maximum dose of rosuvastatin is 10mg, simvastatin 40mg, pitavastatin 2mg, pravastatin 20mg, lovastatin 20mg, and fluvastatin 40mg per day (49).

 

For additional information on the treatment of dyslipidemia in patients with renal disease see the chapter entitled “Dyslipidemia in Chronic Kidney Disease” (49).

 

CONGESTIVE HEART FAILURE

 

In the Corona study 5011 patients with systolic heart failure were randomly assigned to receive 10 mg of rosuvastatin or placebo per day (50). While rosuvastatin treatment reduced LDL cholesterol levels by 45% compared to placebo, rosuvastatin did not decrease death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke. Similarly, the GISSI-HF trial randomized 4574 patients with congestive heart failure to 10mg of rosuvastatin or placebo (51). The primary endpoints were time to death, and time to death or admission to hospital for cardiovascular reasons and these were similar in the statin and placebo groups Why statin treatment was not beneficial in patients with congestive heart failure is unknown.

 

LIVER DISEASE

 

Many patients with liver disease, particularly those with nonalcoholic fatty liver disease (NAFLD), are at high risk for cardiovascular disease and therefore require statin therapy (52). There have been concerns that these patients would not tolerate statin therapy and that statin therapy would worsen their underlying liver disease. Fortunately, there are now studies of statin therapy in patients with abnormal liver function tests and underlying liver disease at baseline (52-54). With a variety of statins, studies have demonstrated no significant worsening of liver disease and in fact several studies have suggested improvement in liver function tests with statin therapy (54). This is true for patients with hepatitis C, NAFLD/NASH, and primary biliary cirrhosis. Additionally, in the GREACE trial, statin treatment reduced cardiovascular events in patients with moderately abnormal liver function tests (transaminases < 3x the upper limit of normal) (55). Thus, in patients with mild liver disease without elevations in bilirubin or abnormalities in synthetic function, statins are safe and reduce the risk of cardiovascular disease.

 

For additional information on the treatment of dyslipidemia in patients with liver disease see the chapter entitled “Lipid and Lipoprotein Metabolism in Liver Disease” (52)

 

Statin Side Effects

 

DIABETES

 

After many years of statin use it was recognized that statins increase the risk of developing diabetes. In a meta-analysis of 13 trials with over 90,000 subjects, there was a 9% increase in the incidence of diabetes during follow-up among subjects receiving statin therapy (56). All statins appear to increase the risk of developing diabetes. In comparisons of intensive vs. moderate statin therapy, Preiss et al observed that patients treated with intensive statin therapy had a 12% greater risk of developing diabetes compared to subjects treated with moderate dose statin therapy (57). Older subjects, obese subjects, and subjects with high glucose levels were at a higher risk of developing diabetes while on statin therapy (43). Thus, statins may be unmasking and accelerating the development of diabetes that would have occurred naturally in these subjects at some point in time. In patients without risk factors for developing diabetes, treatment with statins does not appear to increase the risk of developing diabetes.

 

In patients with diabetes, an analysis of 9 studies with over 9,000 patients with diabetes reported that the patients randomized to statin therapy had a 0.12% higher A1c than the placebo group indicating that statin therapy is associated with only a very small increase in A1c levels in patients with diabetes that is unlikely to be clinically significant (58). Individual studies, such as CARDS and the Heart Protection Study, have also shown only a very modest effect of statins on A1c levels in patients with diabetes (59,60).

 

The mechanism by which statins increase the risk of developing diabetes is unknown. A study has demonstrated that a polymorphism in the gene for HMG-CoA reductase that results in a decrease in HMG-CoA reductase activity and a small decrease in LDL levels is also associated with an increase in body weight and plasma glucose and insulin levels (61). Additionally, a cross sectional study that compared the change in BMI in individuals on statins to individuals not on statins observed an increased BMI in the subjects taking statins (+1.3 in stain users vs. + 0.4 in non-users over a 10 year period; p=0.02) (62). These observations suggest that the inhibition of HMG-CoA reductase per semay be leading to the statin induced increased risk of diabetes via weight gain. However, a large number of studies have now shown that polymorphisms in a variety of different genes that lead to a decrease in LDL cholesterol levels are associated with an increase in diabetes suggesting that decreases in LDL cholesterol levels per sealter glucose metabolism and increase the risk of diabetes (63). How decreased LDL cholesterol levels effect glucose metabolism is unknown. Clearly further studies are required to understand the mechanisms by which statins increase the risk of developing diabetes.

 

In balancing the benefits and risks of statin therapy it is important to recognize that an increase in plasma glucose levels is a surrogate marker for an increased risk of developing micro and macrovascular disease (i.e. an increase in plasma glucose per seis not an event but rather increases the risk of future events). In contrast, statin therapy is preventing actual clinical events that cause morbidity and mortality. Furthermore, it may take many years for an elevated blood glucose to induce diabetic complications while the reduction in cardiovascular events with statin therapy occurs relatively quickly. Patients on statin therapy, particularly those with risk factors for the development of diabetes, should be periodically screened for the development of diabetes with measurement of fasting glucose levels or A1c levels.

 

CANCER

 

Analysis of 14 trials with over 90,000 subjects by the Cholesterol Clinical Trialists did not demonstrate an increased risk of cancer or any specific cancer with statin therapy (30). An update with an analysis of 27 trials with over 174,000 participants also did not observe an increase in cancer incidence or death (32). Additionally, no differences in cancer rates were observed with any particular statin.

 

COGNITIVE DYSFUNCTION

 

Several randomized clinical trials have examined the effect of statin therapy on cognitive function and have not indicated any increased risk (64-66). The Prosper Trial was designed to determine whether statin therapy will reduce cardiovascular disease in older subjects (age 70-82) (67). In this trial cognitive function was assessed repeatedly and no difference in cognitive decline was found in subjects treated with pravastatin compared to placebo (67,68). In the Heart Protection Study over 20,000 patients were randomized to simvastatin 40mg or placebo and again no significant differences in cognitive function was observed between the statin vs. placebo group (69). Additionally, a Cochrane review examined the effect of statin therapy in patients with established dementia and identified 4 studies with 1154 participants (70). In this analysis no benefit or harm of statin therapy on cognitive function could be demonstrated in this high-risk group of patients. Thus, while the FDA has mandated warnings regarding statin induced cognitive dysfunction, randomized clinical trials do not indicate a significant association.

 

LIVER DISEASE

 

It was in initially thought that statins induced liver dysfunction and it was recommended that liver function tests be routinely obtained while patients were taking statins. However, studies have now shown that the risk of liver function test abnormalities in patients taking statins is very small (53). For example, in a survey of 35 randomized studies involving > 74,000 subjects, elevations in transaminases were seen in 1.4% of statin treated subjects and 1.1% of controls (71). Similarly, in a meta-analysis of > 49,000 patients from 13 placebo controlled studies, the incidence of transaminase elevations greater than three times the upper limit of normal was 1.14% in the statin group and 1.05% in the placebo group (72). Moreover, even when the transaminase levels are elevated, repeat testing often demonstrates a return towards normal levels (73). The increases in transaminase levels with statin therapy are dose related with high doses of statins leading to more frequent elevations (74). At this time, routine monitoring of liver function tests in patients taking statins is no longer recommended. However, obtaining baseline liver function tests prior to starting statin therapy is indicated (53). If liver function tests are obtained during statin treatment, one should not be overly concerned with modestly elevated transaminase levels (less than 3x the upper limit of normal) (53). If the transaminase is greater than 3x the upper limit of normal the test should be repeated and if it remains > 3x the upper limit of normal, statin therapy should be stopped and the patient evaluated (53).

 

A more clinically important issue is whether statins lead to an increased risk of liver failure. Studies have suggested that the incidence of liver failure in patients taking statins is very similar to the rate observed in the general population (approx. 1 case per 1 million patient years) (75,76). Thus, statin therapy causing serious liver injury is a very rare event.

 

MUSCLE

 

The most common side effect of statin therapy is muscle symptoms. These can range from life threatening rhabdomyolysis to myalgias (Table 3) (77).

 

Table 3: Spectrum of Statin Induced Muscle Disorders (Adapted from J. Clinical Lipidology 8:S58-71, 2014)
Myalgia- aches, soreness, stiffness, tenderness, cramps with normal CK levels
Myopathy-muscle weakness with or without increased CK
Myositis- muscle inflammation
Myonecrosis-mild (CK >3x ULN); moderate (CK> 10x ULN); severe (CK> 50x ULN)
Rhabdomyolysis-myonecrosis with myoglobinuria or acute renal failure

 

Many patients will discontinue the use of statins due to muscle symptoms. Risk factors associated with an increased incidence of statin associated muscle symptoms are listed in Table 4 (78,79).

 

Table 4: Risk factors for Statin Myopathy
Medications that alter statin metabolism
Older age
Female
Hypothyroidism
Excess alcohol
Vitamin D deficiency
History of muscle disorders
Renal disease
Liver disease
Personal or family history of statin intolerance
Low BMI
Polymorphism in SLCO1B1 gene
High dose statin
Drug-drug interactions

 

In most randomized clinical trials, the incidence of myalgia was similar in the statin and placebo groups (see table 5) (80). For example, in the AFCAPS/TexCAPS trial in the 3,301 subjects treated with 20-40mg lovastatin, 2,053 reported musculoskeletal symptoms (81). However, in the 3,301 subjects in the placebo group 1,971 also reported musculoskeletal symptoms.Similarly, in the Jupiter trial, where 18,902 subjects were randomized to rosuvastatin 20mg per day or placebo 16% of the subjects treated with rosuvastatin had muscle symptoms (82). However, in the placebo group muscle symptoms occurred in 15.4% of subjects.

        

Table 5: Muscle Disorders in Randomized Controlled Statin Trials
  Myalgia % Myopathyb%      Rhabdomyolysisc%

 

Trial Drug Dose Sa Pa S P S P
4S Simva 20-40 3.7 3.2 0.05 0 0 0
WOSCOPS Prava 40 3.5 3.7 0 0 0 0
HPS Simva 40 NR NR 0.07 0.02 0.04 0.01
PROSPER Prava 40 1.2 1.1 0 0 0 0
CARDS Atorva 10 4.0 4.8 0 0 0 0
ASPEN Atorva 10 3.0 1.6 0 0 0.08 0.08
SPARCL Atorva 80 5.5 6.0 0.3 0.3 0.1 0.1
JUPITER Rosuva 20 7.9 6.9 0.1 0.1 0.01 0

a- S= statin, P= placebo; b- muscle pain of weakness with CK > 10x ULN; c- myopathy with CK > 40x ULN and/or renal impairment; d- HPS asked about muscle symptoms at each visit

 

While the results of the randomized trials suggest that muscle symptoms are not induced by statin therapy, in typical clinical settings a significant percentage of patients are unable to tolerate statins due to muscle symptoms (in many studies as high as 5-25% of patients) (83-85). Recently there was a randomized trial that explored the issue of myopathy with statin therapy in great detail (86). In this trial the effect of atorvastatin 80mg a day vs. placebo for 6 months on creatine kinase (CK), exercise capacity, and musclestrength was studied in 420 healthy, statin-naive subjects. Atorvastatin treatment led to a modest increase in CK levels (20.8U/L) with no change observed in the placebo group. None of the subjects had an elevation of CK > 10x the upper limits of normal. There were no changes in muscle strength or exercise capacity with atorvastatin treatment. However, myalgia was reported in 19 subjects (9.4%) in the atorvastatin group compared to 10 subjects (4.6%) in the placebo group (p=0.05).  In this study “myalgia” was considered to be present if all of the following occurred: (1) subjects reported new or increased muscle pain, cramps, or aching not associated with ex­ercise; (2) symptoms persisted for at least 2 weeks; (3) symptoms resolved within 2 weeks of stopping the study drug; and (4) symp­toms reoccurred within 4 weeks of restarting the study medication. Notably these myalgias were not associated with elevated CK levels. In the atorvastatin group the myalgias tended to occur soon after therapy (average 35 days) whereas in the placebo group myalgias occur later (average 61 days). In the atorvastatin group the symptoms were predominantly localized to the legs and included aches, cramps, and fatigue, whereas in the placebo group they were more diverse including whole body fatigue, foot cramps, worsening of pain in previous injuries, and groin pain. A number of conclusions can be reached from this study. First, statin treatment does in fact increase the incidence of myalgias. Second, a substantial number of patients treated with placebo will also develop myalgias. Third, clinically differentiating statin induced myalgias from placebo induced myalgias is difficult, as there are no specific symptoms, signs, or biomarkers that clearly distinguish between the two.  It should be recognized that the patient population typically treated with statins (patients 50-80 years of age) often have muscle symptoms in the absence of statin therapy and it is therefore difficult to be certain that the muscle symptoms described by the patient are actually due to statin therapy.

 

In a very small study in the Annals of Internal Medicine eight patients with “statin related myalgia” were re-challenged with statin or placebo and there were no statistically significant differences in the recurrence of myalgias on the statin or placebo (87). This approach has been expanded upon in two larger studies. In 120 patients with “statin induced myalgia” patients were randomized in a double blinded crossover trial to either simvastatin 20mg per day or placebo and the occurrence of muscle symptoms was determined (88). Only 36% of these patients were confirmed to actually have statin induced myalgia (presence of symptoms on simvastatin without symptoms on placebo). In a similar study, Nissen and colleagues studied 491 patients with “statin induced myalgia” treating with either atorvastatin 20mg per day or placebo in a double-blind crossover trial (89). In this trial 42.6% of patients were confirmed to have statin induced muscle symptoms. Thus, while statin induced myalgias are a real entity careful studies have shown that in the majority of patients with “statin induced muscle symptoms” the symptoms are not actually due to statin therapy. In the clinic it is difficult to be certain whether the muscle symptoms are actually due to true statin intolerance or to other factors. The approach to treating these patients will be discussed later in this chapter (Treatment of Stain Intolerant Patients). While some patients will not tolerate statin therapy due to myalgias, this side effect does not appear to result in serious morbidity or long-term consequences.

 

Fortunately, the more serious muscle related side effects of statin therapy are rare (table 5). In a meta-analysis of 21 statin vs. placebo trials there was an excess risk of rhabdomyolysis of 1.6 patients per 100,000 patient years or a standardized rate of 0.016/patient years (73). Other studies report a rate of rhabdomyolysis between 0.03- 0.16 per 1,000 patient years (90). Similarly, the risk of statin induced myositis (muscle symptoms with an increase in CK 10 times the upper limits of normal) is also very low (table 5). In an analysis of 21 randomized trials myositis occurred in only 5 patients per 100,000 person years or 0.05/1000 patient years (73). The higher the dose of statin used the greater the risk of myositis and rhabdomyolysis. In a comparison of five trials that compared high dose statin vs. low dose statin there was an excess risk of rhabdomyolysis of 4 per 10,000 people treated (31). The likely basis for an increased risk of myositis or rhabdomyolysis is elevated statin blood levels, which are more likely to occur with high doses of statins. In the development of statins, manufacturers have studied higher doses that are not approved for clinical use. For example, simvastatin and pravastatin at 160mg per day were studied but discontinued due to an increased incidence of muscle side effects (91, 92). The use of simvastatin 80mg per day, a previously approved dose, was restricted due to an increased risk of muscle side effects. Similarly, pitavastatin at doses greater than 4mg per day was investigated, but development was abandoned when an increased risk of rhabdomyolysis was observed. Along similar lines, in many of the patients that develop rhabdomyolysis, the etiology can be linked to the use of other drugs that alter statin metabolism thereby increasing statin blood levels (79). For example, prior to drug interactions being recognized the use of cyclosporine, gemfibrozil, HIV protease inhibitors, and erythromycin in conjunction with certain statins was linked with the development of rhabdomyolysis (79). Finally, common variants in SLCO1B1, which encodes the organic anion-transporting polypeptide OATP1B1, are strongly associated with an increased risk of statin-induced myopathy (93). OATP1B1 facilitates the transport of statins into the liver and certain polymorphisms are associated with an increased risk of developing statin induced muscle disorders, due to the decreased transport of statins into the liver resulting in increased blood levels (94).  The exact mechanism by which elevated blood levels induce muscle toxicity remains to be elucidated.

 

Recently it has been recognized that a very small number of patients taking statins develop a progressive autoimmune necrotizing myopathy, which is characterized by progressive symmetric proximal muscle weakness, elevated CK levels (typically >10x the ULN), and antibodies against HMG-CoA reductase (95). It is estimated that this occurs in 2 or 3 per 100,000 patients treated with a statin (95). This myopathy may begin soon after initiating statin therapy or develop after a patient has been on statins for many years (95).Muscle biopsy reveals necrotizing myopathy without severe inflammation (95). In contrast to the typical muscle disorders induced by statin therapy, the autoimmune myopathy progresses despite discontinuing therapy. Spontaneous improvement is not typical and most patients will need to be treated with immunosuppressive therapy (glucocorticoids plus methotrexate, azathioprine, or mycophenolate mofetil) (95). It should be recognized that this disorder can occur in individuals that have not been exposed to statin therapy (96). Statins likely potentiate the development of this disorder in susceptible individuals, perhaps by increasing HMG-CoA reductase levels.

 

From the above certain conclusions can be reached. First, the risk of serious muscle disorders due to statin therapy is very small, particularly if one is aware of the potential drug interactions that increase the risk. Second, the muscle toxicity is usually linked to elevated statin blood levels and the higher the dose of the statin the more likely the chance of developing toxicity. Third, myalgias in patients taking statins are very common and can be due to statin treatment. However, in the individual patient, it is very difficult to know if the myalgia is actually secondary to statin therapy and in many patients the myalgias are not due to the statin therapy. Fourth, the muscle symptoms that occur in association with statin treatment are a major reason why patients discontinue statin use and therefore better diagnostic algorithms and treatments are required to allow patients to better comply with these highly effective treatments to reduce cardiovascular disease.

 

Contraindications

 

Statins are contraindicated in pregnant women or lactating women. In women of child bearing age birth control should be discussed and statins should be discontinued prior to conception. In addition, liver function tests should be obtained prior to initiating statin treatment and moderate to severe liver disease is a contraindication to statin therapy (53).

 

Summary

 

An enormous data base has accumulated which demonstrates that statins are very effective at reducing the risk of cardiovascular disease and that statins have an excellent safety profile. The risk benefit ratio of treating patients with statins is very favorable and has resulted in this class of drugs being widely utilized to lower serum lipid levels and to reduce the risk of cardiovascular disease and death.

 

EZETIMBE

 

Introduction

 

Ezetimibe (Zetia) inhibits the absorption of cholesterol by the intestine thereby resulting in modest decreases in LDL cholesterol levels (97). Ezetimibe is primarily used in combination with statin therapy when statin treatment alone does not lower LDL levels sufficiently. It may also be used as monotherapy to lower LDL cholesterol levels in patients with statin intolerance. Finally, it is the drug of choice in patients with the rare genetic disorder sitosterolemia (discussed in detail in the chapter “Rare Genetic Disorders Altering Lipoproteins”) (98).

 

Effect of Ezetimibe on Lipid and Lipoprotein Levels

 

Pandor and colleagues have published a meta-analysis of ezetimibe monotherapy that included 8 studies with 2,722 patients (99). They reported that ezetimibe decreased LDL cholesterol levels by 18.6%, decreased triglyceride levels by 8.1%, and increased HDL cholesterol levels by 3% compared to placebo. In a pooled analysisby Morrone and colleagues of 27 studies with 11, 714 subjects treated with ezetimibe in combination with statin therapy similar results were observed (100). Specifically, LDL cholesterol levels were decreased by 15.1%, non-HDL cholesterol levels by 13.5%, triglycerides by 4.7%, apolipoprotein B levels by 10.8%, and HDL cholesterol levels were increased by 1.6%. The combination of a high dose potent statin plus ezetimibe can lower LDL cholesterol levels by 70% (101). The effect of ezetimibe on lipid parameters occurs quickly and can be seen after 2 weeks of treatment. In patients with Heterozygous Familial Hypercholesterolemia who have marked elevations in LDL cholesterol levels, the addition of ezetimibe to statin therapy resulted in similar changes with a further 16.5% decrease in LDL cholesterol levels (102). Thus, in comparison with statins, ezetimibe treatment produces modest decreases in LDL cholesterol levels (15-20%). In addition to these changes in lipid parameters, ezetimibe in combination with a statin decreased hs-CRP by 10-19% compared to statin monotherapy (103, 104). However, ezetimibe alone does not decrease hs-CRP levels (104).

 

Mechanisms Accounting for the Ezetimibe Induced Lipid Effects

 

NPC1L1 (Niemann-Pick C1-like 1 protein) is highly expressed in the intestine with the greatest expression in the proximal jejunum, which is the major site of intestinal cholesterol absorption (105, 106). Knock out animals deficient in NPC1L1 have been shown to have a decrease in intestinal cholesterol absorption (105). Ezetimibe binds to NPC1L1 and inhibits cholesterol absorption (97, 105, 106). In animals lacking NPC1L1, ezetimibe has no effect on intestinal cholesterol absorption, demonstrating that ezetimibe’s effect on cholesterol absorption is mediated via NPC1L1 (97, 106). Thus, a major site of action of ezetimibe is to block the absorption of cholesterol by the intestine (97, 106). Cholesterol in the intestinal lumen is derived from both dietary cholesterol (approximately 25%) and biliary cholesterol (approximately 75%); thus the majority is derived from the bile (106). As a consequence, even in patients that have very little cholesterol in their diet, ezetimibe will decrease cholesterol absorption. While ezetimibe is very effective in blocking intestinal cholesterol absorption it does not interfere with the absorption of triglycerides, fatty acids, bile acids, or fat-soluble vitamins including vitamin D and K.

 

When intestinal cholesterol absorption is decreased the chylomicrons formed by the intestine contain less cholesterol and thus the delivery of cholesterol from the intestine to the liver is diminished (107). This results in a decrease in the cholesterol content of the liver, leading to the activation of SREBPs, which enhance the expression of LDL receptors resulting in an increase in LDL receptors on the plasma membrane of hepatocytes (Figure 1) (107). Thus, similar to statins the major mechanism of action of ezetimibe is to decrease the levels of cholesterol in the liver resulting in an increase in the number of LDL receptors leading to the increased clearance of circulating LDL (107). In addition, the decreased cholesterol delivery to the liver may also decrease the formation and secretion of VLDL (107).

 

In addition to NPC1L1 expression in the intestine this protein is also expressed in the liver where it mediates the transport of cholesterol from the bile back into the liver (108). The inhibition of NPC1L1 in the liver will result in the increased secretion of cholesterol in bile and thereby could also contribute to a decrease in the cholesterol content of the liver and an increase in LDL receptor expression and a decrease in VLDL production.

 

Pharmacokinetics and Drug Interactions

 

Following absorption by intestinal cells ezetimibe is rapidly glucuronidated. The glucuronidated ezetimibe is then secreted into the portal circulation and rapidly taken up by the liver where it is secreted into the bile and transported back to the intestine (97). This enterohepatic circulation repeatedly returns ezetimibe to its site of action (note glucuronidated ezetimibe is a very effective inhibitor of NPC1L1) (97). Additionally, this enterohepatic circulation accounts for the long duration of action of ezetimibe and limits peripheral tissue exposure (97). Ezetimibe is not significantly excreted by the kidneys and thus the dose does not need to be adjusted in patients with renal disease.

 

Ezetimibe is not metabolized by the P450 system and does not have many drug interactions (97). It should be noted that cyclosporine does increase ezetimibe levels.

 

Effect of Ezetimibe Therapy on Clinical Outcomes

 

There have been a limited number of ezetimibe clinical outcome trials. Two have studied the effect of ezetimibe in combination with a statin vs. placebo making it virtually impossible to determine if ezetimibe per sehas beneficial effects. However, one study has compared ezetimibe plus a statin vs. a statin alone.

 

SEAS TRIAL

 

The SEAS Trial was a randomized trial of 1873 patients with mild-to-moderate, asymptomatic aortic stenosis (109). The patients received either simvastatin 40mg per day in combination with ezetimibe 10mg per day vs. placebo daily. The primary outcome was a composite of major cardiovascular events, including death from cardiovascular causes, aortic-valve replacement, non-fatal myocardial infarction, hospitalization for unstable angina pectoris, heart failure, coronary-artery bypass grafting, percutaneous coronary intervention, and non-hemorrhagic stroke. Secondary outcomes were events related to aortic-valve stenosis and ischemic cardiovascular events. Simvastatin plus ezetimibe lowered LDL cholesterol levels by 61% compared to placebo. There were no significant differences in the primary outcome between the treated vs. placebo groups. Similarly, the need for aortic valve replacement was also not different between the treated and placebo groups. However, fewer patients had ischemic cardiovascular events in the simvastatin plus ezetimibe treated group than in the placebo group (hazard ratio, 0.78; 95% CI, 0.63 to 0.97; P=0.02), which was primarily accounted for by a decrease in the number of patients who underwent coronary-artery bypass grafting. The design of this study does not allow for one to determine if the beneficial effect on ischemic cardiovascular events typically produced by statin therapy was enhanced by the addition of ezetimibe.

 

SHARP TRIAL

 

The SHARP Trial was a randomized trial of 9270 patients with chronic kidney disease (3023 on dialysis and 6247 not on dialysis) with no known history of myocardial infarction or coronary revascularization (110). Patients were randomly assigned to simvastatin 20 mg plus ezetimibe 10 mg daily vs. placebo. The primary outcome was first major atherosclerotic event (non-fatal myocardial infarction or coronary death, non-hemorrhagic stroke, or any arterial revascularization procedure). Treatment with simvastatin plus ezetimibe resulted in a decrease in LDL cholesterol of 0·85 mmol/L (~34mg/dl). This decrease in LDL cholesterol was associated with a 17% reduction in major atherosclerotic events. In patients on hemodialysis there was a 5% decrease in cardiovascular events that was not statistically significant. Unfortunately, similar to the SEAS Trial, it is impossible to tell whether the addition of ezetimibe improved outcomes above and beyond what would occur with statin treatment alone.

 

IMPROVE-IT TRIAL

 

The IMPROVE-IT Trial tested whether the addition of ezetimibe to statin therapy would provide an additional beneficial effect in patients with the acute coronary syndrome(111). The IMPROVE-IT Trial was a large trial with over 18,000 patients randomized to simvastatin 40mg per day vs. simvastatin 40mg per day + ezetimibe 10mg per day. On treatment LDL cholesterol levels were 70mg/dl in the statin alone group vs. 54mg/dl in the statin + ezetimibe group. There was a small but significant 6.4% decrease in major cardiovascular events (cardiovascular death, MI, documented unstable angina requiring rehospitalization, coronary revascularization, or stroke) in the statin + ezetimibe group (HR 0.936 CI (0.887, 0.988) p=0.016). Cardiovascular death, non-fatal MI, or non-fatal stroke were reduced by 10% (HR 0.90 CI (0.84, 0.97) p=0.003). There was a significant 21% reduction in ischemic stroke (HR, 0.79; 95% CI, 0.67-0.94; P=0.008) and a nonsignificant increase in hemorrhagic stroke (HR, 1.38; 95% CI, 0.93-2.04; P=0.11) (112). Patients with a prior stroke were at a higher risk of stroke recurrence and the risk of a subsequent stroke was reduced by 40% (HR, 0.60; 95% CI, 0.38-0.95; P=0.030) with ezetimibe added to simvastatin therapy (112). In patients with diabetes or other high risk factors the benefits of adding ezetimibe to statin therapy was enhanced (113). In fact, patients without DM and at low or moderate risk demonstrated no benefit with the addition of ezetimibe to simvastatin (113). Similarly, patients who also had peripheral arterial disease or a history of cerebral vascular disease also had the greatest absolute benefits from the addition of ezetimibe (114). Thus, the addition of ezetimibe to statin therapy is of greatest benefit in patients at high risk (for example patients with diabetes, peripheral vascular disease, cerebrovascular disease, etc.).

 

The results of this study have a number of important implications. First, it demonstrates that combination therapy may have benefits above and beyond statin therapy alone. Second, it provides further support for the hypothesis that lowering LDL per sewill reduce cardiovascular events. The reduction in cardiovascular events was similar to what one would predict based on the Cholesterol Treatment Trialists results. Third, it suggests that lowering LDL levels into the 50s will have benefits above and beyond lowering LDL levels to the 70mg/dl range in patients with diabetes or other factors that result in a high risk for cardiovascular events. These results have implications for determining goals of therapy and provide support for combination therapy.

 

Side Effects

 

Ezetimibe has not demonstrated significant side effects. In monotherapy trials, the effect on liver function tests was similar to placebo. In a meta-analysis by Toth et al. of 27 randomized trials in > 20,000 participants evaluating statin plus ezetimibe vs. statin alone the incidence of liver function test abnormalities was slightly greater in the combination therapy group (statin alone- 0.35% vs. statin plus ezetimibe 0.56%) (115). In contrast, Luo and colleagues in a meta-analysis of 20 randomized with > 14,000 subjects did not observe a difference in liver function tests in the ezetimibe plus statin vs. statin alone group (116). With regards to muscle side effects, a meta-analysis of seven randomized trials by Kashani and colleagues found that monotherapy with ezetimibe or ezetimibe in combination with a statin did not increase the risk of myositis compared to placebo or monotherapy with a statin (117). Similarly, Luo et al also did not observe that combination therapy with ezetimibe and a statin increased the risk of myositis (116). In a meta-analysis by Savarese et al. of 7 randomized long term studies including SEAS, SHARP, and IMPROVE-IT, the incidence of cancer was similar in patients treated with ezetimibe vs. patients not treated with ezetimibe (118). This confirms a previous study that also did not demonstrate an increased cancer risk in the three largest ezetimibe trials (119). Ezetimibe does not appear to have adverse effects on fasting glucose levels or A1c levels (120).

 

Thus, over many years of use ezetimibe has been shown to be a very safe drug without major side effects.

 

Contraindications

 

Ezetimibe is contraindicated in patients with active liver disease. The use of ezetimibe during pregnancy and lactation has not been studied.

 

Summary

 

Ezetimibe has a modest ability to lower LDL cholesterol levels and can be a very useful adjunct to statin therapy. When added to statin therapy it will lower the LDL cholesterol by an additional 15-20% which is equivalent to three titrations of the statin dose (for example adding ezetimibe is equivalent to increasing atorvastatin from 10mg to 80mg per day). Additionally, the combination of a high dose of a potent statin (rosuvastatin 40mg per day) with ezetimibe was able to lower the LDL by approximately 70%, which will allow many patients to reach their LDL goal (104). In patients intolerant of statins who either cannot take a statin or can only take low doses of a statin, ezetimibe is extremely useful in further lowering LDL cholesterol. The ease of taking ezetimibe and the lack of serious side effects make it an obvious second choice drug after statins to lower LDL.

 

BILE ACID SEQUESTRANTS 

 

Introduction

 

There are three bile acid sequestrants approved for use in the United States. The first bile acid sequestrant, cholestyramine (Questran), was developed in the 1950s and was the second drug available to lower cholesterol levels (niacin was the first drug). Colestipol (Colestid) was developed in the 1970s and is very similar to cholestyramine. In 2000, Colesevelam was approved. Colesevelam has enhanced binding and affinity for bile acids compared to cholestyramine and colestipol and therefore can be given in much lower doses reducing some side effects (121). Because these drugs work by binding bile acids they are most effective when taken with meals.

 

Effect of Bile Acid Sequestrants on Lipid and Lipoprotein Levels

 

The major effect of bile acid sequestrants is to lower LDL cholesterol levels in a dose dependent fashion. Depending upon the specific drug and dose the decrease in LDL cholesterol ranges from approximately 5 to 30% (121-123). The effect of monotherapy with bile acid sequestrants on LDL cholesterol levels observed in various studies is shown in table 6.

 

Table 6: Effect of Bile Acid Sequestrants on LDL Cholesterol
Drug LDL lowering
Cholestyramine 4g/day 7% decrease
Cholestyramine 24g/day 28% decrease
Colestipol 4g/day 12% decrease
Colestipol 16g/day 24% decrease
Colesevelam 3.8g/day 15% decrease
Colesevelam 4.3g/day 18% decrease

 

Bile acid sequestrants are typically used in combination with statins and the addition of bile acid sequestrants to statin therapy will result in a further 10% to 25% decrease in LDL cholesterol levels (121-123). Combination therapy can result in a 60% reduction in LDL cholesterol levels when high doses of potent statins are combined with high doses of bile acid sequestrants. Bile acid sequestrants will also further lower LDL cholesterol levels by as much as 18% when added to statins and ezetimibe (124). This is particularly useful in patients with Heterozygous Familial Hypercholesterolemia who can have very high LDL cholesterol levels at baseline. Additionally, in patients who are statin intolerant, the combination of a bile acid sequestrant and ezetimibe resulted in an additional 10-20% decrease in LDL cholesterol compared to either drug alone  (125, 126). Thus, both in monotherapy and in combination with other drugs that lower LDL cholesterol levels, bile acid sequestrants are very effective in lowering LDL cholesterol levels

 

Bile acid sequestrants have a very modest effect on HDL cholesterol levels, typically resulting in a 3-9% increase (121-123). The effect of bile acid sequestrants on triglyceride levels varies (121-123). In patients with normal triglyceride levels, bile acid sequestrants increase triglyceride levels by a small amount. However, as baseline triglyceride levels increase, the effect of bile acid sequestrants on plasma triglyceride levels becomes greater, and can result in substantial increases in triglyceride levels. In patients with triglycerides > 400mg/dl the use of bile acid sequestrants is contraindicated.

 

Non-Lipid Effects of Bile Acid Sequestrants

 

Bile acid sequestrants have been shown to reduce fasting glucose and hemoglobin A1c levels (127). Colesevelam has been most intensively studied and in a number of different studies colesevelam has decreased A1c levels by approximately 0.5-1.0% in patients also treated with a variety of glucose lowering drugs including metformin, sulfonylureas, and insulin. The Food and Drug Administration (FDA) has approved colesevelam for improving glycemic control in patients with type 2 diabetes.

 

Bile acid sequestrants decrease CRP. For example, Devaraj et al have shown that colesevelam decreases hs-CRP by 18% compared to placebo (128). In combination with a statin, colesevelam reduced hs-CRP levels by 23% compared to statin alone (129).

 

Mechanisms Accounting for Bile Acid Sequestrants Induced Lipid Effects

 

Bile acid sequestrants bind bile acids in the intestine, preventing their reabsorption in the terminal ileum leading to the increased fecal excretion of bile acids (130). This decrease in bile acid reabsorption reduces the size of the bile acid pool, which stimulates the conversion of cholesterol into bile acids in the liver (130). This increase in bile acid synthesis decreases hepatic cholesterol levels leading to the activation of SREBPs that up-regulate the expression of the enzymes required for the synthesis of cholesterol and the expression of LDL receptors (130). The increase in hepatic LDL receptors results in the increased clearance of LDL from the circulation leading to a decrease in serum LDL cholesterol levels (Figure 1). Thus, similar to statins and ezetimibe, bile acids lower plasma LDL cholesterol levels by decreasing hepatic cholesterol levels, which stimulates LDL receptor production and thereby accelerates the clearance of LDL from the blood.

 

The key regulator of bile acid synthesis is FXR (farnesoid X receptor), a nuclear hormone receptor that forms a heterodimer with RXR to regulate gene transcription (131, 132). Bile acids down-regulate cholesterol 7α hydroxylase, the first enzyme in the bile acid synthetic pathway by several FXR mediated mechanisms. In the ileum, bile acids via FXR stimulate the production of FGF19, which is secreted into the portal vein and inhibits cholesterol 7α hydroxylase expression in the liver (131). Additionally, in the liver, bile acids activate FXR leading to the increased expression of SHP (small heterodimer partner), which inhibits the transcription of cholesterol 7α hydroxylase (132). Thus, a decrease in bile acids will lead to the decreased activation of FXR in the liver and intestines and thereby result in an increase in cholesterol 7α hydroxylase expression and the increased conversion of cholesterol to bile acids resulting in a decrease in hepatic cholesterol content.

 

Decreased activation of FXR can also explain the adverse effects of bile acid sequestrants on triglyceride levels (133, 134). Activation of FXR increases the expression of apolipoprotein C-II, apolipoprotein A-V, and the VLDL receptor, proteins that decrease plasma triglyceride levels while decreasing the expression of apolipoprotein C-III, a protein that is associated with increases in plasma triglycerides (133, 134). Thus, activation of FXR would be expected to decrease triglyceride levels as increases in apolipoprotein C-II, apolipoprotein A-V, and the VLDL receptor and decreases in apolipoprotein C-III would reduce plasma triglyceride levels. With bile acid sequestrants the activation of FXR would be reduced and decreases in the expression of apolipoprotein C-II, apolipoprotein A-V, and the VLDL receptor and increased expression of apolipoprotein C-III would increase plasma triglyceride levels.

 

The mechanism by which treatment with bile acid sequestrants improves glycemic control is unclear.

 

Pharmacokinetics and Drug Interactions

 

Bile acid sequestrants are not absorbed and not altered by digestive enzymes and thus their primary effects are localized to the intestine (121-123). It should be noted that bile acid sequestrants can indirectly have systemic effects by decreasing the reabsorption of bile acids and thereby reducing the exposure of cells to bile acids, which are biologically active compounds.

 

Unfortunately, in the intestine bile acid sequestrants can impede the absorption of many other drugs (121-123). This is particularly true for cholestyramine and colestipol which are used in large quantities (maximum doses- cholestyramine 24 grams per day; colestipol 30 grams per day). In contrast, colesevelam, which requires a much lower quantity of drug because of its high affinity and binding capacity for bile salts, has less of an effect on the absorption of other drugs (recommended dose of colesevelam 3.75 grams/day). Of particular note colesevelam does not interfere with absorption of statins, fenofibrate, or ezetimbe. A list of some of the drugs whose absorption is affected by cholestyramine or colestipol is shown in table 7 and a list of drugs whose absorption is affected by colesevelam is shown in table 8.

 

Table 7:  Some of the Drugs Affected by Cholestyramine/Colestipol
Statins Ezetimibe Gemfibrozil Fenofibrate
Thiazides Furosemide Spironolactone Digoxin
Warfarin L-thyroxine Corticosteroids Vitamin K
Cyclosporine Raloxifine NSAIDs Sulfonylureas
Aspirin Beta blockers Tricyclic

 

Table 8: Some of the Drugs Affected by Colesevelam
L-thyroxine Cyclosporine Glimepiride Glipizide
Glyburide Phenytoin Olmesartan Warfarin
Oral contraceptives

 

It is currently recommended that medications should be taken either 4 hours before or 4 hours after taking bile acid sequestrants. This is particularly important with drugs that have a narrow toxic/therapeutic window, such as thyroid hormone, digoxin, or warfarin. It can be very difficult for many patients, particularly those on multiple medications, to take bile acid sequestrants given the need to separate pill ingestion.

 

Cholestyramine and colestipol may also interfere with the absorption of fat-soluble vitamins. Taking a multivitamin 4 hours before or after these drugs can reduce the likelihood of a vitamin deficiency.

 

Effect of Bile Acid Sequestrants on Clinical Outcomes

 

The Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT) of cholestyramine vs. placebo was the first large drug study to explore the effect of specifically lowering LDL cholesterol on cardiovascular outcomes (135). LRC-CPPT was a multicenter, randomized, double-blind study in 3,806 asymptomatic middle-aged men with primary hypercholesterolemia. The treatment group received cholestyramine 24 grams per day and the control group received a placebo for an average of 7.4 years. In the cholestyramine group total and LDL cholesterol was decreased by 8.5% and 12.6% as compared to the placebo group. In the cholestyramine group there was a 19% reduction in risk (p < 0.05) of the primary end point accounted for by a 24% reduction in definite CHD death and a 19% reduction in nonfatal myocardial infarction. In addition, the incidence rates for new positive exercise tests, angina, and coronary bypass surgery were reduced by 25%, 20%, and 21%, respectively, in the cholestyramine group. The reduction in events correlated with the decrease in LDL cholesterol levels (136). Of note, compliance with cholestyramine 24 grams per day was limited with many patients taking much less than the prescribed doses. These results indicate that lowering LDL cholesterol with bile acid sequestrant monotherapy will reduce cardiovascular disease.

 

In addition to the LRC-CPPT clinical outcome study, two studies have examined the effect of cholestyramine monotherapy on angiographic changes in the coronary arteries. The National Heart, Lung, and Blood Institute Type II Coronary Intervention Study and the St Thomas Atherosclerosis Regression Study reported that cholestyramine decreased the progression of atherosclerosis (137, 138). There are a number of studies that have employed bile acid sequestrants in combination with other drugs and have shown a reduction in the progression of atherosclerosis or an increase in the regression of atherosclerosis but given the use of multiple drugs it is difficult to attribute the beneficial effects to the bile acid sequestrants (139-141). Unfortunately, there are no clinical outcome studies comparing statins alone vs. statins plus bile acid sequestrants.

 

Side Effects

 

Bile acid sequestrants do not have major systemic side effects as they are not absorbed and remain in the intestinal tract. However, they do cause gastrointestinal (GI) side effects (121-123). Constipation is a very common side effect and can be severe. In addition, patients will often complain of bloating, abdominal discomfort, and aggravation of hemorrhoids. Because of GI distress, a significant number of patients will discontinue therapy with bile acid sequestrants. These GI side effects are much more common with cholestyramine and colestipol compared to colesevelam, which is much better tolerated. One can reduce or ameliorate these GI side effects by increasing hydration, adding fiber to the diet (psyllium), and using stool softeners. Notably, bile acid sequestrants do not cause liver or muscle problems.

 

One should also be aware that bile acid sequestrants can be difficult for many patients to take. Colestipol and colesevelam pills are large and can be difficult for some patients to swallow. Additionally, patients need to take a large number of these pills (colesevelam- 6 pills per day; colestipol- as many as 16 pills per day). The granular forms of cholestyramine and colestipol do not dissolve and are ingested as a suspension in liquid. Many patients find mixing with water leads to an unpalatable mixture that is difficult to take. Sometimes mixing with fruit juice, apple sauce, mash potatoes, etc. make the mixture more palatable. The suspension form of colesevelam with either 1.875 or 3.75 grams is preferred by many patients.

 

As noted, earlier bile acid sequestrants can increase triglyceride levels, particularly in patients with elevated baseline triglyceride levels.

 

Contraindications

 

Bile acid sequestrants usually should be avoided in patients with pre-existing GI disorders. Bile acid sequestrants are contraindicated in patients with recent or repeated intestinal obstruction and patients with plasma triglyceride levels > 400mg/dl. In contradistinction from other lipid lowering drugs, bile acid sequestrants are not contraindicated during pregnancy or lactation (category B)(142). In women of child bearing age who are planning to become pregnant bile acid sequestrants can be a good choice to lower LDL levels.

 

Summary

 

Bile acid sequestrants are useful secondary drugs for the treatment of elevated LDL cholesterol levels. They are typically used in combination with statin therapy as a second line drug or as an addition to statin plus ezetimibe therapy as a third line drug. In statin intolerant patients the combination of ezetimibe and a bile acid sequestrant is frequently employed. Bile acid sequestrants can be difficult drugs for patients to take due to GI side effects, difficulty taking the medication, and the need to avoid taking these drugs with other medications. To improve compliance with these drugs the clinician needs to spend time educating the patient on how to take these drugs and how to avoid side effects.

 

PCSK9 INHIBITORS

 

Introduction

 

In 2015 two monoclonal antibodies that inhibit PCSK9 (proprotein convertase subtilisin kexin type 9) were approved for the lowering of LDL cholesterol levels. Alirocumab (Praluent) is produced by Regeneron/Sanofi and evolocumab (Repatha) is produced by Amgen (143, 144). Alirocumab is administered as either 75mg or 150mg subcutaneously every 2 weeks or 300mg once a month while evolocumab is administered as either 70mg subcutaneously every 2 weeks or 420mg subcutaneously once a month.

 

Effect of PCSK inhibitors on Lipid and Lipoprotein Levels

 

There are a large number of studies that have examined the effect of PCSK9 inhibitors on lipid and lipoprotein levels. A meta-analysis of 24 studies comprising 10,159 patients reported a reduction in LDL cholesterol levels of approximately 50% and in an increase in HDL of 5-8% (145). Notably, in 12 RCTs with 6,566 patients, Lp(a) levels were reduced by 25-30% (145). It should be recognized that most LDL cholesterol lowering drugs (statins, ezetimibe, and bile acid sequestrants) do not lower Lp(a) levels. PCSK9 inhibitors have not been shown to decrease hs-CRP levels (146).

 

MONOTHERAPY

 

Both alirocumab and evolocumabhave been studied as monotherapy vs. ezetimibe. In the Mendel-2 study patients were randomly assigned to evolocumab, placebo, or ezetimibe (147). In the evolocumab group, LDL cholesterol levels decreased by 57% while in the ezetimibe group LDL cholesterol levels decreased by 18% compared to placebo. Additionally, non-HDL cholesterol was decreased by 49%, apolipoprotein B by 47%, triglycerides by 5.3% (NS), and Lp(a) by 18.5% while HDL levels increased by 5.5% in the evolocumab treated subjects. In a study of alirocumab vs. exetimibe, LDL cholesterol levels were reduced by 47% in the alirocumab group and 16% in the ezetimibe group (148). In addition, alirocumab decreased non-HDL cholesterol by 41%, apolipoprotein B by 37%, triglycerides by 12%, and Lp(a) by 17% and increased HDL by 6%. Thus, PCSK9 monoclonal antibodies are very effective in lowering pro-atherogenic lipoproteins when used in monotherapy and have a more robust effect than ezetimibe.

 

IN COMBINATION WITH STATINS

 

In the Odyssey Combo I study, patients on maximally tolerated statin therapy were randomized to alirocumab or placebo (149). Similar to monotherapy results, when alirocumab was added to statin therapy there was a further decrease in LDL cholesterol levels by 46%, non-HDL cholesterol by 38%, apolipoprotein B by 36%, and Lp(a) by 15% with an increase in HDL of 7% and no change in triglyceride levels. In the Odyssey Combo II study, patients on maximally tolerated statin therapy were randomized to alirocumab vs. ezetimibe (150). Alirocumab reduced LDL levels by 51% while ezetimibe reduced LDL by 21%, demonstrating that even when added to statin therapy, alirocumab has a significantly greater ability to reduce LDL cholesterol levels than ezetimibe. In Odyssey Combo II, non-HDL cholesterol levels were decreased by 42%, apolipoprotein B by 41%, triglycerides by 13%, and Lp(a) by 28% while HDL increased by 9% in the alirocumab treated group. In the Laplace-2 study, evolocumab was added to various statins used at different doses (151). It didn’t make any difference which statin was being used (atorvastatin, rosuvastatin, or simvastatin) or what dose (atorvastatin 10mg or 80mg; rosuvastatin 5mg or 40mg); the addition of evolocumab resulted in an approximately 60% further decrease in LDL cholesterol levels beyond statin alone. Additionally, the Laplace-2 trial also showed that evolocumab was much more potent than ezetimibe when added to statin therapy (evolocumab resulted in an approximately 60% decrease in LDL vs. while ezetimibe resulted in an approximately 20-25% reduction).

 

IN COMBINATION WITH STATINS AND EZETIMIBE

 

When evolocumab was added to patients receiving atorvastatin 80mg and ezetimibe 10mg there was 48% further reduction in LDL cholesterol levels indicating that even in patients on very aggressive lipid lowering therapy the addition of a PCSK9 inhibitor can still result in a marked reduction in LDL cholesterol (152). In addition to decreasing LDL cholesterol there was also a 41% decrease in non-HDL cholesterol, a 38% decrease in apolipoprotein B, and a 19% decrease in Lp(a) when evolocumab was added to statin plus ezetimibe therapy.

 

PATIENTS WITH HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA

 

Both alirocumab and evolocumab have been tested in patients with Heterozygous Familial Hypercholesterolemia (153, 154). In the Rutherford-2 trial, evolocumab lowered LDL cholesterol by 60%, non-HDL cholesterol by 56%, apolipoprotein B by 49%, Lp(a) by 31%, and triglycerides by 22% while increasing HDL by 8% (153). In the Odyssey FH I and FH II studies, alirocumab lowered LDL cholesterol by approximately 55%, non-HDL cholesterol by ~50%, apolipoprotein B by ~43%, Lp(a) by ~19% and triglycerides by ~14% while increasing HDL by ~7% (154). Thus, in these difficult to treat patients PCSK9 monoclonal antibodies were still very effective at lowering pro-atherogenic lipoproteins.

 

PATIENTS WITH HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA

 

Evolocumab resulted in a 21-31% decrease in LDL levels compared to placebo in patients with Homozygous Familial Hypercholesterolemia(155, 156). The response to therapy appears to be dependent on the underlying genetic cause. Patients with mutations in the LDL receptor leading to the expression of defective receptors respond to therapy whereas patients with mutations leading to negative receptors have a poor response (155-157). Given the mechanism by which PCSK9 inhibitors lower LDL cholesterol levels it is not surprising that patients that do not have any functional LDL receptors will not respond to therapy (see section on Mechanism of Lipid Lowering)

 

STATIN INTOLERANT PATIENTS

 

A number of studies have examined the effect of PCSK9 monoclonal antibodies in statin intolerant patients (myalgias) and compared the response to ezetimibe treatment (89, 158, 159). As expected, treatment with a PCSK9 inhibitor was more effective in lowering LDL cholesterol levels than ezetimibe. Importantly, muscle symptoms were less frequent in the PCSK9 treated patients than those treated with ezetimibe, indicating that PCSK9 monoclonal antibodies will be an effective treatment choice in statin intolerant patients with myalgias.

 

PATIENTS WITH DIABETES

 

A meta-analysis of three trials with 413 patients with type 2 diabetes found that in patients with type 2 diabetes evolocumab caused a 60% decrease in LDL cholesterol compared to placebo and a 39% decrease in LDL cholesterol compared to ezetimibe treatment (160). In addition, in patients with type 2 diabetes, evolocumab decreased non-HDL cholesterol 55% vs. placebo and 34% vs. ezetimibe) and Lp(a) (31% vs. placebo and 26% vs. ezetimibe). These beneficial effects were not affected by glycemic control, insulin use, renal function, and cardiovascular disease status. Thus, PCSK9 inhibitors are effective therapy in patients with type 2 diabetes and the beneficial effects on pro-atherogenic lipoproteins is similar to what is observed in non-diabetic patients.

 

PATIENTS WITH HYPERTRIGLYCERIDEMIA

 

There are no studies that have examined the effect of PCSK9 monoclonal antibodies in patients with marked elevations in triglyceride levels (>400mg/dl).

 

 

Mechanism Accounting for the PCSK9 Inhibitor Induced Lipid Effects

 

The linkage of PCSK9 with lipoprotein metabolism was first identified by Abifadel and colleagues in 2003, when they demonstrated that certain mutations in PCSK9 could result in the phenotypic appearance of Familiar Hypercholesterolemia (161). Subsequent studies demonstrated that gain of function mutations in PCSK9 are an uncommon cause of Familiar Hypercholesterolemia (143, 144, 162). In 2005 it was shown that loss of function mutations in PCSK9 resulted in lower LDL cholesterol levels and this decrease in LDL cholesterol levels is associated with a reduction in the risk of cardiovascular events (163, 164).

 

The main route of clearance of clearance of plasma LDL is via LDL receptors in the liver (165). When the LDL particle binds to the LDL receptor the LDL particle- LDL receptor complex is taken into the liver by endocytosis (165). The LDL particle and the LDL receptor then disassociate and the LDL lipoprotein particle is delivered to lysosomes where it is degraded and the LDL receptor returns to the plasma membrane (Figure 2) (165). After endocytosis LDL receptors recirculate back to the plasma membrane over 100 times.

 

PCSK9 is predominantly expressed in the liver and secreted into the circulation. Once extracellular, PCSK9 can bind to the LDL receptor and alter the metabolism of the LDL receptor (166, 167). Instead of the LDL receptor recycling to the plasma membrane the LDL receptor bound to PCSK9 remains associated with the LDL particle and is delivered to the lysosomes where it is also degraded (Figure 2)(166, 167). This results in a decrease in the number of plasma membrane LDL receptors resulting in the decreased clearance of circulating LDL leading to elevations in plasma LDL cholesterol levels.

 

The PCSK9 monoclonal antibodies bind PCSK9 preventing the PCSK9 from interacting with LDL receptors and thereby preventing PCSK9 from inducing LDL receptor degradation (166, 167). The decreased LDL receptor degradation results in an increase in hepatic LDL receptors on the plasma membrane leading to the increased clearance of LDL and decreases in plasma LDL cholesterol levels (168, 169). Thus, similar to statins, ezetimibe, and bile acid sequestrants, PCSK9 inhibitors are reducing plasma LDL cholesterol levels by up-regulating hepatic LDL receptors. The difference is that PCSK9 inhibitors are decreasing the degradation of LDL receptors while statins, ezetimibe, and bile acid sequestrants stimulate the production of LDL receptors.

Figure 2: PCSK9 Directs LDL Receptor to Degradation in Lysosome

 

The expression of PCSK9 is stimulated by SREBP-2 (166, 167). Statins and other drugs that lower hepatic cholesterol levels lead to the activation of SREBP-2 and thereby increase plasma PCSK9 levels (166, 167). Inhibition of PCSK9 with monoclonal antibodies is more effective in lowering plasma LDL cholesterol levels in patients on statin therapy due to the higher levels of plasma PCSK9 in these individuals.

 

The mechanism by which PCSK9 inhibitors reduce Lp(a) levels is unclear. It has been postulated that increasing hepatic LDL receptor levels in the setting of marked reductions in circulating LDL levels will result in the clearance of Lp(a) by liver LDL receptors (170). Whether this entirely explains the decrease in Lp(a) is unclear given that in patients with Homozygous Familiar Hypercholesterolemia the absolute LDL cholesterol levels are not profoundly reduced but Lp(a) levels still decrease.

 

Pharmacokinetics and Drug Interactions

 

PCSK9 monoclonal antibodies are eliminated primarily by cellular endocytosis, phagocytosis, and target-mediated clearance. They are not metabolized or cleared by the liver or kidneys and therefore there is no need to adjust the dose in patients with either liver or kidney disease. There are no interactions with the cytochrome P450 system or transport proteins and thus the risk of drug-drug interactions is minimal. Currently there are no reported drug-drug interactions with PCSK9 monoclonal antibodies.

 

Effect of PCSK9 Inhibitors on Clinical Outcomes

 

There are four large outcome trials with PCSK9 inhibitors (Table 9).

 

Table 9: PCSK9 Outcome Trials

Number Drug Patient Population
ODYSSEY 18,924 Alirocumab Post-acute coronary syndrome; LDL>70
FOURIER 27,564 Evolocumab Stable cardiovascular disease; LDL>70
SPIRE-1 16,817 Bococizumab High risk patients; LDL 70-100
SPIRE-2 10,621 Bococizumab High risk patients; LDL > 100

 

FOURIER TRIAL

 

The FOURIER trial was a randomized, double-blind, placebo-controlled trial of evolocumab vs. placebo in 27,564 patients with atherosclerotic cardiovascular disease and an LDL cholesterol level of 70 mg/dl or higher who were on statin therapy (171). The primary end point was cardiovascular death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization and the key secondary end point was cardiovascular death, myocardial infarction, or stroke. The median duration of follow-up was 2.2 years. Baseline LDL cholesterol levels were 92mg/dl and evolocumab resulted in a 59% decrease in LDL levels (LDL cholesterol level on treatment approximately 30mg/dl). Evolocumab treatment significantly reduced the risk of the primary end point (hazard ratio, 0.85; 95% confidence interval (CI), 0.79 to 0.92; P<0.001) and the key secondary end point (hazard ratio, 0.80; 95% CI, 0.73 to 0.88; P<0.001). The results were consistent across key subgroups, including the subgroup of patients in the lowest quartile for baseline LDL cholesterol levels (median, 74 mg/dl). Of note, a similar decrease in cardiovascular events occurred in patients with diabetes treated with evolocumab and glycemic control was not altered (172). Additionally, in patients with peripheral arterial disease evolocumab also reduced cardiovascular events (173). Further analysis has shown that in the small number of patients with a baseline LDL cholesterol level less than 70mg/dl, evolocumab reduced cardiovascular events to a similar degree as in the patients with an LDL cholesterol greater than 70mg/dl (174). The lower the on-treatment LDL cholesterol levels (down to levels below 20mg/dl), the lower the cardiovascular event rate, suggesting that greater reductions in LDL cholesterol levels will result in greater reductions in cardiovascular disease (175). Finally, the relative risk reductions with evolocumab for the cardiovascular events tended to be greater in high-risk subgroups (20% for those with a more recent MI, 18% with multiple prior MI, and 21% with residual multivessel coronary artery disease), whereas the relative risk reduction was 5% to 8% in patients without these risk factors (176). This observation suggests that certain groups of patients will derive greater benefit from the addition of a PCSK9 inhibitor.

 

It should be noted that that the duration of the FOURIER trial was very short and it is well recognized from previous statin trials that the beneficial effects of lowering LDL cholesterol levels takes time with only modest effects observed during the first year of treatment. In the FOURIER trial the reduction of cardiovascular death, myocardial infarction, or stroke was 16% during the first year but was 25% beyond 12 months. Thus, long-term benefit may be greater than observed during the study.

 

SPIRE TRIALS

 

Two trials examined the effects of bococizumab, another PCSK9 inhibitor, on cardiovascular outcomes (177, 178). In one trial patients with cardiovascular disease or at high risk for cardiovascular disease with LDL cholesterol levels greater than 70mg/dl on statin therapy were randomized to bococizumab or placebo (SPIRE 1; n= 16,817)). In the second trial, similar patients were studied except LDL cholesterol levels were greater than 100mg/dl and some patients were statin intolerant (SPIRE 2; n= 10,621). The primary end point was nonfatal myocardial infarction, nonfatal stroke, hospitalization for unstable angina requiring urgent revascularization, or cardiovascular death. The trials were stopped early after a median follow-up of 7 months in SPIRE 1 and 12 months in SPIRE 2 due to high rates of development of antidrug antibodies, which markedly reduced the magnitude and durability of the decrease in LDL cholesterol levels (177, 178). In SPIRE 1 baseline LDL cholesterol levels were 94mg/dl while in SPIRE 2 LDL cholesterol levels were 133mg/dl. At 14 weeks LDL cholesterol levels were reduced by approximately 55% in the bococizumab treated groups. In patients with lower baseline LDL cholesterol levels (SPIRE 1) bococizumab treatment did not reduce cardiovascular events (hazard ratio, 0.99; 95% CI 0.80 to 1.22; P=0.94). However, in patients with higher LDL cholesterol levels (SPIRE 2) cardiovascular disease was reduced by bococizumab treatment (hazard ratio, 0.79; 95% CI, 0.65 to 0.97; P=0.02). In patients with Familiar Hypercholesterolemia a similar magnitude of risk reduction for cardiovascular events occurred with the PCSK9 inhibitor (179). Thus, in patients with higher LDL cholesterol levels who were treated for 12 months, lowering LDL cholesterol levels with a PCSK9 inhibitor decreased cardiovascular outcomes. Due to high rates of antidrug antibodies further development of bococizumab is not planned.

 

ODYSSEY TRIAL

 

The ODYSSEY trial was a multicenter, randomized, double-blind, placebo-controlled trial involving 18,924 patients who had an acute coronary syndrome 1 to 12 months earlier, an LDL cholesterol level of at least 70 mg/dl, a non-HDL cholesterol level of at least 100 mg/dl, or an apolipoprotein B level of at least 80 mg/dl while on high intensity statin therapy or the maximum tolerated statin dose (180). Patients were randomly assigned to receive alirocumab 75 mg every 2 weeks or matching placebo. The dose of alirocumab was adjusted to target an LDL cholesterol level of 25 to 50 mg/dl. The primary end point was a composite of death from coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization. During the trial LDL cholesterol levels in the placebo group was 93-103mg/dl while in the alirocumab group LDL cholesterol levels were 40mg/dl at 4 months, 48mg/dl at 12 months, and 66mg/dl at 48 months (the increase with time was due to discontinuation of alirocumab or a decrease in dose). The primary endpoint was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.78 to 0.93; P<0.001). In addition, total mortality was reduced by 15% in the alirocumab group (HR 0.85; 95% CI 0.73 to 0.98). The absolute benefit of alirocumab was greatest in patients with a baseline LDL cholesterol level greater than 100mg/dl. In patients with an LDL cholesterol level > than 100mg/dl the number needed to treat with alirocumab to prevent an event was only 16. It should be noted that similar to the other PCSK9 outcome trials the duration of this trial was very short (median follow-up 2.8 years) which may have minimized the beneficial effects. Additionally, because alirocumab 75mg every 2 weeks was stopped if the LDL cholesterol level was < 15mg/dl on two consecutive measurements the beneficial effects may have been blunted (7.7% of patients randomized to alirocumab were switched to placebo).

 

SUMMARY OF OUTCOME TRIALS

 

It should be noted that that the duration of the PCSK9 outcome trials were relatively short and it is well recognized from previous statin trials that the beneficial effects of lowering LDL cholesterol levels takes time with only modest effects observed during the first year of treatment. In the FOURIER trial the reduction of cardiovascular death, myocardial infarction, or stroke was 16% during the first year but was 25% beyond 12 months. In the ODYSSEY trial the occurrence of cardiovascular events was similar in the alirocumab and placebo group during the first year of the study with benefits of alirocumab appearing after year one. Thus, the long-term benefits of treatment with a PCSK9 inhibitor may be greater than that observed during these relatively short-term studies.

 

GLAGOV TRIAL

 

While not an outcome trial the GLAGOV trial provides further support for the benefits of further lowering of LDL cholesterol levels with a PCSK9 inhibitor added to statin therapy (181). This trial was a double-blind, placebo-controlled, randomized trial of evolocumab vs. placebo in 968 patients presenting for coronary angiography. The primary efficacy measure was the change in percent atheroma volume (PAV) from baseline to week 78, measured by serial intravascular ultrasonography (IVUS) imaging. Secondary efficacy measures included change in normalized total atheroma volume (TAV) and percentage of patients demonstrating plaque regression. As expected, there was a marked decrease in LDL cholesterol levels in the evolocumab group (Placebo 93mg/dl vs. evolocumab 37mg/dl; p<0.001). PAV increased 0.05% with placebo and decreased 0.95% with evolocumab (P < .001) while TAV decreased 0.9 mm3 with placebo and 5.8 mm3 with evolocumab (P < .001). There was a linear relationship between achieved LDL cholesterol and change in PAV (i.e. the lower the LDL cholesterol the greater the regression in atheroma volume down to an LDL cholesterol of 20mg/dl). Additionally, evolocumab induced plaque regression in a greater percentage of patients than placebo (64.3% vs 47.3%; P < .001 for PAV and 61.5% vs 48.9%; P < .001 for TAV).

Side Effects

 

The major side effect of PCSK9 monoclonal antibodies has been injection site reactions including erythema, itching, swelling, pain, and tenderness. Allergic reactions have been reported and as with any protein there is potential immunogenicity. In general side effects have been minimal, which is not surprising, as monoclonal antibodies do not typically have off target side effects. Since PCSK9 does not appear to have important functions other than regulating LDL receptor degradation, it is not surprising that inhibiting PCSK9 function has not resulted in major side effects.

 

A meta-analysis of 20 randomized controlled trials with 68,123 subjects found a very modest effect on fasting glucose (mean difference 1.88 mg/dL) and A1c levels (mean difference 0.032%) and did not observe an increased risk of developing diabetes (182). It should be recognized that the duration of these trials was relatively short (median follow-up 78 weeks) and therefore further long-term studies are required.

 

In the large outcome trials (ODYSSEY and FOURIER) there was no significant difference between the PCSK9 treated group vs. the placebo group with regard to adverse events (including new-onset diabetes and neurocognitive events). The only exception was the expected increase in injection-site reactions in the patients treated with a PCSK9 inhibitor. Additionally, in a subgroup of patients from the FOURIER trial a prospective study of cognitive function (EBBINGHAUS Study) was carried out and no significant differences in cognitive function was observed over a median of 19 months in the PCSK9 treated vs. placebo group (183). It should be recognized that while short-term treatment with PCSK9 inhibitors have not demonstrated any significant side effects it is possible that long-term use could lead to unexpected side-effects.

 

An issue of concern is whether lowering LDL to very low levels has the potential to cause toxicity. In a number of the PCSK9 studies a significant number of patients have had LDL cholesterol levels < 25mg/dl. For example, in the Odyssey long term study 37% of patients on alirocumab had two consecutive LDL cholesterol levels below 25mg/dl and in the Osler long term study in patients treated with evolocumab 13% had values below 25mg/dl (184, 185). In these short term PCSK9 studies, toxicity from very low LDL cholesterol levels has not been observed. Additionally, in patients with Familial Hypobetalipoproteinemia LDL levels can be very low and these patients do not have any major disorders (98). Similarly, there are rare individuals who are homozygous for loss of function mutations in the PCSK9 gene and they also do not appear to have major medical issues (144). Finally, in a number of statin trials there have been patients with very low LDL cholesterol levels and an increased risk of side effects has not been  consistently observed in those patients (186-188). Thus, with the limited data available there does not appear to be a major risk of markedly lowering LDL cholesterol levels.

 

Contraindications

 

Other than a history of a hypersensitivity to these drugs there are currently no contraindications.

 

Summary

 

PCSK9 monoclonal antibodies robustly reduce LDL cholesterol levels when used as monotherapy, in combination with statins, or when added to the combination of statins + ezetimibe. In distinction to most other cholesterol lowering drugs the PCSK9 inhibitors also decrease Lp(a) levels. Outcome studies have clearly demonstrated that decreasing LDL cholesterol levels with PCSK9 inhibitors reduces cardiovascular events. The side effect profile appears to be very favorable and there are no drug-drug interactions. The major limitation is the very high expense of these drugs, which has limited their widespread use.

 

LOMITAPIDE

 

Introduction

 

Lomitapide (Juxtapid), a selective microsomal triglyceride transfer protein inhibitor, was approved in December 2012 for lowering LDL cholesterol levels in adults with Homozygous Familial Hypercholesterolemia (189-191). As will be discussed below it lowers LDL cholesterol levels by an LDL receptor independent mechanism.

 

Effect on Lomitapide on Lipid and Lipoprotein Levels

 

The effect of lomitapide on lipid and lipoprotein levels has been studied in patients with Homozygous Familial Hypercholesterolemia. The pivotal study was a 78 week single arm open label study in 29 patients receiving treatment for Homozygous Familial Hypercholesterolemia(192). Lomitapide was initiated at 5mg per day and was up-titrated to 60mg per day based on tolerability and liver function tests. On an intention to treat basis, LDL cholesterol was decreased by 40% and apolipoprotein B by 39%. In patients who were taking lomitapide, LDL cholesterol levels were reduced by 50%. In addition to decreasing LDL cholesterol levels, non-HDL cholesterol levels were decreased by 50%, Lp(a) by 15%, and triglycerides by 45%. Interestingly HDL and apolipoprotein A-I levels were decreased by 12% and 14% respectively in this study.

 

The effect of lomitapide has also been studied in patients without Homozygous Familial Hypercholesterolemia. A study by Samaha and colleagues compared the effect of ezetimibe and lomitapide in patients with elevated cholesterol levels (193). Patients were treated with ezetimibe alone, lomitapide alone, or the combination of ezetimibe and lomitapide. Ezetimibe monotherapy led to a 20–22% decrease in LDL cholesterol levels, lomitapide monotherapy led to a dose dependent decrease in LDL-cholesterol levels (19% at 5.0 mg, 26% at 7.5 mg and 30% at 10 mg). Combined therapy produced a larger dose-dependent decrease in LDL cholesterol levels (35%, 38% and 46%, respectively).  Additionally, lomitapide decreased triglycerides by 10%, non-HDL cholesterol by 27%, apolipoprotein B by 24%, and Lp(a) by 17%.

 

The above studies demonstrate that lomitapide decreases LDL cholesterol, non-HDL cholesterol, triglycerides, and Lp(a) levels.

 

Mechanism Accounting for the Lomitapide Induced Lipid Effects

 

Lomitapide is a selective inhibitor of microsomal triglyceride transfer protein (MTP) (189-191). MTP is located in the endoplasmic reticulum of hepatocytes and enterocytes where it plays a key role in transferring triglycerides onto newly synthesized apolipoprotein B leading to the formation of VLDL and chylomicrons (194). Loss of function mutations in both alleles of MTP results in abetalipoproteinemia, which is characterized by the virtual absence of apolipoprotein B, VLDL, chylomicrons, and LDL in the plasma due to the failure of the liver and intestine to produce VLDL and chylomicrons (98). Lomitapide by inhibiting MTP activity reduces the secretion of chylomicrons by the intestine and VLDL by the liver leading to a decrease in LDL, apolipoprotein B, triglycerides, non-HDL cholesterol, and Lp(a) (189-191).

 

Pharmacokinetics and Drug Interactions

 

Lomitapide is extensively metabolized in the liver by the CYP3A4 pathway (189, 190). Therefore, lomitapide is contraindicated in patients on strong CYP3A4 inhibitors and lower doses should be used in patients on weak inhibitors. Of particular note, in patients on atorvastatin the maximal dose of lomitapide is 30mg per day and lomitapide should not be used in patients taking more than 20mg of simvastatin (189, 190). Lomitapide can increase warfarin levels and therefore close monitoring is required. Finally, given the risk of liver abnormalities (see side effect section) the avoidance of alcohol or a reduction in alcohol intake is prudent.

 

Effect of Lomitapide on Clinical Outcomes

 

There are no clinical outcome trials but it is presumed that lowering LDL cholesterol levels in patients with Homozygous Familial Hypercholesterolemia will reduce cardiovascular events.

 

Side Effects

 

As expected from its mechanism of action lomitapide causes side effects in the GI tract and liver. In the GI tract diarrhea, nausea, vomiting, and dyspepsia occur very commonly (189-191). In the pivotal study in patients with Homozygous Familial Hypercholesterolemia, 90% of the patients developed GI symptoms during drug titration (192). GI side effects are potentiated by high fat meals and it is therefore recommended that dietary fat be limited. Approximately 10% of patients will discontinue lomitapide, mostly from diarrhea. Lomitapide also reduces the absorption of fat soluble vitamins and therefore patients need to take vitamin supplements (189, 190). Additionally, it may also block the absorption of essential fatty acids and it is therefore recommended that supplements of essential fatty acids also be provided (at least 200 mg linoleic acid, 210 mg alpha-linolenic acid (ALA), 110 mg eicosapentaenoic acid (EPA), and 80 mg docosahexaenoic acid (DHA) (189, 190).

 

Blocking the formation of VLDL in the liver can lead to fatty liver with elevated liver enzymes (189-191). Approximately 30% of patients will develop increased transaminase levels but in the small number of patients studied this has not resulted in liver failure. After stopping the drug, the transaminases have returned to normal. Whether long term treatment with lomitapide will lead to an increase in liver disease is unknown. There is a single case of a patient with lipoprotein lipase deficiency who was treated for 13 years with lomitapide who developed steatohepatitis and fibrosis (195). To reduce the risk of liver dysfunction it is important that patients avoid or limit alcohol intake and avoid drugs that inhibit Cyp3A4 activity.

 

Because of the high potential risk of serious complications the FDA has mandated several measures to ensure that patients are closely followed and monitored for liver toxicity ((Risk Evaluation and Mitigation Strategy (REMS) Program) (189, 190). ALT, AST, alkaline phosphatase, and total bilirubin should be measured before initiating treatment. During the first year, liver function tests should be measured prior to each increase in dose or monthly, whichever occurs first. After the first year, liver function tests should be measured at least every 3 months and before any increase in dose.

 

Contraindications

 

Lomitapide should not be used during pregnancy and in patients with moderate or severe liver disease. In addition, it should not be used in patients on strong CYP3A4 inhibitors.

 

Summary

 

Lomitapide is approved only for the treatment of lipid disorders in patients with Homozygous Familiar Hypercholesterolemia. The frequent GI side effects and the potential risk of serious liver disease greatly limit the use of this drug and it should be reserved for the small number of patients in which more benign therapies are not sufficient in lowering LDL cholesterol into a reasonable range. It is used as an adjunct to other lipid lowering therapies in patients with Homozygous Familiar Hypercholesterolemia.

 

MIPOMERSEN

 

Introduction

 

Mipomersen (Kynamro) is a second generation apolipoprotein B antisense oligonucleotide that was approved in January 2013 for the treatment of patients older than 12 years with Homozygous Familiar Hypercholesterolemia (190, 191, 196). It is administered as a 200mg subcutaneous injection once a week (190, 191, 196). As will be discussed below, it lowers LDL cholesterol levels by an LDL receptor independent mechanism.

 

Effect on Mipomersen on Lipid and Lipoprotein Levels

 

In the pivotal trial, 51 patients with Homozygote Familial Hypercholesterolemia on treatment were randomized to additional treatment with mipomersen (n= 34) or placebo (n=17) and followed for 26 weeks (197). Mipomersen lowered LDL cholesterol levels by 21% and apolipoprotein B levels by 24% compared to placebo.  In addition, non-HDL cholesterol was decreased by 21.6%, triglycerides by 17%, and Lp(a) by 23% while HDL and apolipoprotein A-I were increased by 11.2% and 3.9% respectively.

 

Mipomersen has also been studied in patients with Heterozygous Familial Hypercholesterolemia. In a double-blind, placebo-controlled, randomized trial patients on maximally tolerated statin therapy were treated weekly with subcutaneous mipomersen 200 mg or placebo for 26 weeks(198). LDL cholesterol levels decreased by 33% in the mipomersen group compared to placebo. Additionally, mipomersen significantly reduced apolipoprotein B by 26%, triglycerides by 14%, and Lp(a) by 21% compared to placebo with no significant changes in HDL cholesterol levels. In an extension follow-up study the beneficial effects of mipomersen were maintained for at least 2 years (199).

 

In a meta-analysis of 8 randomized studies with 462 subjects with either non-specified hypercholesterolemia or Heterozygous Familial Hypercholesterolemia, Panta and colleagues reported that mipomersen decreased LDL cholesterol levels by 32% compared to placebo (200). Additionally, non-HDL cholesterol was decreased by 31%, apolipoprotein B by 33%, triglycerides by 36%, and Lp(a) by 26% with no effect on HDL levels.

 

Mechanism Accounting for the Mipomersen Induced Lipid Effects

 

Apolipoprotein B 100 is the main structural protein of VLDL and LDL and is required for the formation of VLDL and LDL (165). Familiar Hypobetalipoproteinemia is a genetic disorder due to a mutation of one apolipoprotein B allele that is characterized by very low concentrations of LDL and apolipoprotein B due to the decreased production of lipoproteins by the liver (98). Mipomersen, an apolipoprotein B antisense oligonucleotide, mimics Familiar Hypobetalipoproteinemia by inhibiting apolipoprotein B 100 production in the liver by pairing with apolipoprotein B mRNA preventing its translation (190, 191, 196). This decrease in apolipoprotein B synthesis results in a decrease in hepatic VLDL production leading to a decrease in LDL levels.

 

Pharmacokinetics and Drug Interactions

 

No significant drug interactions have been reported. Given the risk of liver abnormalities (see side effect section) the avoidance of alcohol or a reduction in alcohol intake would be prudent.

 

Effect of Mipomersen on Clinical Outcomes

 

There are no clinical outcome trials but it is presumed that lowering LDL cholesterol levels in patients with Homozygous Familial Hypercholesterolemia will reduce cardiovascular events.

 

Side Effects

 

The most common side effect is injection site reactions, whichoccur in 75-98% of patients and typically consist of one or more of the following: erythema, pain, tenderness, pruritus and local swelling (190, 191, 196).  Additional, influenza like symptoms, which typically occur within 2 days after an injection, occur in 30-50% of patients and include one or more of the following: influenza-like illness, pyrexia, chills, myalgia, arthralgia, malaise or fatigue which result in a substantial percentage of patients discontinuing therapy (190, 191, 196).

 

A major safety concern is liver toxicity(190, 191, 196). By inhibiting VLDL formation and secretion the risk of fatty liver is increased. Fatty liver has been observed in 5-20% of patients treated with mipomersen (190, 191, 196). In 10-15% of patients treated with mipomersen increases in transaminases occur (190, 191, 196). Additionally, liver biopsies from 7 patients after a minimum of 6 months of mipomersen therapy have demonstrated the presence of fatty liver although there was no inflammation despite elevations in liver enzymes (201). Fortunately, when treatment is discontinued liver function tests and fatty liver return to normal.

 

Because of the high potential risk of serious complications the FDA has mandated several measures to ensure that patients are closely followed and monitored for liver toxicity (Risk Evaluation and Mitigation Strategy (REMS) Program) (190, 191, 196). Liver function should be measured prior to initiating therapy and monthly during the first year and every 3 months after the first year.

 

Contraindications

 

Mipomersen is contraindicated in patients in patients with liver disease or severe renal disease. Mipomersen is not recommended for use during pregnancy or lactation. In animal studies mipomersen has not resulted in fetal abnormalities.

 

Summary

 

Mipomersen is approved only for the treatment of lipid disorders in patients with Homozygous Familiar Hypercholesterolemia. The potential risk of serious liver disease greatly limits the use of this drug and therefore it should be reserved for the small number of patients in which more benign therapies are not sufficient in lowering LDL cholesterol into a reasonable range. It is used as an adjunct to other lipid lowering therapies in patients with Homozygous Familiar Hypercholesterolemia.

 

APPROACH TO TREATING PATIENTS WITH HYPERCHOLESTEROLEMIA

 

Introduction

 

The issues of deciding who to treat, how aggressive to treat, and the goals of therapy are discussed in detail in the chapter “Risk Assessment and Guidelines for the Management of High Blood Cholesterol” and therefore will not be addressed in this chapter (2). Additionally, the role of life style changes to lower LDL cholesterol is discussed in great depth in chapter “Lifestyle Changes: Effect of Diet, Exercise, Functional Food, and Obesity Treatment, on Lipids and Lipoproteins” and therefore will also not be addressed here (1). Rather we will focus on how to use the drugs discussed in this chapter to treat various categories of patients. The factors to consider when deciding which drugs are appropriate to use for lowering plasma LDL cholesterol levels are; the efficacy in lowering LDL cholesterol levels, the effect on other lipid and lipoprotein levels, the ability to reduce cardiovascular events, the side effects of drug therapy, the ease of complying with the drug regimen, and the cost of the drugs. Many statins and ezetimibe are generic drugs and therefore they are relatively inexpensive. PCSK9 inhibitors are expensive (approx. $5-14,000 per year) while mipomersen, and lomitapide are very expensive drugs (>$100,000 per year).

 

 

Isolated Hypercholesterolemia with Cardiovascular Disease

 

In patients with isolated hypercholesterolemia and cardiovascular disease, initial drug therapy should be high dose statin therapy (atorvastatin 40-80mg or rosuvastatin 20-40mg). In patients with cardiovascular disease one should aim to lower the LDL cholesterol to below 70mg/dl or by > than 50%. Some experts based on studies comparing statin alone vs. statin + ezetimibe or statin + a PCSK9 inhibitor would recommend a more aggressive LDL goal in high risk patients (LDL cholesterol <55mg/dl). If statin therapy alone is not sufficient adding ezetimibe is a reasonable next step. Ezetimibe is easy to take, has few side effects, will modestly lower LDL cholesterol, and has been shown in combination with statins to further reduce cardiovascular events. High dose statin and ezetimibe will lower LDL cholesterol by as much as 70%, which will lower LDL cholesterol to goal in the vast majority of patients who do not have a genetic basis for their elevated LDL cholesterol levels. If the combination of statin plus ezetimibe does not lower the LDL to goal one can add a third drug. If the LDL is close to goal one could add a bile acid sequestrant such as colesevelam. If the LDL is not very close to goal one could instead use a statin plus a PCSK9 inhibitor, which together will result in marked reductions in LDL cholesterol levels. If the patient has diabetes with a moderately elevated A1c level using a bile acid sequestrant such as colesevelam instead of ezetimibe or in combination with ezetimibe could improve both glycemic control and further lower LDL levels. If the cost of PCSK9 inhibitors decrease the earlier use of these drugs will become feasible.

 

Isolated Hypercholesterolemia in Primary Prevention

 

In patients with isolated hypercholesterolemia (LDL cholesterol < 190mg/dl) without cardiovascular disease initial drug therapy is with a generic statin. The statin dose should be chosen based on the percent reduction in LDL required to lower the LDL level to below the target goal (typically < 100mg/dl) or to decrease LDL cholesterol by the desired percent. As discussed earlier, the side effects of statin therapy increase with higher doses so one should not automatically use high doses, but instead should choose a dose balancing the benefits and risks. Generic statins are inexpensive drugs and are very effective in both lowering LDL cholesterol levels and reducing cardiovascular events. Additionally, they have an excellent safety profile. If the initial statin dose does not lower LCL cholesterol sufficiently, one can then increase the dose. If the maximal statin dose does not lower LDL cholesterol sufficiently adding ezetimibe is a reasonable next step if the LDL cholesterol level is in a reasonable range and an additional 20-25% reduction in LDL will be sufficient. High dose statin and ezetimibe will lower LDL cholesterol by as much as 70%, which will lower LDL cholesterol to goal in the vast majority of patients who do not have a genetic basis for their elevated LDL cholesterol levels.If the combination of statin plus ezetimibe does not lower the LDL to goal one can add a third drug, such as colesevelam. If the patient has diabetes with a moderately elevated A1c level using colesevelam instead of ezetimibe or in combination with ezetimibe could improve both glycemic control and further lower LDL levels.

 

Mixed Hyperlipidemia

 

In patients with mixed hyperlipidemia (elevated LDL cholesterol and triglyceride levels) Initial drug therapy should also be a generic statin unless triglyceride levels are greater than 500-1000mg/dl. If triglycerides are > 500-1000mg/dl initial therapy is directed at lowering triglyceride levels (202). In addition to lowering LDL cholesterol levels, statins are also very effective in lowering triglyceride levels particularly when the triglycerides are elevated. If LDL is not lowered sufficiently ezetimibe is a reasonable next step. Bile acid sequestrants are not appropriate drugs in patients with hypertriglyceridemia. The great uncertainty is what to do when the LDL levels are at goal but the triglycerides and non-HDL cholesterol are still elevated. Should one add a fibrate, niacin, or fish oil to lower triglycerides and non-HDL cholesterol levels? At this time experts have diverse opinions but hopefully, future studies will clarify the appropriate approach. This issue is discussed in detail in the chapter on triglyceride lowering drugs (202).

 

Heterozygous Familial Hypercholesterolemia

 

In patients with Heterozygous Familial Hypercholesterolemia or other disorders with very elevated LDL cholesterol levels (>190mg/dl), high doses of a potent statin such as atorvastatin 40-80mg or rosuvastatin 20-40mg are the first step to lower LDL cholesterol levels. In many patients this will not be sufficient. If the LDL cholesterol levels are close to goal then adding ezetimibe is a reasonable next step. However, if the LDL cholesterol still needs to be markedly reduced a PCSK9 inhibitor may be a better choice as these drugs can markedly lower LDL cholesterol levels.

 

Statin Intolerance

 

Statin intolerance is frequently due to myalgias but on occasion can be due other issues, such as increased liver or muscle enzymes, cognitive dysfunction, or other neurological disorders. The percentage of patients who are “statin intolerant” varies greatly but in clinical practice a significant number of patients have difficulty taking statins.

 

As discussed earlier it can be difficult to determine if the muscle symptoms that occur when a patient is taking a statin are actually due to the statin or are unrelated to statin use. The first step in a “statin intolerant patient” is to take a careful history of the nature and location of the muscle symptoms and the timing of onset in relation to statin use to determine whether the presentation fits the typical picture for statin induced myalgias. The characteristic findings with a statin induced myalgia are shown in table 10 and findings that are not typical for statin induced myalgia are shown in table 11. The disappearance of symptoms within a few weeks of stopping statins and the reappearance after restarting statins is very suggestive of the symptoms being due to true statin intolerance. An on-line tool (htpp://too;s.acc.org/statinintolerance/#!/) and an app produced by the ACC/AHA is available. This tool characterizes patients based on 8 criteria into possible vs. unlikely to have statin induced muscle symptoms (table 12)

 

Table 10: Characteristic Findings with Statin Induced Myalgia
Symmetric
Proximal muscles
Muscle pain, tenderness, weakness, cramps
Symptom onset < 4 weeks after starting statin or dose increase
Improves within 2-4 weeks of stopping statin
Cramping is unilateral and involves small muscles of hands and feet
Same symptoms occur with re-challenge within 4 weeks

 

 

Table 11: Symptoms Atypical in Statin Induced Myalgia
Unilateral
Asymmetric
Small muscles
Joint or tendon pain
Shooting pain, muscle twitching or tingling
Symptom onset > 12 weeks
No improvement after discontinuing statin

 

Table 12: Diagnosis of Statin Associated Muscle Symptoms
Symptom timing
Symptom type
Symptom location
Sex
Age
Race/ethnicity
CK elevation > 5 times the upper limit of normal
Known risk factors for statin induced muscle symptoms and non-statin causes of muscle symptoms

 

One should also check a CK level but this is almost always in the normal range. If the CK is not elevated and the symptoms do not suggest a statin induced myalgia one can often reassure the patient and continue statin therapy. This is often successful and studies have shown that many patients that stop taking statins due to “statin induced myalgia” can be successfully treated with a statin. If the CK is elevated it should be repeated after instructing the patient to avoid exercise for 48 hours. Also, the CK levels should be compared to CK levels prior to starting therapy. If the CK remains elevated (3x upper limit of normal) the statin should be discontinued. Similarly, if the CK is normal but the symptoms are suggestive of a statin induced myalgia the statin should also be discontinued.  The next step is to determine if one can identify reversible factors that could be increasing statin toxicity (hypothyroidism, drug interactions).  If none are identified the next step after the myalgias have resolved is to try a low dose of a different statin that is metabolized by a different pathway (for example instead of atorvastatin, which is metabolized by the CYP3A4 pathway, rosuvastatin, which has a different pathway of metabolism). Because statin side effects are dose related, a low dose of a statin may often be tolerated. One can also try several different statins as sometimes a patient may tolerate one statin and not others. A meta-analysis has shown that every other day administration of statins is as effective as daily administration in lowering lipid levels and therefore is a very reasonable strategy (203). In some instances, using a long acting statin (rosuvastatin or atorvastatin) 1-3 times per week can work (we usually start with once per week and then slowly increase frequency as tolerated) (204). In these circumstances (low doses or 1-3 times per week) the reduction in LDL cholesterol may not be sufficient but one can use combination therapy with other drugs such as ezetimibe, bile acid sequestrants, or PCSK9 inhibitors to achieve LDL target goals.

 

Many providers have combined Coenzyme Q10 with statins to prevent statin induced myalgias. However, randomized trials with Coenzyme Q10 supplementation have not consistently shown benefit (205, 206). A recent trial, which carefully screened patients to make sure they actually had statin induced myalgias, failed to show a benefit from Coenzyme Q10 supplementation (88)It has also been recommended that vitamin D supplementation be used to prevent statin induced myalgias but there are no randomized trials demonstrating benefit (207).

 

If after trying various approaches a patient still has myalgias and is unable to tolerate statin therapy one needs to utilize other approaches to lower LDL levels. Similarly, if there are other reasons why a patient cannot take a statin, such as developing muscle pathology, one will also need to utilize other approaches to lower LDL levels. These patients can be treated with ezetimibe, bile acid sequestrants, or PCSK 9 inhibitors either as monotherapy or in combination to achieve LDL goals.

 

There are patients who will refuse statins and other drug therapy because they do not believe in taking pharmaceuticals but will take natural products. In these patients we have employed red yeast rice, which decreases LDL cholesterol because it contains a form of lovastatin (208, 209). It is effective but one should recognize that the quality control is not similar to the standards of pharmaceutical products and that there can be batch to batch variations. Furthermore, there is a risk of drug-drug interactions if used with inhibitors of CYP3A4. However, in this particular patient population, who refuses to take statins or other drugs, this can be a reasonable alternative. If a patient just refuses statins (usually based on a belief that statins are toxic) we will employ other cholesterol lowering drugs.

 

CONCLUSIONS

 

With currently available drugs to lower LDL cholesterol levels we are now able to markedly reduce LDL cholesterol levels and achieve our LDL goals in the vast majority of patients and thereby reduce the risk of cardiovascular disease. Patients with Homozygous Familial Hypercholesterolemia and some patients with Heterozygous Familial Hypercholesterolemia still present major clinical challenges and it can be very difficult in these patients to achieve LDL goals.

 

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Immune System Effects on the Endocrine System

ABSTRACT

 

The immune system is a host defence system that combines multiple mechanisms to protect an organism from several pathogens and the development of disease. The endocrine and immune system crosstalk and multiple immune processes are involved in endocrine diseases. Defects of immune tolerance associated with genetic and environmental factors result to the development of autoimmune diseases such as autoimmune thyroiditis and diabetes mellitus type 1. In addition, a mixture of immune cells and mediators is considered to be implicated in thyroid cancer development and progression. Multiple cytokines and the evolving inflammatory process are involved in the pathogenesis of insulin resistance and diabetes mellitus type 2 whereas immune mechanisms related to lymphocytes and cytokine circuits may play a role in the pathogenesis of osteoporosis. Several studies have recently been published regarding the therapeutic implications of immune system mechanisms on endocrine diseases.

 

INTRODUCTION

 

The immune system is a host defense system that integrates many biological structures and processes to protect an organism from invading pathogens. Overall, the immune response is a combination of multiple mechanisms that include innate immunity (phagocytosis by macrophages, neutrophils, monocytes and dendritic cells, or cytotoxicity by natural killer cells) and adaptive immunity (antibody-dependent complement or cell mediated cytotoxicity by T-cells that recognize heat shock proteins and cytotoxicity by CD4 or CD8 T cells) (1). Briefly, antigens taken up by antigen presenting cells (APCs) are presented to T-cells through binding with major histocompatibility complex (MHC) molecules in the APCs surface and co-stimulation by several critical molecules. Activated CD4 helper T-cells stimulate the release of cytokines such as interleukin-2 (IL-2) that induce T-cell proliferation and activation, killer cell activity in CD8 suppressor T-cells and stimulation of B-cells to differentiate to plasma cells and produce antibodies. Cytokines are small molecules that assist the immune system by triggering, maintaining and amplifying immune responses (1). In addition, immunosurveillance is a process during which immune cells recognize and eliminate cancer cells andthere is a dynamic interaction between immune system and cancer cells that influences all stages of tumorigenesis. (2)

 

Naïve T-cells differentiate in two subsets that produce different cytokines and regulate distinct immune functions. T-helper 1 (Th1) cells produce mainly interferon-γ(IFN-γ), tumor necrosis factor α(TNF-α) and IL-12 to regulate cell mediated responses while T-helper 2 (Th2) cells secrete IL-4, IL-5 and IL-13 in order to provide help for antibody production (3). Type 1 cytokines are pro-inflammatory and have been implicated in the pathogenesis of several autoimmune diseases, whereas type 2 cytokines are anti-inflammatory. However, newly discovered T-cells subsets such as Th22 and Th17 may also play a role in autoimmune diseases (4).

 

The immune system is constantly confronted with various molecules and recognize them as foreign substances that need to be destroyed or as self-components that should not trigger an immune response. In order to ensure that no self-molecules cause an immune response, multiple mechanisms are involved to maintain central and peripheral B and T-cell tolerance (1). Disorders of the immune system can lead to autoimmune diseases, inflammatory diseases, or cancer.

 

Multiple studies have shown that the endocrine system is under the regulation of immune processes. This has been observed for autoimmune endocrine diseases such as autoimmune thyroiditis, diabetes mellitus type 1 (DM1), and Addison’s disease as well as for endocrine malignancies such as papillary thyroid cancer(5-7).Defects of the processes ensuring immune cell tolerance resulting in an adaptive immune response to a self-antigen are related to the pathogenesis of autoimmune diseases whereas environmental factors are also considered to be involved (8). Furthermore, a mixture of immune cells and mediators such as chemokines and cytokines has been described to play a critical role in thyroid cancer progression and is associated with patient clinical outcome. (9)

 

The purpose of this chapter is to review the effect of immune process on endocrine organs and diseases and comment on possible therapeutic implications of immune mechanismsin endocrine disorders.

 

IMMUNE SYSTEM AND THYROID DISEASE

 

Autoimmune Thyroid Disease

 

Autoimmunity is involved in the pathogenesis of multiple diseases associated with the thyroid gland including Graves’ disease (GD), Hashimoto thyroiditis (HT), and silent or postpartum thyroiditis. HT and GD have a prevalence of approximately 2% (10) and develop as a consequence of loss of tolerance to thyroid antigens due to several factors such as genetic predisposition, dietary iodine, environmental chemicals, interferon alpha(INF-α), molecular mimics, drugs, and selenium (11). There are three major thyroid auto-antigens: thyroglobulin (Tg), thyroid peroxidase (TPO) and thyrotropin (TSH) receptor. Circulating auto-antibodies to these antigens are useful markers of thyroid autoimmunity but it is generally considered that the pathogenesis of thyroid diseases is associated with T-cell immune mechanisms. Thyroid cells ingest antigens (eg. Tg) and present them to T-cells after stimulation by cytokines such as IFN-γand under the control of co-stimulatory signals from APCs. Genetic factors play an important role in thyroid autoimmunity but it is generally considered that external antigens initiate or promote the immune process probably through cross reactivity (5).

 

Autoimmunity to TPO and/or Tg is related to thyroid lymphocytic infiltration and may result to hypothyroidism. In HT the humoral immune response is characterized by the presence of autoantibodies to TPO or Tg. However, the mechanism of development of these autoantibodies is still not completed elucidated. A direct attack of T-cells on the thyroid gland could lead to thyroiditis and exposure of thyroid antigens that trigger subsequently the production of autoantibodies (5, 12). Autoantibodies are themselves cytotoxic or may affect the antigen processing or presentation to T-cells. In iodine-sufficient regions the prevalence of autoantibodies to TPO or Tg is approximately 15-25%, increases with age, and is highest in females (13). Interestingly, anti-TPO and anti-Tg specific clones derived from HT thyroid tissue produce high levels of IFN-γ, a pro-inflammatory cytokine (14). Th1 cells, the predominant T-cell clones found in patients with HT, may also affect autoimmune thyroid disease through induction of thyrocyte apoptosis, which appears to be a major mechanism of thyroid tissue damage, indirectly through IL-1βproduction by activated macrophages (15). In addition, thyrocytes themselves can produce inflammatory cytokines such as TNF-α, TGF-β, IL-1, IL-6, and IL-8, which can also cause thyrocyte destruction.

 

Toll-like receptors (TLR) are a family of cell surface receptors, consisting of more than ten members, that associate with the recognition of molecules that trigger the activation of innate and adaptive immune response. A recent study showed that TLR3 is overexpressed in thyroid cells surrounded by immune system cells in patients with HT, suggesting a potential role of TLR3 in the pathogenesis of HT and immune cell infiltration (16).

 

In patients with Graves’ disease, the predominant antibodies are directed against the thyroid-stimulating hormone (TSH) receptor (TSH-R). Thyrotrophin receptor antibodies (TRAb) exist as stimulating or blocking antibodies in the serum; however, neutral TRAbare also identified. Thyroid stimulating antibodies (TSAbs) that activate the TSH-R are the cause of Graves’ disease. Rarely, TSH blocking antibodies that competitively inhibit the ligand activation of TSH receptor cause hypothyroidism and thyroid atrophy (5). However, GD, as already mentioned for HT, is also considered to be primarily related to a T-cell abnormality.

 

It is generally believed that thyroid autoimmunity is related to an imbalance between Th1 and Th2 response. In HT an immune response deviation towards Th1 has been observed while in GD there is a shift towards Th2 differentiation.  However, recent studies have shown that newly recognized T cells subsets such as Th17 and Th22 and their cytokines (IL-17, IL-23 and IL-22) may contribute to the pathogenesis of thyroid autoimmune diseases (3).

 

Both in GD and HT, thyroid cells are exposed to complement attack, with subsequent release of prostaglandin E2, IL-1α, and IL-6, which promote infiltration by lymphocytes leading to cell destruction (17). In GD, inflammatory mediators, such as interleukins and TNF-α, stimulate the production of external thyroid-stimulating antibodies that bind the TSH-R.  In thyroid tissue, Th1 recruited lymphocytes may be responsible for enhanced IFN-γand TNF-αproduction, which in turn stimulate C-X-C motif chemokine 10 (CXCL10:the prototype of the IFN-γ-inducible Th1 chemokines) secretion from the thyroid cells, creating an amplification feedback loop, that initiates and perpetuates the autoimmune process(18).

 

In thyroid-associated ophthalmopathy, fibrocytes which are precursor cells of bone-marrow-derived monocyte lineage expressing the hematopoietic cell antigen CD34 (CD34+ fibrocytes), also express the TSHR. TSHR-expressing fibrocytes in which the receptor is activated by its ligand generate extremely high levels of several inflammatory cytokines. Acting in concert with TSHR, the insulin-like growth factor 1 receptor (IGF-1R) expressed by orbital fibroblasts and fibrocytes may participate in TSHR-dependent cytokine production, as anti-IGF-1R blocking antibodies attenuate these pro-inflammatory TSH actions leading to GD ophthalmopathy (19)

 

Postpartum thyroiditis (PPT) is characterized by the development of postpartum thyroid dysfunction (PPTD), which may occur up to 12 months after delivery. Postpartum exacerbation of autoimmunity may reflect an imbalance in specific regulatory T cells, which is caused by the rapid fall in the numbers of these cells after delivery,and is associated with fluctuations in transforming growth factor-beta1 (TGF-β1) serum levels (17,20)

 

Usually, PPT presents as transient hyperthyroidism (median time of onset, 13 weeks post-delivery) followed by transient hypothyroidism (median time of onset, 19 weeks post-delivery). In the majority of patients later restoration of normal thyroid function is observed (21). A plausible explanation for the development of postpartum thyroiditis is that during pregnancy there is a shift from Th1 to Th2 cytokine production followed by a "rebound" shift back to Th1 after delivery (Figure 1). The pathogenesis of the disease has an autoimmune basis as anti-TPO and anti-Tg antibodies are found in almost all patients although anti-TPO are those best correlating with the development of PPT.Postpartum thyroiditis occurs in up to 50% of women who are found to have anti-TPO antibodies at the end of the first trimester of gestation (i.e. before thyroid antibody titers start to decline during pregnancy). Furthermore, there is evidence that the TPO antibody titer at 16 weeks of gestation is related to the severity of the PPTD (22). In addition, activation of complement is also thought to play a role in the development of PPT (23).

Figure 1. Th1/Th2 Balance During Pregnancy and Postpartum. IUGR: Intrauterine growth retardation

Euthyroid Sick Syndrome

 

The term "Euthyroid Sick Syndrome" (ESS) has been used for more than thirty years to describe a pattern of thyroid hormone alterations during non-thyroidal illness. Conditions associated with ESS include systemic inflammation, myocardial infarction, starvation, sepsis, surgery, trauma, chronic degenerative diseases, malignancy and every other conditionassociated with severe illness. The characteristic laboratory abnormalities of the ESS include low triiodothyronine (T3) and/or free T3 (fT3), elevated reverse T3 (rT3), normal or low TSH, and normal or low serum thyroxine (T4) or free T4 (fT4) concentrations. These abnormalities develop as a result of cytokine action on several pathways of thyroid hormonal synthesis and/or degradation while the more severe the illness is the more extensive the hormonal alterations are. In particular, thyroid hormone changes are the result of suppression ofthyrotropin-releasing hormone(TRH) and TSH release, and inhibition of hepatic type-1 5Άdeiodinase (D1) that facilitates conversion of T4 to T3 and of rT3 to diiodothyronine (24). Thus, the cause of the decreased T3 concentration in ESS is decreased T3 production, whereas the cause of the increased rT3 concentration is the result of attenuated degradation. Prolonged and severe illness is marked by a decrease in circulating total T4 along with low T3 and high rT3; furthermore, very low T4 levels carry a poor prognosis and have been associated with an increased mortality rate (24). Cytokines including IL-1α, IL-1β, IL-6,IFN-γ, TNF-α, and TGF-β2, exert an inhibitory role on sodium–iodine symporter (NIS) protein expression and NIS gene transcription, an intrinsic membrane protein that facilitates the active transport of iodine into the thyroid cell  (25-27). In addition to the effects on iodide uptake, cytokines have also been shown to decrease thyrocyte growth (28), iodide organification  (29, 30), thyroglobulin synthesis  (31, 32), and thyroid hormone release in vitro (33).

Cytokines can also affect hepatic deiodinase type 1 D1 activity (Figure 2). The main role of D1 is to peripherally convert T4 to T3 and rT3 to diiodothyronine. In ESS, there is a decrease in D1 activity leading to decreased T3 and increased rT3 concentrations. However, the exact mechanism of decreased D1 activity in ESS still remains unclear. In vitro studies, evaluating the effects of cytokines IL-1β, IL-6, and TNF-αon D1 levels in rat thyroid FRTL-5 and liver cells, have produced controversial results. The D1 activity in rat thyroid FRTL-5 was inhibited by these cytokines (34), whereas liver D1 activity was surprisingly increased (35).

 

Figure 2. IL-6 Inhibition of 5΄Deiodinase-I Resulting in Decrease in T3 and Increase in rT3 Concentration

While the in vitrostudies of cytokine effect on D1 activity are controversial, in vivostudies have revealed that cytokines can inhibit D1 activity either directly or indirectly. To delineate the effects of IL-6 on D1 activity, IL-6 knockout mice (36)were used in whom Listeria monocytogenesinfection or turpentine injection induced a ESS state. The decrease in serum T3 concentration was attenuated in the IL-6 knockout mice compared to wild-type animals. This was associated with only a modest decrease in hepatic D1 activity (compared to wild-type animals), implying that IL-6 played a significant role in the pathogenesis of ESS in that model.

 

In exploring the effects of cytokines on the hypothalamic-pituitary unit, in vitro studies demonstrated that IL-1βand TNF-αcan inhibit TSH release from the pituitary through stimulation of K+-mediated release of somatostatin from the hypothalamus (Figure 3) (37). IL-6 exhibited no effect on TSH secretion or somatostatin release, implying that this cytokine had no direct effect on the hypothalamic-pituitary unit of the thyroid axis (38).

In animal studies, administration of TNF-αto rats had a similar effect (39, 40). After IL-6 was administered to rats, TSH decreased without any change in hypothalamic pro-TRH mRNA levels, or in stored β-TSH in the pituitary (41). These data, along with the lack of any IL-6 effect on TSH release in vitro, suggest that the observed decrease of circulating TSH in vivofollowing IL-6 administration was the result of an indirect rather than a direct action on the TRH-TSH unit.

 

The central role of cytokines in the pathophysiology of ESS has been further elucidated in studies involving cytokine administration to humans. Following TNF-αadministration to healthy volunteers a decrease in serum T3 and an increase in serum rT3 concentration was found (42). Unlike IL-6, serum TNF-αlevels did not correlate with any of the typical thyroid parameters such as low T3, increased rT3, or decreased TSH levels, as seen in ESS (43, 44), suggesting that the changes of thyroid hormonal profile following TNF-αadministration might be indirect (i.e. through TNF-αincrease in circulating IL-6 levels) rather than direct. Furthermore, both IL-6 and TNF-αcan regulate type 2 iodothyronine 5’-deiodinase in the anterior pituitary, affecting TSH release, thus contributing to the development of the non-thyroidal illness syndrome (45, 46).

 

A link between leptin and pro-inflammatory cytokines such as TNF-αleading to the development of ESS, has also been suggested following the finding that TNF-αlevels were associated with increased leptin levels in patients with chronic obstructive pulmonary disease(47). Moreover, serum leptin levels were increased and significantly associated with IL-6 levels and disease activity in men with ankylosing spondylitis (48). It has been suggested that the primary action of leptin on the hypothalamic-pituitary-thyroid (HPT) axis is alteration of the set point for feedback sensitivity of hypophysiotropic TRH producing neurons in the paraventricular nucleus (PVN) of the hypothalamus to thyroid hormones (mainly T3) through lowering of the set point when leptin levels are suppressed during fasting (49). Two anatomically distinct and functionally antagonistic populations of neurons in the arcuate nucleus of the hypothalamus, α-melanocortin-stimulating hormone (α-MSH) producing neurons that co-express cocaine and amphetamine-regulated transcript, and neuropeptide Y (NPY) producing neurons that co-express agouti-related peptide (AGRP), are responsible for the actions of leptin on hypophysiotropic TRH. It is thought that the inhibitory effect of AGRP on TRH gene expression is the result of antagonizing the activating effects of α-MSH at the melanocortin 4 receptor on the surface of hypophysiotropic TRH neurons, whereas the inhibitory effect of NPY occurs by reducing cAMP levels (0). A direct action of leptin on hypophysiotropic TRH neurons has also been proposed (51). These data suggest that leptin can act via two different and independent mechanisms (cytokine dependent and directly) in seriously ill patients, affecting the thyroid function as a whole.

 

Amiodarone-Induced Thyroid Disease

 

Amiodarone, a benzofuran derivative with a similar structure to thyroid hormones, is a highly effective antiarrhythmic agent widely used in the treatment of various types of tachyarrhythmias (supraventricular and ventricular arrhythmias). Amiodarone contains two iodine atoms per molecule which is approximately 37,5% iodine by molecular weight (52). Administration of amiodarone is associated with complex changes in thyroid physiology. The iodine load results in an inhibition of thyroid hormone synthesis and metabolism, particularly 5’-monodeiodination of T4 to T3. Such alterations occur in all patients and although the majority remain clinically euthyroid, approximately 14% of amiodarone-treated patients develop thyroid dysfunction (52-54). Treatment with amiodarone may be related with an increase in lymphocyte subsets leading to an exacerbation of pre-existing autoimmunity (52, 55, 56). The relative proportion of patients developing either thyrotoxicosis or hypothyroidism depends on the iodine content of the local diet and pre-existing thyroid autoimmunity. In relatively iodine replete areas, approximately 25% of patients with amiodarone-induced thyroid dysfunction become thyrotoxic, accounting for approximately 3% of amiodarone treated individuals (544).

 

Amiodarone-induced hypothyroidism is attributed to an increased susceptibility to the inhibitory effect of iodide in thyroid hormone synthesis and/or to a failure to escape the Wolff-Chaikoff effect (52, 57). HT is the most common risk factor for amiodarone-induced hypothyroidism and it is considered the most likely reason for the female preponderance of this clinical entity (58). Female patients with positive Anti-TPO and Anti-Tg autoantibodies have a relative risk of 13.5 for developing amiodarone-induced hypothyroidism compared with men without thyroid autoantibodies (59). A probable explanation for this could be an exacerbation of the autoimmune response by amiodarone’s iodide while excess iodide could also confer to the damage caused by an underlying autoimmune disease by inducing non-specific thyroid injury (52).

 

The pathogenesis of amiodarone-induced thyrotoxicosis (AIT)is complex although two distinct forms, type 1 and type 2, are recognized; Type 1 develops in patients with latent thyroid disease, predominantly nodular goiter in whom the amiodarone iodine load triggers increased synthesis of thyroid hormones. Type 2 is the result of a destructive thyroiditis in a previously normal gland, with leakage of preformed thyroid hormones despite a reduction in hormone synthesis (52-55). A cross-sectional study in patients with AIT revealed that serum IL-6 concentration was elevated in such patients without a goiter or circulating thyroidal autoantibodies (AIT-) compared to patients with AIT in the presence of a goiter or thyroidal autoantibodies (AIT+) (60). AIT- patients had a very low (<3%) 24-hour thyroidal radioiodine uptake suggesting that a subacute thyroiditis-like mechanism was responsible for the thyrotoxicosis. To determine if plasma IL-6 concentration was elevated in other destructive processes besides AIT, serum IL-6 concentration was measured in patients undergoing fine needle aspiration of the thyroid, percutaneous ethanol injections into thyroid nodules, or radioactive iodine treatment. Serum IL-6 concentration increased significantly following any of these procedures, suggesting that IL-6 could be used as a marker of any thyroid destructive process regardless of the etiology (61).

 

Differentiating between the two types of AIT is an essential step in their management as treatment of each type is different (54). Type 1 usually responds to thionamide therapy that blocks hormone synthesis and perchlorate that blocks active transport of iodine into the thyroid, whereas type 2 responds to high-dose of corticosteroids (54, 55,61,62). Nevertheless, several studies now suggest that these two types should be treated concomitantly, and thus patients with AIT receive both anti-thyroid drugs and prednisolone.In resistant to medical treatment cases and/or in patients with severe cardiac diseases who cannot interrupt amiodarone or require quick amiodarone reintroduction, total thyroidectomy may be offered after rapid correction of thyrotoxicosis followingcombination treatment with thionamides, KClO4, corticosteroids, and a short course of iopanoic acid(63).

 

Thyroid Cancer

 

Thyroid cancer is the most common endocrine cancer and its incidence has steadily increased over the past decades (64). The association of chronic inflammation and thyroid cancer has long been recognized and a mixture of immune cells and mediators frequently observed within or surrounding the tumor is considered to play a role in tumor progression and clinical outcome (9).

 

Immunosurveillance is a process during which the immune system recognizes and eliminates the development of tumor cells but if this mechanism fails, acquisition of new mutations may facilitate evasion from immunological mechanisms of surveillance and result to uncontrolled tumor growth and cancer development. This process is called immunoediting. As a consequence, current research has been focused on developing immunotherapies that enhance tumor specific immune responses or counteract the immunosuppression caused by tumor cells (4).

 

Currently, there are a lot of studies that support the presence of multiple types of leukocytes in thyroid cancers, which is considered to be associated with positive or negative clinical outcome. The number of tumor-associated macrophages has been found to be increased in thyroid cancer and is related to de-differentiation, lymph node metastases, larger tumors and reduced survival (9, 65-69). Myeloid derived suppressor cells are associated with aggressive characteristics of differentiated thyroid cancer and related to poor prognosis (9, 70). The dendritic cells, that play a critical role in antigen presentation, are also increased in papillary thyroid cancer while neutrophils are found in more aggressive thyroid cancers (poorly differentiated or anaplastic). In addition, natural killer cells that display an important role in immunosurveillance are increased in papillary thyroid cancer and are negatively correlated with tumor stage while lymphocytic infiltration is associated with better overall survival and low recurrence rate (7, 65, 71).

 

Cytokines may be produced by thyroid follicular cells as well as by immune cells infiltrating thyroid tumors and are related to tumor development. IL-1 and IL-6 stimulate thyroid cell proliferation and tumor growth while TGF-βwhich is a suppressive cytokine is overexpressed in aggressive cancers (7). A recent study found a correlation of ionizing radiation and oxidative stress with IL-13 while IL-17 has tumorigenic and anti-tumor effects (72-74). In addition, multiple chemokines may be secreted by thyroid cancer or immune cells and affect chemiotaxis, angiogenesis, and lymphangiogenesis (7).

The mixture of immune cells and mediators observed in thyroid tumors may be related to cancer development and growth through production of proangiogenic and lymphangiogenic molecules. Research regarding targeting these pathways or blocking immunosuppressive molecules may lead to development of immunotherapy for thyroid cancer as for other cancer types (65).

 

IMMUNE SYSTEM AND DIABETES MELLITUS

 

Over recent years our understanding of the etiology of diabetes mellitus and its vascular complications has widened considerably. Diabetes mellitus type 1 (DM1) is considered an autoimmune disease while inflammation plays an important role in the pathogenesis of diabetes mellitus type 2 (DM2). In addition, an interplay between inflammatory and metabolic abnormalities leads to tissue damage in patients with diabetes and results to various chronic complications that increase morbidity and mortality.

 

Type 1 Diabetes Mellitus

 

DM1 is immune mediated in more than 95% of cases and it is considered an organ specific autoimmune disease that is characterized by lymphocytic infiltration and inflammation that leads to βcell destruction and absolute insulin deficiency. Both genetic and environmental factors are involved in the pathogenesis but the precise mechanism behind the initiation and progression of the disease remains to be elucidated (75). Two animal models, the nonobese diabetic (NOD) mouse and the bio breeding (BB) rat, have been extensively used to study the pathophysiology of DM1 (76, 77).

 

The susceptibility to develop DM1 is associated with multiple alleles of the MHC I and II locus. More than 90% of patients with DM1 express either HLA DR3, DQ2 or DR4, DQ8 (78). On the other hand, HLA haplotype DR2, DQ6 is protective against DM1 development. In addition, multiple environmental factors may trigger the autoimmune process leading to DM1 in patients genetically susceptible. These factors include dietary compounds (e.g., cow’s milk) or viruses (e.g., Coxsackie B4, mumps, rubella) but further investigation is required to establish the exact mechanism of their action (79, 80).

 

Three different mechanisms have been proposed for the pathogenesis of DM1. One mechanism involves molecular mimicry-activated T-cell proliferation. This mechanism is based on the assumption that epitopes of proteins expressed by infectious agents can be shared by unrelated molecules encoded by host genes (81). A second mechanism that triggers molecular mimicry-activated T-cell proliferation is "by stander" T-cell proliferation. This mechanism involves the stimulation of non-antigen-specific T cells by various cytokines during infection simply because they are in the area. The cytokines thought to be involved in this nonspecific stimulation are IFN-αand IFN-β(82). A third theory involving a superantigen-mediated T-cell proliferation mechanism proposes that auto-reactive T-cells can be inappropriately primed to react against self-structures through an encounter with a superantigen (83).

 

Most patients diagnosed with DM1 have circulating islet cell autoantibodies directed against βcell proteins. Multiple types of autoantibodies have been identified: islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies to glutamic acid decarboxylase 65 (GAD), tyrosine phosphatase IA2 (ICA512), and zinc transporter 8 (ZnT8). These autoantibodies may be detected a long time before the onset of hyperglycemia and usually decline during the course of the disease (84, 85).

 

Despite the fact that the detection of autoantibodies may be useful for DM1 diagnosis and prediction, there is evidence that the cellular immune system infiltrates the islets and causes βcell destruction. Particularly, autoreactive T cells are involved in disease initiation and progression. Studies in NOD mice have shown that an initial failure of immune regulation results in an amplification of autoreactive CD4and CD8T cells, autoantibody producing B lymphocytes, macrophages, and dendritic cells that ultimately damage islets. Further βcell destruction may lead to self-antigen presentation and subsequent amplification of the immune response (75, 86, 87).

 

While CD4 T helper cells are required for the development of the autoimmune process in the pancreatic islets, CD8 T cytotoxic are probably the responsible cells for βcell destruction. In addition, studies in NOD mice suggest that the Th1 subset plays a crucial role in DM1 pathogenesis. It has been observed that IL-21, a cytokine produced by CD8 T cells, is required for the development of DM1 while TNF-αmay also be involved in the disease (88, 89). However, further studies are needed to specify the role of other cytokines such as IFN-γ, IL-12, IL-17 and IL-23. On the contrary, it has been observed that IL-4, a cytokine produced by Th2 cells, protected NOD mice from developing insulinitis or diabetes (75, 83, 88-91).In addition, IL-6plays an important role in the pathogenesis of vitiligo-associatedDM1and is likely to gain favor as a therapeutic target in these patients(92). IL-6may also contribute to DM1and increased albumin-to-creatinine ratio as well as to poor glycemic control and hyperlipidaemia(93).

 

Understanding both the pathophysiology and the regulatory mechanisms involved in DM1 may be a useful tool in an attempt to develop antigen-specific, β-cell directed, immunomodulatory or cellular treatment modalities (94).

 

Type 2 Diabetes Mellitus

 

DM2 is one of the most common metabolic disorders that accounts for 90% of cases of diabetes worldwide (95, 96). DM2 is a heterogeneous disorder that is associated with insulin resistance (IR) in the presence of an impairment in insulin secretion. The increasing prevalence of DM2 has been largely attributed to unhealthy lifestyle and development of obesity and overweight around the world.  Obesity is strongly related to DM2 mainly through inducing IR which is the impaired ability of insulin to effectively induce glucose uptake by cells. DM2 is also associated with hypercholesterolemia, atherosclerosis, hypertension, kidney disease, and coronary artery disease.

 

The concept that a smoldering inflammatory process is important in the pathogenesis ofDM2 (92)has recently attracted much attention and is supported by evidence of inflammation in islets, adipose tissue, liver, and muscle that can provoke IR and β-cell dysfunction(97-99). Adipose tissue is characterized by infiltration by macrophages and other immune cells that produce cytokines and chemokines and contributeto the development of local and systemic chronic low-grade inflammation. This inflammatory milieu is considered to be the link between obesity, IR and diabetes mellitus (100-101).

 

Initially nonspecific indicators of inflammation such as white cell count and fibrinogen were found to be predictive of diabetes (102-103). Subsequently, elevated plasminogen activator inhibitor-1 (PAI-1), CRP, and fibrinogen levels were shown to be independent predictors (104). These observations are supported by a number of prospective studies, in which tissue plasminogen activator (tPA), another marker of reduced fibrinolysis (1055), and von Willebrand factor (vWf), a marker of endothelial injury, were also shown to be predictive(106). There have been many studies demonstrating an association between CRP and/or IL-6 and incident DM2 that was independent of adiposity or IR (104,107). In addition, the early markers of inflammation, monocyte chemotactic protein-1 (MCP-1), IL-8, and interferon-γ-inducible protein-10, were also found to predict the development of DM2 with MCP-1 being independent of traditional risk factors (108).

 

The visceral adipose tissue appears to be a major source of circulating IL-6 in humans. The secretion of IL-6 by immune cells such as macrophages is triggered by other cytokines such as TNF-αand IL-1 and reduced by IL-4 and Il-10. Plasma IL-6 concentration correlates well with body mass index (BMI=kg/m2) (109, 110), and has been  found  to be elevated in obese people with IR (111,112). In addition, Il-6 levels may predict the development of DM2 (113).

 

TNF-αhas also received considerable attention with regard to IR and may be a key mediator of its pathogenesis. TNF-αhas been shown to be significantly increased in obese individuals with IR ( 114), and is thought to play a major role in the pathogenesis of obesity-linked DM2 (115).TNF-αis associated with increased release of free fatty acids by adipose tissue and leads to impaired insulin secretion and signalling (116, 117).

 

At the molecular level, chronic exposure of adipocytes to low doses of TNF-αled to a dramatic decrease in the insulin-stimulated auto-phosphorylation of the insulin receptor and the phosphorylation of insulin receptor substrate 1 (IRS-1) (118). Furthermore, IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in the presence of TNF-αhas been demonstrated. Treatment of cultured murine adipocytes with TNF-αhas been shown to induce serine phosphorylation of IRS-1 and convert IRS-1 into an inhibitor of the IR tyrosine kinase activity in vitro (119). It was concluded that TNF-αplays an inhibitory role on the insulin-stimulated tyrosine kinase phosphorylation cascade. The question of whether TNF-αinduces IR directly or indirectly through inhibitors of tyrosine kinase or counter-regulatory hormones on muscle, fat, and liver in vivo needs further investigation. TNF-αhas also been shown to down-regulate glucose transporter GLUT4 mRNA levels in adipocyte and myocyte cultures as well (120-122).

 

TNF-αmay also play a role in the hyperlipidemia observed in DM2 as it has been shown to have profound effects on whole body lipid metabolism (123-125). Circulating triglycerides and very low density lipoproteins in rats and humans are increased after administration of TNF-α(123, 124). Moreover, an animal study has provided evidence of increased TNF-αlevels in animals receiving a high-fat diet (126).In addition to TNF-α, IL-1 and IFN-γalso stimulate fatty acid synthesis whereas IL-6 influences fat metabolism as well. Several studies have suggested that certain polymorphisms in the promoter region of the IL-6 gene can affect lipid levels through changes in IL-6 gene transcription and ultimately IL-6 production (127). IL-6 has been proposed to cause an increase in circulating lipid levels probably through a decrease in peripheral lipoprotein lipase activity (128).

 

Oxidative stress, as a result of increased cytokine levels in DM2, is also thought to play an important role in activating inflammatory genes (129, 130). It is possible that oxidative stress markers do not adequately reflect the impact of increased reactive oxygen species (ROS) on β-cells or insulin signaling. Inflammatory, pro-coagulant or endothelial dysfunction markers are more specific because they may be more proximate to the pathophysiology of hyperglycemia (129, 130). Recently, Hasnain et al showed that islet-endogenous and exogenous IL-22, by regulating oxidative stress pathways, suppresses oxidative and endoplasmic reticulum (ER) stress caused by cytokines or glucolipotoxicity in mouse and human βcells. In obese mice, antibody neutralization of IL-23 or IL-24 partially reduced βcell ER stress and improved glucose tolerance, whereas IL-22 administration modulated oxidative stress regulatory genes in islets, suppressed ER stress and inflammation, promoted secretion of high-quality efficacious insulin and fully restored glucose homeostasis followed by reinstitution of insulin sensitivity(131).

 

In addition, recent studies have shown that the chemokine system is associated with obesity and IR. MCP-1 that acts on monocytes, macrophages, T cells and NK cells has been found to be increased in obese compared to lean patients and is related to non-alcoholic fatty liver disease and other lipid overload states (132-136). Furthermore, a recent clinical report showed significant benefit in glycemic and lipid profile along with a decrease of MCP-1 levels after a 4-month program of lifestyle improvement (137).

 

These findings support the investigation of new therapeutic approaches that target inflammation to ameliorate diabetes and its complications. Multiple studies investigate the effect of salicylates, TNF-αinhibitors and IL-1βantagonists on insulin sensitivity, glucose and lipid control, and cardiovascular disease (,138). A recent trial showed improvement of glycemia and secretory function of βcells after treatment with anakinra, a competitive antagonist of the IL-1βreceptor IL-1Ra (139). Monoclonal antibodies against IL-1βmay reduce inflammatory proteins CRP, IL-6 and fibrinogen in patients with DM2 and high cardiovascular risk (140). In addition, a recent study investigating tocolizumab, a monoclonal antibody against IL-6 receptor showed a significant inhibition of migration of smooth muscle cells under hyperglycemic conditions suggesting it could be a useful therapeutic candidate for atherosclerosis in diabetic patients (141).

 

OSTEOPOROSIS AND THE IMMUNE SYSTEM

 

Osteoporosis is a condition of low bone mass and microarchitectural disruption which is associated with an increased risk of fracture in response to low velocity force. It is more frequent in postmenopausal women and in older men or women due to age-related bone loss while it is classified in primary and secondary osteoporosis according to the presence of precipitating factors. Recently, there is growing evidence regarding the effect of immune system on bone metabolism leading to the emergence of the new field of osteo-immunology (142). States of immune dysfunction such as immunodeficiency, inflammatory diseases or immune response to infections may lead to osteoporosis and increased risk fracture. These states are associated with an increased bone resorption from osteoclasts compared to bone formation from osteoblasts resulting to net bone loss (143, 144).

 

Osteoclasts originate from the same myeloid precursor that derive macrophage and dendritic cells and are specialized in bone degradation (145). Osteoblasts are the main bone forming cells and are derived from mesenchymal stem cells. Osteoclast formation and differentiation is regulated by macrophage colony stimulating factor (M-CSF) and the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) produced by osteoblasts. RANKL is also expressed by fibroblasts and immune cells, including antigen-stimulated T cells and dendritic cells (142, 146). In addition, osteoprotegerin (OPG) which is produced by osteoblasts, B lymphocytes and dendritic cells binds to RANKL preventing its association with RANK andinhibits osteoclast formation and differentiation (147, 148).

 

Activated T cells increase the production of TNF-αand RANKL and stimulate osteoclastogenesis during inflammation (142, 149-151). Multiple cytokines may promote osteoclastogenesis mainly by regulating the RANK/RANKL/OPG axis. TNF-α, IL-1, IL-6, IL-7, IL-11, Il-17 and IL-23 promote osteoclast differentiation while IFN-α, IFN-γ, IL-3, IL-4, IL-10, IL-27 and IL-33 are considered anti-osteoclastogenic cytokines that protect bone integrity (148). Th17 cells are considered an osteoclastogenic subset of T cells as they enhance osteoclastogenesis by secreting IL-1, IL-6, Il-17, RANKL, TNF-αand IFN-γ. It has been observed that Th17 cells are increased in patients with osteoporosis and could be used as a potential marker (151, 152). Activation of Th2 leads to enhanced production of PTH and promotes the anabolic activity of osteoblasts in several inflammatory states. Furthermore, Th2 lymphocytes are associated with a low RANKL/OPG ratio and inhibition of bone loss (152). In addition, B cells produce RANKL and OPG and may influence bone formation and absorption while it has been observed that in HIV infected patients there is an altered B cell RANKL/OPG ratio that is inversely correlated with BMD (153).

 

Interleukin 6 (IL-6) plays a major role in osteoclast development and function. IL-6 is produced by both stromal and osteoblastic cells in response to stimulation by systemic hormones such as parathyroid hormone (PTH), PTH-related peptide (PTH-rP), thyroid hormones and 1,25-dihydroxyvitamin D3 while TGF-βand other cytokines such as IL-1 and TNF-αincrease IL-6 production (154). IL-6 has been shown to stimulate osteoclast formation and bone resorption in fetal mouse bone in vitro and along with IL-1 also stimulates bone resorption in vivo (155, 156). Furthermore, IL-6 has been shown to play a role in the abnormal bone resorption observed in patients with multiple myeloma (157), Paget's disease (158), rheumatoid arthritis (159), and Langerhans cell histiocytosis(160). Effects of increased osteoclast-induced bone resorption are not solely attributed to IL-6, but to all IL-6 family cytokines such as leukemia-inhibitory factor (LIF). It appears that LIF acts on osteoclasts indirectly via stimulating IL-6 release by osteoblasts, resulting in an increase in bone resorption (161).

 

TNF-αhas also been shown to induce bone resorption and plays an important role in inflammatory bone diseases (160, 162). TNF-αpromotes RANK expression in osteoclast precursors and the formation of multinucleated osteoclasts in the presence of M-CSF. Furthermore, TNF-αmay indirectly induce osteoclastogenesis by increasing RANKL and M-CSF expression in osteoblasts, stromal cells and T lymphocytes while it has been observed that RANKL can also enhance TNF-αmediated osteoclastogenesis (148). IL-1βincreases RANKL expression and stimulates osteoclast formation and bone resorption while promotes also TNF-αinduced osteoclastogenesis (163-164). The Th17 cytokine IL-17 is associated with RANKL increase as well as with stimulation of the osteoclastogenic cytokines TNF-α, IL-1, IL-6 and IL-8 and promotes bone resorption.

 

Estrogen deficiency is associated with an increased rate of bone resorption relative to bone formation resulting to net bone loss (143). Estrogen loss is associated with an expansion of T and B lymphocytes that could be related with the enhanced bone resorption (165-166). Furthermore, studies have shown that there is an increased production of RANKL by T and B lymphocytes in postmenopausal compared to premenopausal women (167). It has been observed that TNF-αlevels are significantly increased in ovariectomized women and mice and a model of enhanced bone resorption due to TNF-αmediated RANKL increase has been proposed (143, 168-169). It has also been shown that after ovariectomy IL-17 levels are increased while studies in ovariectomized mice have revealed that anti-IL-17 antibodies or IL-17 gene deletion may reduce bone loss (170-172). In addition, B lymphocytes may partially contribute to trabecular bone loss (173).

 

Regarding thyrotoxicosis-induced osteoporosis, it appears that IL-6 and IL-8 play a major role.  as they have been found to be increased in patients with thyrotoxicosis due to GD or toxic multinodular goiter (172-175). Siddiqi A et al. have shown that patients with thyroid carcinoma on TSH suppressive therapy had significantly raised circulating levels of IL-6 and IL-8 compared to controls (175). In both groups, plasma levels of IL-6 and IL-8 correlated with serum T3 and free T4 concentrations. Both IL-6 and IL-8 have also been shown to be released by human bone marrow stromal cell cultures containing osteoblast progenitor cells in response to T3 (174). TNF-αelevations due to low TSH signaling in human hyperthyroidism contribute also to the bone loss that has traditionally been attributed solely to high thyroid hormone levels (176). Hyperthyroid mice lacking TSHR had greater bone loss and resorption than hyperthyroid wild-type mice, thereby demonstrating that the absence of TSH signaling contributes to bone loss (177).

 

Bone resorption in primary hyperparathyroidism (PHP) also appears to be related to immune system effects. Circulating levels of IL-6 and TNF-αhave been found to be significantly elevated in patients with PHP and return to normal after successful treatment. In addition, it has been observed a significant correlation of these cytokines with biochemical markers of resorption suggesting they may play a role in the pathogenesis of osteoporosis in PHP (178).The hypothesis that IL-6 mediates the catabolic effects of parathyroid hormone (PTH) on the skeleton has been further strengthened by the findingthat neutralizing IL-6 in vivo attenuates PTH-induced bone resorption in mice while the resorptive response to PTH was also reduced in IL-6 knockout mice (179). Furthermore, it has been observed that transplantation of parathyroid from humans with hyperparathyroidism to mice lacking T cells was not associated with bone loss suggesting a possible role of T lymphocytes in PTH related osteoporosis (180). A recent study has shown a direct action of PTH on T lymphocytes as deletion of PTH receptor from T cells failed to induce bone loss (181). It has been proposed that PTH action on T cells results to secretion of TNF-αand in combination with RANKL increase and OPG suppression guides their differentiation to Th17 subsets with subsequent IL-17 secretion and further RANKL amplification (182).

 

EFFECTS OF THE IMMUNE SYSTEM ON THE STRESS SYSTEM

 

The Hypothalamo-Pituitary-Adrenal (HPA) Axis

 

Acute stress increases the expression of cytokines and other inflammatory-related factors in the central nervous system (CNS), plasma, and endocrine glands.  Activation of inflammatory signaling pathways within the HPA axis may play a key role in prevention of the self-damaging effects of the immune system. Data on this topic have been provided by a series of experiments that characterize stress effects on members of the IL-1βsuper-family and other inflammatory-related genes in key structures comprising the HPA axis.(183).

 

The HPA-axis is activated in states of inflammation or infection. This activation is mediated by the inflammatory cytokines, TNF-α, IL-1, and IL-6, which are secreted in tandem in response to various infectious and non-infectious stimuli. The inflammatory cytokines are produced by a variety of cells, including monocytes, macrophages, astrocytes, endothelial cells, and fibroblasts and lead to an increase of corticotropin releasing hormone (CRH), adrenocorticotroph hormone (ACTH) and finally glucocorticoids (38, 184). The underlying mechanisms are not very well understood but Toll like receptor 4 found in immune and endocrine pituitary cells is considered to be related to the induction of local cytokine production (185).

 

The pro-inflammatory cytokine interleukin-1, especially its βform, is probably the most important molecule capable of modulating cerebral functions during systemic and localized inflammation. Systemic IL-1βinjection activates the neurons involved in the control of autonomic functions, and neutralizing antibodies or IL-1 receptor antagonists are capable of preventing numerous responses during inflammatory stimuli (186). Other cytokines implicated in neuroendocrine and febrile responses include TNF-αand IL-6. Similar to IL-1β, intravenous IL-6 stimulates the hypothalamic-pituitary unit leading to the secretion of cortisol by the adrenal glands, and subsequent termination of the inflammatory cascade (187). Although all three inflammatory cytokines (IL-1, IL-6 and TNF-α) have the capacity to activate the HPA-axis, it appears that IL-6 is the critical component of this cascade. Studies in rats have demonstrated that immunoneutralization of IL-6 abolishes the effects (as potent activators) of the other two cytokines on the HPA-axis (188). TNF-αand IL-1 stimulate the production of IL-6 and IL-6 in turn stimulates the HPA-axis. While acute stimulation with IL-6 stimulates the HPA-axis through activation of the hypothalamic CRH neurons, chronic exposure to IL-6 can stimulate directly the corticotroph cells of the pituitary and the adrenal cells (Figure 4).

Figure 4. TNF-α and IL-1 Stimulate the Production of IL-6 and IL-6 in Turn Stimulates the HPA-Axis

Glucocorticoids appear to inhibit IL-6 secretion at the transcriptional level through interaction of the ligand-activated glucocorticoid receptor with nuclear factor-Kappa B. This demonstrates that glucocorticoids and IL-6 participate in a feedback loop, in which IL-6 stimulates glucocorticoid release and glucocorticoids subsequently through negative feedback inhibit IL-6 release. This explains the inverse relationship with diurnal variation between circulating IL-6 and glucocorticoid levels (189-190). Furthermore, acute hypocortisolism has been shown to result in a four to five-fold elevation of circulating IL-6 and TNF-αlevels. In a study involving patients with Cushing’s disease studied before and after transsphenoidal adenectomy, cytokines were measured during the hypercortisolemic, hypocortisolemic, and eucortisolemic (while patients were on glucocorticoid replacement) states (191). When patients were hypocortisolemic, plasma IL-6 concentration increased, while they experienced symptoms of glucocorticoid deficiency, which are part of the "steroid withdrawal syndrome". This syndrome consists of pyrexia, headache, anorexia, nausea, fatigue, malaise, arthralgias, myalgias, and somnolence of variable degree. Interestingly, IL-6 levels did not increase in patients who did not become hypocortisolemic after surgery (and did not develop symptoms consistent with the withdrawal syndrome), indicating that hypocortisolism was necessary for the rise in IL-6. Glucocorticoid replacement was followed by a dramatic decrease of IL-6 levels concomitantly with relief of the observed symptoms (191).

 

Furthermore, increased cortisol turnover is a feature of obese individuals and is exaggerated in upper body (visceral) obesity (192). Some studies indicate that IL-6 directly stimulates adrenal cortisol release in addition to stimulating hypothalamic CRH and pituitary ACTH release (193-195). Adipose tissue IL-6 may, therefore, act as a feed-forward regulator of the hypothalamic-pituitary axis function. Cortisol suppression of adipose IL-6 production may serve as a feedback inhibitor of this regulatory loop. Adrenal cortisol production could be influenced by IL-6 originating from peri-renal adipose tissue that surrounds the adrenal glands.

 

Similar to IL-6,leukemia-inhibitory factor (LIF) can also stimulate the hypothalamic pituitary axis. LIF is a multifunctional cytokine of the IL-6 cytokine family, sharing the common gp130 receptor subunit together with IL-6, interleukin-11, oncostatin-M, ciliary neurotrophic factor and cardiotrophin-1. Both LIF and its receptor have been found to be expressed in the pituitary gland during development (196). Furthermore, LIF binding sites (LIFR) have been found in one third of ACTH-positive cells and approximately 20% of growth hormone (GH)-positive cells of the pituitary. In several tissues, LIF, LIFR, and gp130 mRNA expression is stimulated by various inflammatory stimuli, whereas LIF gene expression is negatively regulated by glucocorticoids. It has been observed that LIF stimulates ACTH secretion in vitro and in vivo.

 

Adrenal Medulla

 

The chromaffin cells of the adrenal medulla are considered to play a role in stress response by secreting catecholamines and various biologically active peptides. Recent studies have shown that cytokines such as TNF-α, IL-1 and IFN-γact directly to chromaffin cells (197-200). It has also been demonstrated that cytokines regulate the secretion of various peptides that are co-secreted with catecholamines such as vasoactive intestinal peptide (VIP), galanin and secretogranin II, enkephalin and neuropeptide Y (198, 201-204).

 

A recent study evaluated the regulation of adrenal chromaffin cells by IL-6. It has been observed that IL-6 directly modulates the secretion of catecholamines and neuropeptides by chromaffin cells and therefore influences the adrenal stress response. It has been hypothesized that medullary peptides may serve as paracrine modulators of glucocorticoid production (205). It has also recently been demonstrated that IL-6 increases intracellular Ca2+concentration and induces catecholamine secretion in rat carotid body glomus cells, a finding which eventually confirms arelationship between IL-6 and catecholamine secretion (206).

 

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Thyroid Nodules and Cancer in the Elderly

ABSTRACT

 

Thyroid nodules and cancer are common in elderly patients and demonstrate age-specific prevalence, malignancy risk, and clinical behavior. Improved risk stratification by ultrasound characteristics and molecular testing of thyroid nodules has reduced the need for diagnostic surgery. Surgery, radioactive iodine ablation, and thyroid hormone suppression remain the cornerstones of differentiated thyroid cancer treatment. Co-morbid conditions and patient preference should inform management of these entities in the elderly, with particular attention to the risks of surgery and medication adverse effects. The mechanisms underlying the distinct clinical behavior of thyroid cancer found in older patients, including the drivers of more advanced stage at presentation, higher recurrence risk, and greater mortality, remain poorly understood. Patients with advanced thyroid cancer may benefit from recently developed chemotherapy and immunotherapy.

 

THYROID NODULES IN THE ELDERLY

 

Thyroid nodules are common in clinical practice and present unique management issues in elderly patients. The reported prevalence of thyroid nodules in iodine sufficient regions is 1-6 % as detected by palpation, or as high as 19-68% when detected by ultrasound imaging (1-5). Evaluation of thyroid nodules is increasingly a concern for general internists and endocrinologists due to several factors, including an aging population, increased use of imaging in clinical practice, and rising obesity.

 

Thyroid nodules are more frequent in elderly patients, with a linear increase with age in both the presence of nodules and the absolute number of nodules per patient (6). Approximately 50% of individuals aged 65 years have thyroid nodules detected by ultrasonography (7). A cross-sectional survey of asymptomatic adults in Germany using ultrasonography to detect thyroid nodules demonstrated an even higher prevalence of 80% in women and 74% in men over 60 years old (4). In a prospective study of 6,391 patients referred for thyroid nodules at a large academic center, Kwong et al. showed a linear increase in the number of thyroid nodules per patient with age, rising from an average of 1.55 nodules ≥1 cm in patients age 20–29 years old to a mean of 2.21 nodules ≥1 cm in patients ≥70 years old, demonstrating a 1.6% annual increased risk for multinodularity (6).

 

Another potential contributor to this rising prevalence of thyroid nodules is the increased use of high-frequency ultrasound, CT, and MR imaging in routine clinical care, leading to the detection of asymptomatic, or incidental, thyroid nodules (4,5,7,8). Lastly, changes in population demographics over time, specifically increased rates of obesity, may contribute. Data from several ethnically diverse cohorts has identified parameters independently associated with the development of thyroid nodules, including obesity, female sex, radiation exposure, iodine deficiency, and smoking. These should be noted when evaluating elderly patients for potential thyroid nodules (9).

 

Once identified, thyroid nodules should be evaluated to determine appropriate management. The differential diagnosis of thyroid nodularity includes benign and malignant solitary nodules, multinodular goiter, autonomous functioning nodules, cysts, and inflammation or thyroiditis (10). Nodules causing thyroid dysfunction, compressive symptoms, or harboring malignancy require attention.

 

In the presence of biochemical or clinical signs of hyperthyroidism, a radioiodine uptake and scan should be pursued to distinguish autonomous nodules. Adjunctive data to support a diagnosis of inflammation or autoimmune destruction may include thyroid autoantibodies [anti-thyroid peroxidase (TPO) and anti-thyroglobulin (Tg)]; the presence of thyroid stimulating immunoglobulins can suggest a diagnosis of Graves’ disease in the presence of goiter (11).

 

Nodules without associated thyroid function abnormalities should be further evaluated to determine or exclude the presence of cancer. Guidelines from the American Thyroid Association were recently updated and summarize the management of non-functional thyroid nodules based upon imaging and patient characteristics (12). A general approach to evaluation of thyroid nodules is shown in Figure 1.

Figure 1. Evaluation of a Thyroid Nodule. Hot nodules refer to autonomous, hyperfunctioning thyroid follicular tissue producing thyroid hormone excess. Cold nodules refer to nodules without autonomous production of thyroid hormone.

Briefly, solid, hypoechoic thyroid nodules with suspicious ultrasound features (i.e.irregular margins, microcalcifications, taller than wide shape, rim calcifications, or evidence of extrathyroidal extension) have a high risk of malignancy (70-90%) and should be biopsied when ³1 cm in largest diameter. Solid, hypoechoic nodules without high risk features on imaging, still portend a 10-20% risk of malignancy and should similarly be biopsied when size ³1 cm. Isoechoic or hyperechoic, solid nodules without microcalcification, irregular margin, extrathyroidal extension or taller than wide shape are recommended to biopsy when ³1.5 cm, with a 5-10% risk of malignancy. Spongiform or partially cystic nodules absent the above suspicious features, may be followed by imaging surveillance or biopsied if ³2 cm. The associated risk of malignancy with such lesions is low (<3%). Purely cystic nodules seen on ultrasound do not warrant biopsy given the exceedingly low risk of malignancy (<1%) (12). A summary of thyroid nodule ultrasound characteristics and their relation to malignancy risk is shown in Table 1.

 

Table 1. Ultrasound Features of Thyroid Nodules and Associated Malignancy Risk
Ultrasound Imaging Features
- Cyst - Spongiform

- Partially cystic

- No suspicious features

- Hyperechoic and isoechoic solid nodule

- Partially cystic with eccentric solid area

- No calcifications or comet tail artifact

- Hypoechoic solid and regular margins

- Macro-calcifications

- Micro-calcifications or Interrupted rim calcifications

- Lobulated or irregular margins

- Extrathyroidal extension/invasion

- Taller than wide shape

Estimated Risk of Nodule Malignancy
<1% <3% 5-10% 10-20% >70-90%
Benign Very low suspicion Low suspicion

 

Intermediate suspicion High suspicion

 

 

Fine needle aspiration (FNA) biopsy is the recommended modality for sampling thyroid nodules. Cytology specimens collected by FNA are classified traditionally by the Bethesda System for Reporting Thyroid Cytopathology (13) across six categories: (i) non-diagnostic or unsatisfactory; (ii) benign; (iii) atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS); (iv) follicular neoplasm or suspicious for a follicular neoplasm; (v) suspicious for malignancy; and (vi) malignant. The risk of malignancy determined by surgical pathology is estimated across each category and used to guide decisions about continued clinical observation or treatment with surgical resection (13), summarized in Table 2.

 

Table 2. Bethesda System for Reporting Thyroid Cytopathology and Associated Estimated Risk of Malignancy.
Bethesda category Cytopathology Cytologic descriptions Malignancy risk

- Cancer =NIFTP

- Cancer ¹NIFTP

Typical management
I Non-diagnostic Acellular specimen

Cyst fluid only

Obscuring factors

- 5-10%

- 5-10%

Repeat FNA
II Benign Benign follicular nodule

Chronic lymphocytic thyroiditis

Granulomatous thyroiditis

- 0-3%

- 0-3%

Clinical and ultrasound follow-up
III Atypia of undetermined significance (AUS) or follicular lesion

of undetermined significance (FLUS)

Atypia: Cytologic (focal nuclear changes, extensive but mild nuclear changes, atypical cyst lining cells, or ‘‘histiocytoid’’ cells) and/or architectural (predominantly microfollicles, sparsely cellular); Hurthle cells - 6-18%

- 10-30%

Repeat FNA, molecular testing, or lobectomy
IV Follicular neoplasm or suspicious for a follicular neoplasm Follicular-patterned cases with mild nuclear changes (increased nuclear size, nuclear contour irregularity, and/or chromatin clearing), and lacking true papillae and intranuclear pseudo-inclusions - 10-40%

- 25-40%

Repeat FNA, molecular testing, lobectomy
V Suspicious for malignancy Features suspicious for PTC, MTC, lymphoma, or other malignancy - 45-60%

- 50-75%

Total thyroidectomy   or lobectomy
VI Malignant Features conclusive for malignancy:

- PTC (true papillae, psammoma bodies, nuclear pseudo-inclusions)

- MTC

- Poorly differentiated / ATC

- Non-endocrine malignancy (squamous cell, lymphoma, metastatic)

- 94-96%

- 97-99%

Total thyroidectomy   or lobectomy for thyroid cancers

PTC, papillary thyroid carcinoma. MTC, medullary thyroid cancer. ATC, anaplastic thyroid cancer. NIFTP, noninvasive follicular thyroid neoplasm with papillary-like nuclear features.

 

In situations of non-diagnostic FNA results or indeterminate cytology (i.e.,Bethesda iii or iv), repeat FNA biopsy is recommended. Additionally, three molecular tests for cancer risk stratification are presently available. The ThyroSeq assay (University of Pittsburgh Medical Center and CBL PATH, Pittsburgh, PA) detects the presence of high-risk cancer mutations and was developed as a rule-in test for thyroid cancer (14). An independent evaluation of ThyroSeq v2 performance in a series of indeterminate nodules calculated a sensitivity of 70% and a specificity of 77%, somewhat decreased from the originally reported 90% and 93%, with a positive predictive value (PPV) of 42% and a negative predictive value (NPV) of 91% (14,15). Performance data for ThyroSeq v3, a revised version of the molecular test incorporating 112 genes associated with thyroid cancer, was recently published by Nikiforov et al. with a reported sensitivity of 98% and specificity of 81% for detection of thyroid cancer from FNA samples (16). The Afirma gene expression classifier (GEC) assay (Veracyte, San Francisco, CA, USA) analyzed the expression pattern of mRNA in cytology specimens to classify them as benign or suspicious and was designed as a rule-out test for cancer. The original Afirma GEC had a reported NPV of 94–95% and a PPV of 37–38% (17,18). The next iteration of this test was approved in 2018 as the Afirma gene sequence classifier (GSC) and includes detection of thyroid cancer-associated mutations with a reportedly improved specificity for thyroid cancer (NPV 95%, PPV 47%) (19). Lastly, the combined ThyraMIR microRNA Classifier and ThyGenX Oncogene Panel (Interpace Diagnostics, Parsippany, NJ) is a cancer rule-in test that uses multiplex PCR to identify cancer-associated gene mutations and translocations, done in tandem with evaluation of microRNA expression. The test estimated NPV and PPV are 94% and 74%, respectively (20). As molecular testing continues to evolve, clinicians and patients will have additional tools to aid in treatment decisions.

 

Judicious use of FNA biopsy, improved stratification of nodule cancer risk by ultrasound characteristics, and molecular testing have improved pre-operative determination of malignancy risk in patients with thyroid nodules and reduced the need for diagnostic surgery. However, a significant number of patients who undergo thyroid nodule resection for suspicious nodules are still ultimately found to have benign lesions on surgical histopathology. Particularly in elderly patients with a greater burden of co-morbid medical disease, the risk of unnecessary thyroid surgery is an important consideration.

 

Several recent studies have specifically addressed thyroid cancer risk and nodule management across the age spectrum. Kwong et al.(6) reported the rate of malignancy in a cohort of 6,391 patients referred to a large academic center who underwent thyroid ultrasound and FNA of 12,115 nodules (all ≥1 cm). With advancing age, the prevalence of clinically relevant (>1 cm) thyroid nodules increased, whereas the risk that such nodules were malignant decreased. For patients ages 20–29, 30–39, 40–49, 50–59, 60–69, and >70 years, the cancer prevalence was 22.9, 21.8, 17.1, 13.0, 13.7, and 12.6%, respectively (p<0.001). When the malignancy rate was analyzed “per-nodule,” the youngest cohort (20–29 years) demonstrated a 14.8% malignant risk per nodule at diagnosis in comparison to 5.6% in the oldest cohort (>70 y; p<0.01). Between the ages of 20 and 60 years, each advancing year was associated with a 2.2% reduction in the relative risk that any newly evaluated thyroid nodule was malignant (OR 0.972; p<0.001), and this risk of malignancy stabilized after age 60 years. However, this study also found that despite a lower likelihood of malignancy for nodules in elderly patients, these cancers were more likely to have aggressive phenotypes (6).

 

Further addressing the burden and risk of thyroid nodule evaluation in older patients, Angell and colleagues recently analyzed a large cohort of elderly patients (age 70 years and older) who underwent thyroid nodule evaluation over a 20-year period (21). In this study, 1,129 patients over the age of 70 years with 2,527 nodules ≥1 cm were evaluated. Thyroid cancer-specific mortality was observed in 8% of thyroid cancer patients. All such patients could be recognized during initial evaluation based on the presence of invasive tumor, extensive lymph node metastases, or distant metastases. While FNA was a safe procedure in this age-group and a benign result was obtained in two-thirds of samples, FNA led to surgery in 208 patients, of whom 93 (44.7%) had benign histopathology. These data suggest that while an identifiable group of older patients are at risk for mortality from thyroid cancer warranting aggressive treatment, many patients ≥70 years old derive little benefit or are even harmed by thyroid nodule therapy.

 

DIFFERENTIATED THYROID CANCER IN THE ELDERLY

 

While thyroid nodules are relatively common in elderly patients and the vast majority are benign (21), thyroid cancer is identified in a subset. Patients and their families are often concerned about the implications of this diagnosis and disease outcomes. Several subtypes of thyroid cancer are frequently encountered and increasing information about the underlying biology of these malignancies is now available. Most thyroid cancers are identified incidentally on imaging rather than by palpation on physical examination. Rarely, symptoms of thyroid cancer can include lymphadenopathy, hoarseness from laryngeal nerve involvement, dysphagia, airway compression from mass effect, or pain; when present, these symptoms portend more advanced disease and worse clinical prognosis (22,23). When thyroid cancer is identified, a combination of surgical, radioactive iodine and surveillance strategies are employed and tailored to the individual patient and disease characteristics.

 

Incidence and Prevalence of Thyroid Cancer

 

Thyroid cancer accounts for 3.1% of all new cancers, but only 0.3% of cancer deaths, in the United States annually (24). In 2018, there were an estimated 53,990 new cases of thyroid cancer and over 750,000 people living with thyroid cancer (24). In the general population, the peak occurrence is between ages 51 and 60 years (25). Thyroid cancer is more common in women than men and among those with a family history of thyroid disease (24).

 

The incidence of thyroid cancer has risen over time, with an approximately two-fold increase between 1973 and 2002. Notably, small (<2 cm) papillary thyroid cancers account for the majority of this increase (26), and despite a much higher incidence, thyroid cancer mortality has only increased slightly (27), likely reflecting greater detection of early disease associated with a good prognosis. Debate exists whether the increased detection by high resolution imaging alone has simply identified more incidental thyroid cancers or whether a true increase in the incidence in these cancers is occurring over the past several decades (26-28).

 

With more thyroid cancers being diagnosed, particularly when they may be of limited mortality significance, clinicians must be well-versed in the management options and the particular risks and benefits anticipated for elderly patients.

 

Classification of Thyroid Cancer

 

Thyroid follicular cell-derived cancer is subdivided into several histopathologic types: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), Hurthle cell carcinoma (HCC), and poorly differentiated or anaplastic thyroid cancer (29-31). Other malignancies encountered in the thyroid include medullary thyroid cancer arising from thyroid gland C-cells, lymphoma, and secondary metastasis of other primary cancers.

 

Papillary thyroid cancer (PTC) is the most common type of differentiated thyroid cancer (DTC) accounting for approximately 80 to 85% of all cases (24,29-31). It has a bimodal frequency, with the peak incidence being in the third and sixth decades, and it affects women three times more often than men. These carcinomas arise from the thyroid follicular cells and frequently harbor BRAF V600E mutations, produce thyroglobulin, and express the sodium-iodide symporter (NIS) with resultant radio-iodine avidity (29). A history of radiation exposure increases the risk of PTC (32-34). PTC frequently spreads via the lymphatics to the regional lymph nodes, and bilateral involvement is present in approximately one-third of the cases at diagnosis. In rare cases, metastatic disease occurs in the lungs, brain, and bone (29).

 

Micropapillary thyroid cancer, defined as a PTC less than 1 cm in diameter and confined to the thyroid, is likely to be of minimal clinical significance (35). A prospective, observation study of papillary thyroid microcarcinoma in Japan, found that patients less than age 40 progressed to clinical disease, (defined as significant growth, size >1.2 cm, or lymph node metastases), in contrast to those over age 60, whose disease remained static (36), suggesting that in most elderly patients these lesions can be safely observed.

 

Follicular thyroid cancer (FTC) is the second most common type of DTC and constitutes approximately 10 to 15% of all thyroid cancers (24,29,30). Risk factors include iodine deficiency and female sex (24,37,38). Compared to PTC, FTC less often has cervical lymph node spread but shows a predilection for vascular invasion and distant metastasis (39). Mutations of RAS, an activator of the mitogen-activated protein kinase and PI3K-AKT pathways,and rearrangements of PPAR-γ (e.g.PAX8-PPAR-γ translocation) have been implicated in the tumorigenesis of follicular adenomas and FTC (39,40).

 

Hurthle cell carcinomas (HCCs) account for 5% of DTC and are characterized by an abundance of dysfunctional mitochondria (>75% of cell volume) and tendency for vascular invasion (41,42). These malignancies are more often radio-iodine refractory and aggressive in clinical behavior. Unique genetic drivers of HCC have recently been reported, namely widespread loss of heterozygosity, a high burden of disruptive mutations to protein-coding and tRNA-encoding regions of the mitochondrial genome, and recurrent mutations in DAXX, TP53, NF1, CDKN1A, ARHGAP35, TERTpromoter, and the RTK/RAS/AKT/ mTOR pathway (43,44).

 

Anaplastic thyroid cancer and medullary thyroid cancer are discussed separately.

 

Variation in Histopathology and Tumor Extent by Age

 

Several studies have shown variance in histopathology distribution with rising age. Lin et al. (45) conducted a retrospective analysis of 204 thyroid cancer patients aged 60 years and older; 142 (70%) thyroid cancers were well differentiated and of those 68% were PTC, 30% FTC, and 2% Hurthle cell carcinoma. Fifty-nine (29%) of the thyroid cancers were poorly differentiated (39 anaplastic thyroid, 9 metastatic cancers to the thyroid, 7 lymphoma, 4 squamous cell carcinomas, and 4 without enough cells for interpretation) and 3 (2%) were medullary thyroid cancer. This pattern is significant for fewer PTC and more FTC in elderly patients, as well as more poorly-differentiated tumors.

 

Girardi et al.conducted a retrospective study of thyroid cancer in 596 adults from 2000-2010; their results similarly showed a lower frequency of PTC among elderly patients, with a complementary increase in the frequency of FTC, poorly differentiated and anaplastic thyroid carcinoma (25). This study also demonstrated variability in other presenting features of thyroid cancer in elderly patients (age ³65 years) compared to middle-aged cohorts (25-44 years or 45-64 years); specifically, there was larger primary tumor size (median 2.1 cm for elderly versus 1.5 cm in 25-44 years and 1.1 cm in 45-64 years) and higher rates of extra-thyroidal disease (mean 43% for elderly versus 25.3% in 25-44 years and 28.6% in 45-64 years) (25). Lymph node metastasis was greatest at the extremes of age (<24 and >70 years).

 

Another retrospective analysis of 1,022 patients undergoing thyroidectomy reported by Payne et al.showed that well-differentiated thyroid cancer (i.e.,PTC and FTC), and lymph node metastasis occurred more often in patients younger than 50 years, whereas micropapillary carcinoma was more common in patients 50 years or older (46). Chereau et al. evaluated histopathology and extent of disease at diagnosis in elderly (65-75 years old) and very elderly (>75 years old) patients compared to younger patients in 3,835 patients treated at an academic center from 1978 to 2014 (47). These data were notable for significantly increased primary tumor size, tumor number, extra-capsular invasion, advanced TNM stage, and lymph node and distant metastasis in the very old group (47). Collectively these studies show a pattern of more widespread disease at presentation in elderly patients and a relative increase in the frequency of more aggressive histologic subtypes.

 

Relation of Age to Mortality and Risk of Recurrence

 

Numerous studies have demonstrated increased recurrence and mortality in thyroid cancer with rising age (48-53). Indeed, age is incorporated into current clinical staging systems for differentiated thyroid cancer, including the American Joint Committee on Cancer (AJCC) 8thedition (54); Metastasis, Age, Completeness of resection, Invasion, Size (MACIS) model (55); Age, Grade, Extent, Size (AGES) score; and the Age, Metastasis, Extent, Size (AMES) score (56). In all of these staging systems, advanced age is included as a risk factor predicting worse prognosis.

 

Historic studies by Halnan (57) and Cady et al. (58) established a positive correlation between advanced age and worse prognosis in patients with DTC, later corroborated by Ito et al. (59) in a study of 1,740 patients with PTC and by Sugino et al. (60) in 134 patients with FTC. In many of these studies, worse prognosis has been defined variably as recurrence, decreased disease- or metastasis-free survival, cause-specific mortality, and/or overall mortality. Other reports have shown that the presence of lymph node involvement and extrathyroidal extension may portend a more ominous outcome in older compared to younger patients (58,61-63). Extrathyroidal disease in older patients increased recurrence to 67% and death rates to 60% compared to those with intrathyroidal disease, while in younger patients the relative increases were 12% and 4%, respectively (58). Additionally, the risk of death with distant metastasis is greater in older compared to younger patients (96% versus 63%) (58).

 

Recently, this well-accepted tenet of thyroid cancer has been modified in two important ways, namely that age likely modifies prognosis in a continuous rather than dichotomous manner and that age itself may not be as relevant to thyroid cancer behavior as the accompanying changes in accumulated cell mutations, immune senescence, and hormone changes that accompany it (64).

 

With the 8thedition of AJCC staging for differentiated thyroid cancer, the age threshold for increased risk was raised from 45 to 55 years, based upon several reports suggesting that this increased validity for staging (65,66). More recent data suggest that thyroid cancer mortality and recurrence prediction is more robust when age is modeled as a continuous variable, leading some to suggest the elimination of a specific age cutoff from staging completely (64).

 

In a study of 3,664 patients with differentiated thyroid cancer, Ganly et al. found that disease-specific mortality increased progressively with advancing age, without a threshold age (53). Similarly, evaluation of over 30,000 patients in the Surveillance, Epidemiology, and End Result (SEER) database by Orosco et al.demonstrated a linear association with age and thyroid cancer death (52).

 

A recent review by Haymart et al.summarizes possible biologic mechanisms underlying the clinical observations of worse thyroid cancer prognosis in the elderly (50). Briefly, mortality findings may be confounded by greater comorbid nonthyroidal diseases with older age. Higher baseline levels of thyroid-stimulating hormone (TSH) may accelerate tumor cell growth via stimulation of the TSH-receptor. If one presumes that thyroid cancers detected in elderly patients have had a longer time of subclinical growth and evolution compared to cancers detected in younger patients, then such tumors might have had greater opportunity to acquire genetic mutations facilitating cell cycle escape, loss of differentiated features (e.g. loss of sodium-iodine symporter and radioiodine avidity), and metastasis. In summary, there is significant observational evidence that older patients with thyroid cancer have worse clinical outcomes, though the precise effect of increasing age and the etiology of this distinct clinical behavior remain incompletely understood.

 

Treatment of Differentiated Thyroid Cancer

 

The cornerstones of therapy for differentiated thyroid cancer are surgical resection, radioactive iodine ablation, and thyroid hormone suppression of TSH. Serum thyroglobulin trends and follow-up neck ultrasound imaging are used for monitoring patients over time. Patients with progressive or metastatic disease may benefit from repeat surgery, radioactive iodine ablation (RAIA), or chemotherapy (12). A general approach to the treatment of differentiated thyroid cancersis presented in Figure 2. Management will be influenced by patient characteristics, such as age and comorbid conditions, as noted in the figure.

 

Figure 2. Treatment of Differentiated Thyroid Cancers. RAI, radioactive iodine. TSH, thyroid stimulating hormone. VEGFR, vascular endothelial growth factor receptor. TKI, tyrosine kinase inhibitor

SURGERY

 

Patients diagnosed with DTC by FNA, should be referred to a surgeon for thyroid resection. The decisions to pursue surgery and the extent of surgery (i.e.total thyroidectomy versus lobectomy) in an elderly patient require individual evaluation of co-morbid illnesses and life expectancy.

 

The most common complications of thyroidectomy include hypoparathyroidism, recurrent laryngeal nerve injury, hematoma, and wound infection; high-volume thyroid surgeons have minimal to no increase in the risk of surgical complications with increasing age (67,68-72). However, elderly patients are more likely to receive thyroidectomy at community and low-volume sites (73) where the rate of surgical complications may be higher. In population-based studies of thyroidectomy, which may reflect more accurately the experience of many elderly patients, increasing age is associated with longer hospital length of stay (73) and readmissions after thyroidectomy (74).

 

In the cohort of elderly and very elderly patients studied by Chereau et al. (47), the authors found no increase in thyroidectomy-specific complications (i.e.permanent hypocalcemia and recurrent laryngeal nerve palsy) with increasing age, but did find an increase in medical complications surrounding surgery, 2.3-2.7% in those over 65 years of age compared to 0.6% in those under 65 years old.

 

Lobectomy, a more limited surgery, may be considered in select patients with multiple co-morbid conditions and low-risk disease (tumors <2 cm) (12,67). On the other hand, elderly patients more often have aggressive disease features on surgical pathology and higher rates of local recurrence requiring re-operation, potentially arguing for a total thyroidectomy (70).

 

RADIOACTIVE IODINE ABLATION

 

Based upon the extent of primary disease noted on surgical pathology (i.e.tumor size, histologic subtype, extrathyroidal extension, lymph node and vascular spread, and the presence of distant metastasis), a risk of recurrence and recommendation for RAIA with I131can be made. Generally, patients with ATA high and intermediate risk should be considered for RAIA therapy (12) to ablate residual disease and remnant thyroid tissue.

 

Two multicenter studies showed that an ablative dose of 30 mCi (1.1 MBq) I131was as effective as 100 mCi (3.7 MBq); both doses were 90% effective for ablation of residual thyroid tissue (75,76). A long-term follow-up of one of these studies (median 4.5 years) showed that the radioiodine dose did not affect recurrence rate (77). A recent analysis of 21,870 patients with intermediate-risk PTC found that adjuvant radioiodine therapy was associated with a 29% reduced risk of death overall with clear benefit in those over 65 years of age (78). The adverse effects observed following RAIA, transient neck pain and swelling, dry mouth and eyes, and secondary malignancy, correlate positively to higher doses (79).

 

THYROID-STIMULATING HORMONE (TSH) SUPPRESSION

 

Following surgery, and RAIA if indicated, patients are treated with thyroid hormone, usually with a dose of levothyroxine that suppresses serum TSH to subnormal levels. Several special considerations for the goals of thyroid hormone therapy following thyroid cancer arise in elderly patients.

 

Thyroid hormone replacement is titrated to levels sufficient to suppress pituitary secretion of TSH, which is considered a growth-promoting factor for follicular cell-derived thyroid cancers. Revised guidelines from the American Thyroid Association (12) suggest individualized targets for TSH suppression in thyroid cancer, generally targeting a low to low-normal range TSH. Greater TSH suppression in more aggressive disease is balanced with greater cardiac and bone complications in elderly patients.

 

Older patients are more likely to have co-morbid cardiac disease, including arrhythmias, coronary artery disease, and heart failure, which can place them at increased risk for complications from thyroid hormone excess. A population-based study of patients taking levothyroxine for any cause, found a significantly higher risk of cardiac arrhythmias [HR 1.6 (1.10–2.33)] and cardiovascular admission or death [1.37 (1.17–1.60)] in those with a suppressed serum TSH (£0.03 mU/L) compared to those with TSH in the normal reference interval (80). Notably, increased cardiovascular risk was not observed in patients with a low but not fully suppressed TSH (TSH 0.04 – 0.4 mU/L). Specifically, in thyroid cancer patients treated with levothyroxine with modestly suppressed TSH (mean TSH <0.35 mU/L), atrial fibrillation was common (17.5% prevalence) in those patients ³60 years old (80).

 

Longstanding hyperthyroidism is associated with osteoporotic fractures and loss of bone mineral density. Specifically, post-menopausal women (³65yo) with suppressed TSH levels (0.1 mU/L) due to endogenous or exogenous thyroid hormone had significantly higher rates of new hip (OR 3.6, 95% CI 1.0-12.9) and vertebral fractures (OR 4.5, 95% CI 1.3 -15.6) compared to comparable women with normal TSH levels over a 3.7 years follow-up (81). In adult patients on levothyroxine therapy, a suppressed TSH (£0.03 mU/L) was associated with a two-fold increase in risk [HR 2.02 (1.55–2.62)] of new osteoporotic fracture compared to similar patients treated with levothyroxine with a TSH maintained in the normal reference interval (80). Studies evaluating thyroid cancer patients are limited in outcome evaluation of bone mineral density (BMD) rather than fracture incidence, but generally support similar conclusions regarding lower BMD with suppressive-dose levothyroxine therapy (82-84). In elderly patients receiving TSH-suppression therapy, dual-emission X-ray absorptiometry (DEXA) monitoring of BMD should be considered based upon age and other risk factors for osteoporosis. There are no guidelines to suggest the optimal interval for DEXA screening; osteoporosis once identified should be treated using standard therapies (such as bisphosphonates or RANKL inhibitor) unless otherwise contraindicated (85).

 

Peripheral metabolism of thyroid hormone and clearance decreases with advanced age so that a lower medication dose is needed to achieve comparable serum levels (86,87). Levothyroxine therapy is complicated further by polypharmacy in elderly patients, where commonly prescribed medications (e.g.calcium, iron) can decrease gut absorption of levothyroxine (88) or change drug metabolism (e.g.rifampicin, phenytoin, carbamazepine, amiodarone) (89).

 

In summary, as suggested by society guidelines (12), TSH goals in thyroid cancer should be individualized and re-evaluated over time. Patients with co-morbid cardiac disease and/or osteoporosis with intermediate risk disease recurrence, might best be managed with TSH targets in the low but not fully suppressed range to minimize adverse effects of therapy.

 

THYROGLOBULIN MONITORING

 

Follow-up by measurement of serum thyroglobulin (Tg) at intervals of 4 to 6 months on thyroxine suppression therapy is recommended (12). At one-year post treatment completion, a stimulated Tg measurement may provide a more sensitive evaluation for persistent or recurrent disease (90). A Tg level >0.2 ng/mL, a stimulated Tg level >2- 5 ng/mL, a rising Tg level, or the persistence of Tg antibodies, warrant further evaluation (12, 91). Diagnostic imaging studies such as neck ultrasound, whole body radioiodine uptake, and PET imaging should beperformed to locate the residual thyroid tissue. Identification of abnormal lymph nodes or tumor mass can then be further evaluated for possible further treatment with radioactive iodine, surgery, or targeted therapy.

 

TARGETED THERAPY

 

Older cytotoxic drugs have shown little benefit for progressive, advanced, or metastatic papillary or follicular thyroid cancer while causing significant side effects. Improved understanding of the pathogenesis of these cancers is leading to the development of new agents aimed at specific oncogenic mechanisms (e.g. RET, BRAF). Currently two tyrosine kinase inhibitors are approved for therapy of metastatic, radio-resistant differentiated thyroid cancer: sorafenib and lenvatinib.

 

Sorafenib, an oral multi-kinase inhibitor, inhibits vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, and VEGFR-3), RET kinase (including RET/PTC), BRAF V600E, and platelet-derived growth factor receptor (PDGFR) beta. In the DECISION phase 3 multicenter placebo-controlled trial of 416 patients, 409 had distant metastases: 86% in the lungs, 51% in lymph nodes, and 27% in bone (92). The group treated with sorafenib had longer progression-free survival (10.8 months) compared to the placebo group (5.8 months). At disease progression, 71% of patients in the placebo group crossed over to receive open-label sorafenib; as a consequence, overall survival did not differ between the two groups. Twenty percent of patients in the sorafenib group received other cancer therapy after the trial. The most frequent adverse events in the active drug group were palmar-plantar erythrodysesthesia, diarrhea, alopecia, rash, weight loss, hypertension, anorexia, oral mucositis and pruritus. Side effects were relieved by dose reduction.

 

Lenvatinib is a tyrosine kinase inhibitor of the VEGFRs 1, 2, and 3; fibroblast derived growth factor receptor (FGFR)s 1 through 4; PDGFRα; RET; and KIT signaling pathways. The SELECT phase 3 trial randomly assigned 261 patients to receive lenvatinib and 131 patients to receive placebo; the median age of patients in the trial was over 60 years (93). The median duration of follow-up was 17 months; 114 patients assigned placebo had progression, and 109 of them elected to receive lenvatinib. Disease progression occurred in 36% in the lenvatinib group compared to 83% in the placebo group. Median progression free survival was 18.3 months with lenvatinib versus 3.6 months with placebo. Disease response rate was 66% with lenvatinib compared with 1.5% with placebo. The benefit appeared in all subgroups, including all histologic types of tumor. Adverse events occurred in 97% of patients taking lenvatinib and in 60% taking placebo; the main adverse events were hypertension, diarrhea, fatigue, decreased appetite, palmar-plantar erythrodysesthesia, proteinuria, renal failure, and thromboembolic events. While not without side effects, lenvatinib and sorafenib demonstrated efficacy in patients with metastatic, radioiodine-refractory differentiated thyroid cancer and should be considered in elderly patients with sufficient performance status and potential benefit.

 

For differentiated thyroid cancer that progresses despite these therapies, additional treatment with external beam radiation, off label use of BRAF inhibitors, and clinical trials of immunotherapy are sometimes utilized. These modalities are discussed below in the context of anaplastic thyroid cancer.

 

ANAPLASTIC THYROID CANCER

 

Anaplastic thyroid carcinoma (ATC) is a rare and aggressive subtype of thyroid cancer that accounts for <1% of all thyroid cancers (24,29). It more commonly affects the elderly, with a mean age at diagnosis of 65 years and more than 90% patients with ATC are over age 50 (29). Despite recent advances, the median overall survival remains poor, around 3–5 months, with a 1-year survival of approximately 20% (94).Aldinger et al.reported a five-year survival rate of only 7.1% with a mean survival period of 6.2 months from the time of tissue diagnosis and 11.8 months from the time of onset of symptoms (95).

 

The most frequent presenting complaint in patients with ATC is a rapidly growing mass with tightness in the neck (95). Patients may also complain of dysphagia, hoarseness, dyspnea, neck pain, sore throat, and cough. Examination of the neck usually reveals a fixed, large, firm mass, which may impair the ability to detect lymphadenopathy on clinical examination. Hemorrhage and necrosis within the tumor may result in soft, fluctuant masses. Rarely, patients with massive tumor extension into the mediastinum or lungs may present with superior vena cava syndrome or dyspnea.

 

Unfortunately, most patients with ATC present with advanced stage disease. In a retrospective study of thyroid cancers in 204 elderly (age >60 years) patients by Lin et al.(45), 75% of patients diagnosed with ATC had distant metastases to the lung, bone, mediastinum, and peritoneum at presentation. Similarly, in the cohort reported by Aldinger et al., 78 of 84 (93%) patients with ATC presented with advanced stage III and stage IV disease (95). Additional patient factors associated with worse prognosis in ATC include advanced age (>60–70 years), male gender, presence of leukocytosis (>10,000), and symptoms related to tumor mass effect, such as neck pain, dysphagia, rapidly growing neck mass. Regarding older age as a poor prognostic factor, in a cohort of 516 patients with ATC, Kebebew et al.reported a 28% greater mortality in patients over 60 years of age compared to those less than 60 years determined by multivariate analysis (94).

 

ATC often, but not always, arises from pre-existing differentiated thyroid cancer, with 20% of patients with antecedent DTC and another 20-30% with concurrent DTC (co-existent on histopathology). There is also a higher incidence of ATC in patients with endemic goiter. These associations are relevant for the treatment of ATC because driver mutations such as BRAF and RAS may be retained in the anaplastic tumor cells and can be targeted with therapy (29,95).

 

Treatment of Anaplastic Thyroid Cancer

 

While the prognosis of ATC remains poor, treatment options to slow the progression of disease, palliate symptoms, and, in rare cases, attempt cure, are available as approved therapies and in clinical trials.

 

EXTERNAL RADIATION

 

External radiation to the neck region is appropriate for patients with aggressive cancers that cannot be completely resected surgically (12). Schwartzet al.reported limited success in the treatment of I131-refractory patients with extrathyroidal spread, positive surgical margins, or gross residual disease with a mean of 60 Gy (38-72 Gy); survival was less in patients with high-risk pathology, metastases, and gross residual disease (96). In the context of ATC, disease is often assumed to be radioiodine refractory, and external beam radiation may be used for preservation of vital neck structures.

 

TARGETED THERAPY AND IMMUNOTHERAPY

 

Most patients with ATC have rapidly progressive disease and should be evaluated for clinical trials when feasible as new treatments continue to be developed. Targeted therapy with inhibitors to specific gene mutations and fusions has shown some success and is the focus of numerous ongoing clinical trials. Therapies include inhibitors of RET (discussed below under medullary thyroid cancer treatment), BRAF, MEK, NTRK, and ALK. Combination treatment with BRAF inhibitor dabrafenib and MEK inhibitor trametinib was recently approved for the treatment of BRAFV600E mutated, unresectable/locally advanced ATC, following a 69% overall response rate in a phase II open label trial of 16 patients with ATC (97).

 

Immunotherapy reagents target the impaired immune responses and immune suppression that arise in cancer allowing malignant cells to grow and spread. Checkpoint inhibitors are a kind of immunotherapy that block immune regulatory pathways with the goal of increasing anti-tumor immune responses and producing tumor killing by host leukocytes. Two primary classes of immunotherapy being evaluated for advanced thyroid cancer are inhibitors of cytotoxic T lymphocyte A (CTLA)-4 (such as ipilimumab) and inhibitors of programmed cell death (PD) receptor/ligand interactions (nivolumab, pembrolizumab, atezolizumab). Currently, immune checkpoint inhibitors are being evaluated alone and in combination with targeted therapies for ATC (97).

 

MEDULLARY THYROID CANCER

 

Medullary thyroid cancer (MTC) constitutes approximately 2-5% of all thyroid malignancies, but it is responsible for up to 13.4% of all deaths from thyroid cancer (27,98). It is a well-differentiated type of tumor that arises from the parafollicular C cells of the thyroid gland, and therefore it is categorized as a neuroendocrine tumor. In 80% of patients, medullary thyroid cancer occurs sporadically, but in about 20% of patients there is a family history of medullary carcinoma. Familial MTC is inherited in an autosomal dominant pattern with nearly complete penetrance. A germline mutation in the RETproto-oncogene, which encodes a transmembrane tyrosine kinase receptor, predisposes individuals to develop hereditary MTC. In the sporadic form, the tumor occurs as a result of a mutation involving only the somatic cells. Sporadic forms of MTC are more common in older patients (mean age at presentation 47 years), while the hereditary forms of MTC are more common in younger patients (98). The prevalence of MTC is nearly equal in males and females.

 

Parafollicular cells secrete calcitonin, and in MTC this protein is greatly elevated and serum level correlates directly with the burden of disease (99). Other neuroendocrine cell products, including histamine, serotonin, prolactin, vasoactive intestinal polypeptide, and prostaglandin, can be elevated in patients with MTC and lead to systemic symptoms such as diarrhea or flushing (99). In some cases, Cushing’s syndrome may develop as a result of ectopic adrenocorticotrophic hormone (ACTH) secretion from the tumor. The typical presentation of MTC is a palpable nodule in the upper part of the thyroid lobe, and the presence of systemic symptoms is almost universally associated with distant metastases (37). In the retrospective report of 104 patients with MTC by Kebebew et al., 74% of the patients in the sporadic group presented with a thyroid mass, 16% had local symptoms (dysphagia, dyspnea, or hoarseness), and 10% had systemic symptoms (bone pain, flushing, and/or diarrhea) attributable to the cancer (98).

 

Within MTC, older age at diagnosis has been associated with a worse prognosis. Kebebew et al.followed patients with MTC for a mean time of 8.6 years and found that advanced age and stage at diagnosis were independent predictors of worse survival (98). The 5-year survival rates by stage were 100% (stage I), 90% (stage II), 86 % (stage III), and 55% (stage IV). The highest survival was seen in female patients under age 45 with MTC confined to the thyroid (98). Saad et al.similarly reported that patients younger than 40 years old at diagnosis had a significantly better survival rate in MTC (99). Scopsi et al.reported a worse prognosis in patients with sporadic MTC who had extrathyroidal tumor invasion, distant metastases, or age greater than 60 years at the time of diagnosis (100). Interestingly, a more recent study that adjusted for baseline age-related mortality in the general population found no significant association with age and prognosis in MTC (101). This raises similar questions to those posed recently for differentiated thyroid cancer as to whether age truly has an independent role in prognosis for these thyroid cancers apart from the general increase in morbidity and mortality with aging.

 

Treatment of Medullary Thyroid Cancer

 

The standard treatment for MTC is surgical resection (total thyroidectomy) with regional lymph node dissection, with routine bilateral central neck dissection and consideration of lateral neck dissection in patients with large primary tumors (>1 cm) or pre-operative imaging with involved nodes. Successful complete surgical resection is associated with improved prognosis. In patients with disease restricted to the thyroid gland and without nodal involvement, the risk of recurrence and mortality is very low, compared to those with nodal disease at presentation (102).

 

Serum calcitonin and CEA levels are trended post-operatively to monitor for residual or recurrent disease, beginning around 2-3 months after surgery. A rise in either tumor marker should prompt imaging to look for recurrent disease. Radioactive iodine is not indicated in the treatment of MTC as parafollicular cells do not express NIS or concentrate iodine. Additionally, thyroid hormone replacement is required following thyroidectomy, with TSH targeted to the normal range rather than suppression (103). TSH does not stimulate the growth of parafollicular cells.

 

In patients with progressive or metastatic disease not amenable to surgery, two tyrosine kinase inhibitors are currently approved for the treatment of MTC: vandetanib and cabozantinib. Vandetanib is an oral inhibitor that targets VEGFR, RET, and epidermal growth factor receptor (EGFR). In the international, randomized controlled phase III ZETA trial of vandetanib 300 mg per day that included over 300 patients with unresectable, locally advanced or metastatic sporadic or hereditary MTC, progression-free survival was significantly greater for patients treated with vandetanib (hazard ratio 0.46, 95% CI 0.31-0.69 versus placebo) (104). Adverse events occurred more commonly with vandetanib compared to placebo, including diarrhea, nausea, palmar-plantar erythrodysesthesia, hypertension, and headache.

 

Cabozantinib (105) is another oral tyrosine kinase inhibitor targeting MET, VEGFR2, and RET signaling pathways. The phase III international, randomized controlled EXAM trial evaluated cabozantinib versus placebo in the treatment of 330 patients with progressive, metastatic MTC, with a primary outcome of progression free survival (PFS). Median PFS was 11.2 months for cabozantinib versus 4.0 months for placebo (hazard ratio, 0.28; 95% CI, 0.19 to 0.40; P <0.001), with benefit seen across all subgroups including age, prior TKI treatment, and RET mutation status (hereditary or sporadic). Response rate was 28% for cabozantinib and 0% for placebo. Common cabozantinib-associated adverse events noted in the trial included diarrhea, palmar-plantar erythrodysesthesia, decreased weight, nausea, and fatigue.

 

Lastly, two ongoing clinical trials of oral RET inhibitors, LOXO-292 and BLU-667, have shown promise in open label phase I clinical trials, with phase II trials currently underway. LOXO-292 was evaluated in the LIBRETTO phase I trial in patients with RET-altered cancers, including RET fusion-positive MTC (29 patients) and DTC (9 patients). Interim results showed an overall response rate of 77% (95% CI 61-89%), with the MTC subset showing a 45% (95% CI 24-68%) response rate, by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria. Additionally, the treatment was well tolerated with diarrhea, constipation, dry mouth, and fatigue as common treatment-emergent side effects and rare grade 3 or 4 toxicities (106,107).

 

In the phase I ARROW trial, RET inhibitor BLU-667 was evaluated for the treatment of 69 patients with RET fusion-positive malignancies, including MTC (35 patients) and PTC (4 patients). The most recent interim analysis from 2018 showed 90% of patients had a radiographic response by RECIST 1.1 criteria, with the overall response rate somewhat lower in the MTC subset of patients (49%). Regarding adverse effects, the most common treatment-emergent events were constipation, increased aspartate aminotransferase, hypertension, anemia, neutropenia, fatigue, and headache; most adverse events were Grade 1 and only 2 of 69 patients discontinued treatment due to treatment-related toxicities (108). Given the poor prognosis of MTC, continued development of new treatment strategies is needed and management at a center experienced with this type of cancer is recommended.

 

CONCLUSION

 

In summary, thyroid nodules and cancer are common in elderly patients and demonstrate age-specific prevalence, malignancy risk, and clinical behavior. Co-morbid conditions and patient preference should inform management of these entities in the elderly, with particular attention to the risks of surgery and medication adverse effects. More research is needed to understand the mechanisms underlying the distinct clinical behavior of thyroid cancer found in older patients, including the drivers of more advanced stage at presentation, higher recurrence risk, and greater mortality.

 

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  64. Wirth L, et al. Clinical activity of LOXO-292, a highly selective RET inhibitor, in patients with RET-altered thyroid cancers [abstract]. 88thAnnual meeting of the American Thyroid Association; 2018 Oct 3-7; Washington, D.C.
  65. Hu M, et al. Clinical activity of selective RET inhibitor, BLU-667, in advanced RET-altered thyroid cancers: updated results from the phase I ARROW study [abstract]. 88thAnnual meeting of the American Thyroid Association; 2018 Oct 3-7; Washington, D.C.

Non-Functioning Pituitary Adenomas

 

ABSTRACT

 

Pituitary adenomas comprise approximately 10-20% of intracranial tumors. Non-functioning pituitary adenomas (NFPAs) are benign adenohypophyseal tumors not associated with clinical evidence of hormonal hypersecretion. NFPAs comprise different histological subtypes, classified according to their immunostaining to different adenohypophyseal hormones and transcription factors. The silent gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (POU1F1) gene lineage and null cell tumors. Patients with NFPAs usually come to medical attention as a result of “mass effects” symptoms such as headaches, visual disorders and/or cranial nerve dysfunctioncaused by lesions large enough to damage surrounding structures. Hypopituitarism, caused by the compression of the normal anterior pituitary and hyperprolactinemia, due to pituitary stalk deviation can also be present. Some cases may be diagnosed incidentally through imaging studies performed for other purposes.Patients with NFPAs should undergo hormonal, clinical, and laboratory evaluation for hyper and hypopituitarism. Assessment of prolactin and IGF-1 levels have been recommended in all patients whereas screening for cortisol excess is suggested in the presence of clinical symptoms.  Impairment of pituitary function should be assessed by baseline hormonal measurements and/or stimulatory tests, if needed.Patients in whom the tumor abuts the optic chiasm should be submitted to visual field perimetry. Surgical resection is the primary treatment for symptomatic patients with NFPAs, i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Visual deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach. Radiotherapy in the postoperative period is not consensual and is generally reserved for cases of tumors not completely resected by surgery, those cases that present progressive tumor growth during follow-up, or for patients who, at diagnosis, already have tumors with aggressive features. For these patients a therapeutic attempt using cabergoline can be made according to clinical judgment in individual cases. For microadenomas and asymptomatic patients a “watch and wait” option is reasonable. Follow up is individualized and should consider tumor size, prior treatments, and clinical symptoms.

INTRODUCTION

 

Pituitary adenomas or, as more recently proposed, pituitary neuroendocrine tumors (PitNETs) (1)are common neoplasms comprising approximately 10-20% of intracranial tumors (2). Non-functioning pituitary adenomas (NFPAs) are benign neoplasms that originate from the adenohypophyseal cells and are not associated with clinical evidence of hormonal hypersecretion (3). They comprise a large and heterogeneous group, representing a sizeable proportion (22% to 54% in different series) of all pituitary adenomas (4-7).

 

NFPAs can be further classified according to their pituitary hormone and transcription factor profile, as defined by the 2017 World Health Organization (WHO) classification for endocrine tumors (8). Tumors that express one or more anterior pituitary hormones or their transcription factors with immunohistochemistry (IHC) but do not secrete hormones at a clinically relevant level can be referred to as silent pituitary adenomas (SPAs) (9,10). As a consequence, the definition of “null cell adenoma” is now limited to an exceptionally rare primary adenohypophyseal tumor that shows immunonegativityfor all adenohypophyseal hormones and a lack of cell-type specific transcription factors (11)- Figure 1.

Figure 1 - Classification of non-functioning pituitary adenomas rely upon clinical features and histopathological data. Reprinted with permission from Drummond et al., 2018

The term “totally silent” has been proposed to be used when a patient with an NFPA presents basal and stimulated serum concentrations of the correspondent hormones within the normal range and there are no clinical signs or symptoms that can be attributed to hormone excess (12). The term “clinically silent” may be used when NFPAs secrete hormonal products that cause an elevation of the serum concentration but do not result in clinical signs or symptoms of hormonal hypersecretion (12). Some cases are referred to as “whispering” adenomas with borderline, mild, often overlooked, clinical symptoms and signs (13,14).

 

EPIDEMIOLOGY

 

The prevalence of NFPAs are variable and are often based upon autopsy or magnetic resonance imaging (MRI) series. Data from Europe, North and South America have estimated that the prevalence of clinically relevant NFPAs is 7–41.3 cases per 100,000 of population (15). This is likely an underestimate of the true prevalence, as many NFPAs go undiagnosed until they are very large and cause mass effect or are accidentally discovered.Data are discordant about gender predominance and the peak occurrence is from the fourth to the eighth decade.

 

CLINICAL PRESENTATION

 

The absence of clinical manifestations of hormonal hypersecretion usually results in significant diagnostic delay and therefore NFPAs are frequently diagnosed when they are large enough to cause mass effects to surrounding structures (3),causing symptomssuch asheadaches, visual disorders and/or cranial nerve dysfunction. Other manifestations are hyperprolactinemia due to pituitary stalk deviation and less frequently pituitary apoplexy (16,17). Additionally, some cases may be diagnosed incidentally through imaging studies performed for other purposes, the so-calledpituitary incidentaloma.

 

Neurologic Manifestations

 

VISUAL IMPAIRMENT

 

Impaired vision, caused by suprasellar extension of the adenoma that compresses the optic chiasm, is the most common neuro-ophthalmologicalsymptom (17). Different types of visual defects depend on the degree and site of nerve compression. Both eyes are usually affected, although asignificant proportion of patients may have unilateral or altitudinal problems in 33 and 16% of the cases, respectively (18). Diplopia, induced by oculomotor nerve compression resulting from parasellar expansion of the adenoma may occur, and the fourth, fifth and sixth cranial nerves may also be occasionally involved (15). Nevertheless, the typical visual field defect associated with pituitary tumors is bitemporal hemianopia, reported in approximately 40% of the patients.

 

HEADACHE

 

Headaches, the second most common neurologic symptom, occur in 19-75% of patients with pituitary tumors, regardless of size (19). In a retrospective case series of incidentally-discovered NFPAs, headache was present in approximately 20% of the cases (20). Although it is not always clear whether the presenting headache is related to the tumor, proposed mechanisms for headache include increased intrasellar pressure, stretching of dural membrane pain receptors, and activation of trigeminal pain pathways (15,21). Cerebrospinal fluid (CSF) rhinorrhea, associated or not with headache, can occur if the tumor causes erosion of the sellar floor and extends inferiorly to the sphenoid sinus.

 

PITUITARY APOPLEXY

 

Pituitary apoplexy (sudden hemorrhage into a pituitary macroadenoma) is rare. It causes acute onset of a severe headache associated with visual disturbances and can occur in all types of pituitary tumors. In a retrospective case series of 485 patients with NFPAs, pituitary apoplexy was the initial presentation in 8% of the cases (22). It has been suggested that the combination of the high metabolism of pituitary adenomas combined with their special blood supply would make them more prone to vascular events (23). Pituitary apoplexy may occur without an identified risk factor, but it has also been reported as beingrelated to pregnancy, use of anticoagulants, surgical procedures, as well as in association with dynamic tests, such as thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and insulin-tolerance stimulation tests (24).

 

The actual long-term risk of apoplexy in NFPAs has not been clearly defined (25). In a Japanese prospective cohort study of 42 asymptomatic patients with NFPAs (mean initial tumor size of 18.3 mm) followed-up for approximately 4 years, pituitary apoplexy was reported in 9.5% of the cases (26).A more recent systematic review and meta-analysis evaluating the outcomes of patients with NFPAs and pituitary incidentalomas who were treated conservatively and followed-up for mean period of 3.9 years showed that the development of pituitary apoplexy was rare (0.2/100 patients-year), although a trend for a greater incidence of pituitary apoplexy was seen in macroadenomas compared with microadenomas, with a reported incidence of apoplexy of 1.1% per year (27).

 

Endocrine Manifestations

 

HORMOMNAL DEFICIENCIES

 

Most patients with macroadenomas present with deficiency of at least one pituitary hormone resulting from the compression of the normal anterior pituitary and/or pituitary stalk, preventing the stimulation of pituitary cells by hypothalamic factors. Hypogonadism can result from either a compressive effect on gonadotropic cells or by hyperprolactinemia. This “disconnection hyperprolactinemia” or non-tumoral hyperprolactinemia, usually <2000mIU/L (95ng/mL) (28), is characterized by compression of the pituitary stalk, which prevents the arrival of dopamine to the anterior pituitary, the main inhibitor of prolactin (stalk effect). GH axis is the most commonly affected of all the pituitary axes,followed by hypogonadism and adrenal insufficiency (15).

 

HORMONAL EXCESS

 

Gonadotroph adenomas are usually considered to be "nonfunctioning" although they can secrete intact gonadotropins, as they do not generally result in a clinical syndrome. Occasionally, gonadotroph adenomas secrete primarily FSH but also LH in quantities high enough to raise serum gonadotropin levels, which in turn, may lead to the development of some specific symptoms, such as ovarian hyperstimulation in young women (29)or, more rarely, precocious puberty or testicular enlargement in men. In addition, low serum LH:FSH ratios (usually < 1.0) have been described in clinically-secreting gonadotroph adenomas (30). Measurement of α-subunit may also contribute to a pre-operative diagnosis in clinically silent but biochemically-secreting NFPAs, as it may be the sole biochemical marker of the gonadotroph subtype in a number of cases. In addition, circulating FSH, LH and α-subunit levels can help the post-operative surveillanceof these patients (30).

 

HISTOPATHOLOGICAL CLASSIFICATION

 

The 2017 WHO classification for endocrine tumors (8)now defines pituitary adenomas, including NFPAs, according to their pituitary hormone and transcription factor profile.It is noteworthy that in this classification system,  IHC forms the basis of the new categorization, in which an adenohypophyseal cell lineage designation of the pituitary adenoma has replaced the concept of a “hormone-producing pituitary adenoma” (31)-Table 1.

 

Table 1 – Classification of Silent Pituitary Adenomas According to Adenohypophyseal Hormones and Transcription Factors.
Cell Lineage Hormone Staining Transcription Factors and Other Co-Factors

Somatotroph adenoma

Sparsely granulated

Densely granulated

GH, α-subunit

Diffuse and strong

Weak and patchy

PIT-1

Lactotroph adenomas

Sparsely granulated

Densely granulated

Acidophilic stem cell adenoma

PRL

Perinuclear

Diffuse PRL

Focal and variable PRL, GH

PIT-1, ERα
Thyrotroph adenomas TSHβ, α-subunit PIT-1, GATA2

Corticotroph adenomas

Densely granulated (type I)

Sparsely granulated (type II)

Crooke-cell

ACTH

Diffuse and strong ACTH

Weak and patchy ACTH

Periphery

TPIT
Gonadotroph adenomas FSHβ, LHβ, α-subunit SF1, GATA2, ERα
Null cell adenomas None None
PIT-1-positive adenomas (plurihormonal) GH, PRL, TSHβ±, α-subunit PIT-1

Adapted from Mete et al, 2017.

 

Indeed, the prevalence of the different histological subtypes of NFPAs is dependent on the extent of the IHC profile. According to a recent large retrospectivecaseseries (10), the gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (GH/ Prolactin/TSH) lineage and null cell - Figure 2.

Figure 2 - Prevalence of silent pituitary adenoma subtypes according to immunochemistry for anterior pituitary hormones and transcription factors. Adapted from Nishioka et al., 2015

Subtypes of NFPAs

 

NULL CELL ADENOMAS AND SILENT GONADOTROPH ADENOMAS

 

Null cell adenomas and silent gonadotroph adenomas (SGAs) were previously misunderstoodto bethe same type of tumor. The addition of immunostainingfor steroidogenic factor 1 (SF1) as a tool in the diagnosis of pituitary lesions has shown that many LH/FSH immunonegative adenomas are in fact SGAs (32), which comprise almost 80% of  resected NFPAs. The distinction between SGAs and null cell adenomas is of clinical relevance because true null celladenomas are likely to be more invasive and aggressive than SGAs (33). Data from a retrospective caseseries of 516 patients with NFPAs have shown that from the 23% of the tumors initially classified as null cell adenomas by using only classical pituitary hormone IHC, only 5%of them remained as true null cell type. Indeed, immunostaining using lineage-specific markers, namely, PIT-1 (coded by the POU class 1 homeobox 1 (POU1F1) gene), SF1, TPIT (coded by the T-Box transcription factor 19 (TBX19) gene)and estrogen receptor-α (ERα) provided tumor reclassification in 95% of cases (10). In patients with SGAs, ERα seems to be a prognostic factor for re-intervention in males - the combination of the absence of ERα expression and young age served as good predictive markers of aggressiveness (34).

 

SILENT CORTICOTROPH ADENOMAS

 

Silent corticotroph adenomas (SCAs) are characterized by the absence of clinical features of Cushing syndrome, along with normal circadian cortisol secretion (totally silent) or elevated ACTH (clinically silent) (5,35-37). They currently account for approximately 15% of NFPAs, an underestimated proportion, since IHC for the transcription factor TPIT, a marker of corticotroph differentiation which regulates the proopiomelanocortin (POMC) lineage giving origin to the corticotrophs, is not widely available(38).

 

SCAs often present themselves as macroadenomas associated with mass-related symptoms. In comparison with SGAs, SCAs show female preponderance, are more frequently giant adenomas, and are more often associated with marked cavernous sinus invasion (37). Importantly, the presence of multiple microcysts in T2-weighted pituitary MRI sequences in a NFPA has a high specificity for the corticotroph subtype (39).

Histologically, SCAs can be further divided into type I (densely granulated) and type 2 (sparsely granulated). Type 1 SCAs show strong ACTH immunoreactivity whereas type 2 SCAs resemble the rare chromophobe corticotroph adenoma and show weak and focal ACTH immunoreactivity.Type 2 SCAs seem to be more common that the type 1 SCA and are likely to display higher expression of migration and proliferation factors compared with type 1 SCAs (5,10,40). Since SCAs are nonfunctional, an important point to note is the absence of Crooke’s hyalinization, as there is no exposure to high circulating glucocorticoid levels to promote hyaline deposits of cytokeratin filaments in the cytoplasm of normal corticotrophs (37).

 

The transformation of a silent corticotroph tumor into Cushing disease has been described, although the mechanism involved in this phenomenon is not yet well understood (41). An attractive and currently most likely hypothesis is that the clinical manifestations of Cushing disease are dependent on the processing of the pro-hormone POMC in corticotrophs. The pro-hormone convertase 1/3 (PC1/3) is involved in the post-translational processing of POMC into mature and biologically active ACTH. SCAs show a decrease in PC1/3 expression associated with a down-regulation of PC1/3 genes compared with corticotroph adenomas associated with Cushing disease (35).

 

SILENT SOMATOTROPH ADENOMAS

 

Silent somatotroph adenomas are PIT1 and GH-immunoreactive tumors without clinical and biological signs of acromegaly. They represent approximately 2% of all pituitary adenomas in surgical case series (42). Patients with silent somatotroph adenomas usually present with normal pre-operative GH and IGF-1 levels but there have been few reports of "clinically silent" cases, with non-suppressible serum GH and elevated IGF-1 levels (43).

 

Similar to secreting somatotroph adenomas, silent somatotroph adenomas are classified into densely granulated and sparsely granulated types, based on the presence and pattern of the low molecular weight cytokeratin staining. Unlike clinically functioning somatotroph tumors, the silent ones are more frequently sparsely granulated with more aggressive behavior and a lower response to somatostatin analog therapy (44). Furthermore, silent somatotroph adenomas are more frequent in females, present at a younger age, are larger, more invasive, and recur earlier and more frequently than their secreting counterparts (45).

 

SILENT THYROTROPH ADENOMAS

 

Silent thyrotroph adenomas usually present TSHβ, α-subunit, and PIT-1 expression on IHC in a variable manner (31). They are slightly more frequent when compared with their functioning counterparts and seem to behave similarly regarding treatment outcomes and recurrence rates (46).

 

SILENT LACTOTROPH ADENOMAS

 

Clinically silent lactotroph adenomas are rare. The positive prolactin staining by IHC with no clinical signs of hyperprolactinemia is usually encountered concomitantly with GH positive staining (silent mixed somatotroph-lactotroph adenoma) (44). They can also express ERα on immunostaining (8).

 

PIT-1 POSITIVE PLURIHORMONAL ADENOMAS

 

These monomorphous PIT-1 positive adenomas, usually negative for pituitary hormones in the serum, were previously called silent adenomas type 3 (8,31). PIT1-positive plurihormonal adenomas are a distinct entity, with reportedly aggressive behavior. A single-center retrospective case series reported a prevalence of 0.9% for ‘silent adenomas type 3’ among resected pituitary tumors over a period of 13 years. All tumors were macroadenomas, aggressive, invasive, with a high rate of persistent/recurrent disease (47). Interestingly, these tumors may present clinical symptoms of hormone excess, such as acromegaly, hyperthyroidism or marked hyperprolactinemia (48).

 

EVALUATION

 

According to current practice guidelines on incidentally discovered sellar masses, all patients, including those without symptoms, should undergo hormonal, clinical, and laboratory evaluation for hyper and hypopituitarism (49). The extent of the evaluation is still debatable and has commonly relied on clinical experience - assessment of prolactin and IGF-1 levels have been recommended, whereas screening for cortisol excess (i.e. overnight 1mg dexamethasone and/or late-evening salivary cortisol and/or urinary-free cortisol) is suggested in the presence of clinical symptoms. Measurement of ACTH levels is not routinely recommended although some experts propose this assessment as it could perhaps aid in the pre-operative identification of SCAs (49).

 

Patients with macroadenoma, especially those > 3 cm and with normal or slightly elevated prolactin, must have their serum prolactin diluted in order to exclude the "hook effect" (50,51). This effect occurs when excessively high levels of this hormone interfere with the formation of the complex antibody-antigen-antibody sandwich, and a prolactinoma might be mistaken for aNFPA.

 

It is also mandatory to check for pituitary hormone deficiencies in patients with non-functioning tumors, irrespective of symptoms. Macroadenomas can cause impairment of pituitary function by the involvement of the normal gland or pituitary stalk compression, and the risk of hypopituitarism is directly related to tumor volume. Microadenomas between 6 and 9mm can eventually lead to pituitary dysfunction(49). The assessment of pituitary function includes baseline hormonal measurements and eventually stimulatory tests. Current guidelines suggest that serum cortisol levels at 8-9 AM should be done as the first-line test for diagnosing central adrenal insufficiency (52). Despite well recognized limitations of most commercial cortisol immunoassays (53), baseline cortisol levels <3 µg/dL (83 nmol/L) are suggestive of central adrenal insufficiency, while baseline cortisol levels >15 µg/dL (414 nmol/L) likely exclude it, and levels between 3 and 15 µg/dL require a corticotropin/insulin stimulation test. Central hypothyroidism is assessed by measuring serum TSH and free T4 (fT4), and fT4 level below the laboratory reference range in conjunction with a low, normal, or mildly elevated TSH in the setting of pituitary disease usually confirms the diagnosis (52). Diagnosing GH deficiency (GHD) is relatively straightforward in patients with IGF1 concentration lower than the gender- and age-specific lower limit of normal and organic pituitary disease. Low IGF1 in patients with non-functioning tumors who have at least three pituitary hormone deficiencies is also suggestive of GHD (25).Nevertheless, some adults with suspected GHD may have normal IGF-1, in such cases GH stimulation testing may be useful. Central hypogonadism in males, manifests with low serum testosterone, low or inappropriately normal FSH/LH and features of testosterone deficiency. For females, pre-menopausal women with oligomenorrhea or amenorrhea should be screened with serum E2, FSH, and LH. In postmenopausal women, if the patient is not in hormonal therapy, the absence of high serum FSH and LH is sufficient for a diagnosis of central hypogonadism (52).

 

Sellar MRI with gadoliniumis the best imaging study for suspected adenomas not only because it provides images of high resolution of the mass but also its relation with surrounding structures. Pituitary adenomas usually appear hypo or isointense compared to normal pituitary tissue in T1 images on MRI. In addition, while the normal pituitary tissue enhances earlier with contrast, pituitary adenomas commonly present with low contrast uptake (54). Patients in whom the tumor abuts the optic chiasm should have visual field perimetry, preferably by the Goldmann method, to assess for visual deficits (49).

The pre-operative differential diagnosis of NFPAs includes several additional primary non-hormonal-secreting lesions (55). This distinction is challenging in many cases because the clinical presentations and radiological aspects may be similarbetween adenomas and other pituitary lesions. The presence of diabetes insipidus (DI) is common in tumors of non-pituitary origin and indicates that the sellar mass is most likely not a pituitary adenoma (55). An increase in αsubunit of the glycoprotein hormones (elevated in 30% of NFPA patients) can help in the differential diagnosis although normal values do not rule them out (56).

 

TREATMENT

 

In spite of current knowledge of definite NFPAs pathologic subtypes, initial treatment strategies are similar regardless of the subset. A small number of studies have reported treatment outcome results taking into consideration adenohypophyseal hormone immuno-profile (57-59)and, for the future, precise pathologic characterization utilizing adenohypophyseal hormones and transcription factors may potentially aid in predicting the response to specific adjunctive therapies.

 

Surgery

 

Surgical resection is the primary treatment for symptomatic patients with NFPAs (60), i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Surgery is also urgently indicated for patients with apoplexy who develop neuro-ophthalmologic complaints. In some experts’ opinion, tumors larger than 2cm should also be considered for surgery due to their propensity for growth (61).Mortality rate is low and reported surgical complication rates are acceptable (62)but accomplishment of total or near-total resection can be challenging and varies in different series, ranging from 20% to 80% (63,64). Visual deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach (62,65).

 

Pituitary function should be reassessed 1-3 months after surgery and treatment of hypopituitarism introduced according to hormone deficiencies. Whether or not GH deficiency should be replaced requires a thoughtful and individualized evaluation of risks and benefits (66,67). Current data supports the absence of any stimulation of remnant or induction of recurrence by growth hormone replacement in patients with NFPAs solelytreatedby surgical removal (68).In a retrospective series, tumorregrowth occurred in 38/107 (36%) of non-GH treated subjects and in 8/23 (35%) of GH-treated subjects, followed up for a mean period of 6.8 years. The Cox regression analysis showed that after adjusting for sex, age at tumor diagnosis, cavernous sinus invasion at diagnosis, and type of tumor removal, GH treatment was not a significant independent predictor of recurrence.

 

A sellar MRI should be obtained three to six months after surgery to assess the extent of tumor resection. Also, as asignificant number of patients with NFPAs may develop tumor re-growth, long-term imaging surveillance is recommended. In a retrospective analysis of 155 patients with NFPAs treated solely by surgery and followed-up for a mean period of 6 years (twenty-nine were followed up for more than 10 years), re-growth was reported in 34.8% of the cases, with 20% of relapse/re-growth occurring after 10 years (63). The risk of re-growth was associated with a pituitary tumor remnant on the first post-operative scan and with younger age at initial surgery. Likewise, in patients with NFPAs who present with classical apoplexy, tumor re-growth rate is not negligible. In a retrospective series of thirty-two patients with NFPAs who underwent surgery for pituitary apoplexy, tumor re-growth was reported in 11% of the cases at just over 5 years (69). Five patients who had received adjuvant radiotherapy presented no recurrences and were excluded from the analysis. In the remaining 27 cases, there were 3 recurrences - all of them had residual tumor on the post-operative scan. Therefore, it is advisable that patients with NFPAs, particularly those with post-operative tumor remnants, be closely monitored following surgery, and follow-up surveillance needs to certainly be continued for more than 10 years.

 

Radiation Therapy

 

Radiation therapy (RT) has been shown to be effective as an adjunct to surgical resection in cases of post-operative residual tumor or recurrence. However, it carries a major long-term risk of hypopituitarism (60,70,71). Stereotactic techniques such as stereotactic radiosurgery or fractionated stereotactic radiotherapy have been developed with the purpose of delivering more localized irradiation and reducing long-term side-effects. Both techniques provide excellent tumor control in patients with NFPAs, ranging from 85% to 95% at 5 to 10 years (72). However, at the present time, there is no consensus concerning the systematic use of RT in the postoperative period for patients with incompletely resected NFPAs (25)and whether an earlier approach would be preferable over conservative management. In general, RT is reserved for cases of tumors not completely resected by surgery and for those cases that present progressive tumor growth during follow-up (60). Adjuvant RT may also be considered for patients who, at diagnosis, already present aggressive tumors, such as those invading parasellar structures or with extensive positive immunostaining for Ki-67, a proliferative index significantly associated with recurrence in NFPAs (8,73). Furthermore, NFPAsubtype may be a relevant factor in the expected response to radiotherapy. A recent retrospective multicenter study has demonstrated that overall tumor control rate after radiosurgery was lower in SCAs compared to other NFPA subtypes (74). Therefore, it has been suggested that in SCAs an elevated margin dose may be considered in order to achieve a better chance of tumor control.

 

Asymptomatic Tumors

Medical Therapy

 

Current evidence does not support primary treatment of NFPAs with medical therapy (60). Small series have demonstrated significant tumor volume stabilization under medical treatment in non-operated patients with NFPAs due to contraindications or refusal (77), whereas other studies have revealed tumor volume progression in the long term (78). The main medical agents that have been evaluated in NFPAs are dopamine agonists (DA) and somatostatin analogues (SAs). Figure 3 shows a suggested algorithm for the management of patients with NFPAs.

Figure 3 - Suggested algorithm for the management of patients with NFPAs. CBA: cabergoline; NFPA: nonfunctioning pituitary adenoma, MRI: magnetic resonance imaging; RT: radiotherapy

DOPAMINE AGONISTS

 

Dopamine receptor type 2 (D2R) expression has been demonstrated in patients with NFPAs.  In a small series of 18 patients with immuno-negative ACTH, GH, PRL and TSH NFPAs, two thirds of them (12/18) expressed D2R by real-time polymerase chain reaction. Patients who presented residual tumor (9/18) were treated with cabergoline up to 3mg per week. After 12 months of treatment, tumor shrinkage was observed in 56% of the patients and tumor reduction was significantly greater in those expressing D2R (79). A historical cohort analysis on the adjunctive role of DA in adult patients with GH and ACTH negative NFPAs was recently published (58). The treatment group consisted of patients who were either initiated on DA upon post-surgical residual tumor detection or when tumor growth was subsequently detected on follow-up, while the control group received no medication after surgical treatment. Tumor control was significantly superior in patients who were treated upon detection of post-operative residual tumor, compared to those who were treated after presenting tumor progression or the control group, 87% x 58% x 47%, respectively. The requirement for additional treatment (surgery and/or radiotherapy) during follow-up was significantly decreased from 47% to 16% with adjunctive DA therapy. In this series, there were no correlations between clinical response to DA and D2R expression, the isoform type or their expression levels.

 

Randomized controlled trials assessing the effects of cabergoline on certain NFPAs subtypes are in progress to evaluate the effects of cabergoline on the change in tumor volume, both as primary treatment and as an adjuvant treatment for post-surgical residual or progressive disease. Meanwhile, although cabergoline has not been a consensual treatment for patients with NFPAs, a therapeutic attempt can be made according to clinical judgment in individual cases with postoperative residual tumor, especially those with extrasellar extension, a potential regrowth predictor. Cabergoline doses are variable, but usually started at 0.5 mg weekly, increasing 0.5 mg each week until a maximum dose of 3.0 mg/week is reached. For these patients, tumor shrinkage is not the major target although it can occur in 38% of them; otherwise, prevention of tumor growth is the main treatment goal. Tumor progression can be assessed by a sellar MRI every 6 months for the first 2 years, and annually thereafter. Once stability is achieved without significant side effects, the drug can be maintained indefinitely, taking into account individual cost-benefits (57,80).

 

SOMATOSTATIN ANALOGUES

 

The finding of somatostatin receptors (SSTRs) expression by NFPAs has raised the possibility that the use of SAs could be an effective treatment strategy (81,82). The SA octreotide which binds with high affinity to SSTR2 was not effective in controlling tumor size nor improving visual field in a small group of patients with NFPAs (83). More recently, a case-control study evaluated the results of long-acting octreotide in patients harboring post-surgical NFPA residues and it demonstrated tumor remnant stabilization in 81% (21/26) of patients in the treated group compared to 47% (6/13) of patients in the control group after a mean follow-up of 37 months (84). However, neither visual field nor pituitary function changed in any of the groups. This cohort of 39 NFPAs consisted of a heterogeneous group as IHC revealed positivity for pituitary hormones in 28 cases (20 of those showing positivity for FSH and/or LH) whereas the remaining 11 cases were negative for adenohypophyseal hormones. SSTR5 was the predominant SSTR expressed (84%), followed by SSTR3 (61%), while SSTR2 was expressed in 46% of the cases.

 

The expression of SSTRs and zinc finger protein 1 (ZAC1), a protein regulating apoptosis and cell cycle arrest, were assessed in a group of NFPAs (SGAs and hormone-negative adenomas), active somatotroph adenomas, and normal pituitary. SSTR2 and ZAC1 expression was reduced whereas SSTR3 expression was increased in SGAs compared to active somatotroph adenomas and normal pituitary (85). Likewise, other studies have suggested that SSTR3 is the predominant SSTR expressed in most NFPAs, both by IHC studies (34,82)and mRNA levels (34,86). However, a few other studies have demonstrated higher SSTR2 expression than SSTR3 or SSTR5 expression in SGAs and hormone-negative adenomas (73,87).

 

In a recent review of silent somatotroph adenomas, SSTR2 and SSTR5 expression was recently demonstrated in all the cases, although SSTR2 was significantly less expressed compared to its secreting counterpart, and SSTR5 expression was similar in both groups (42). Clinical data regarding the therapeutic effect of SA on silent somatotroph adenomas are not currently available. Immunoreactivity for SSTR2 and SSTR5 has been shown to be positive in 89% and 78% of silent thyrotroph adenomas, respectively, which was not significantly different from those hormonally-active (88).

 

Pasireotide is a universal SA with action on SSTR1, SSTR2, SSTR3 and STR5 subtypes and therefore seems more attractive than octreotide as an alternative medical treatment for NFPAs. There are several ongoing phase 2 randomized controlled trials evaluating pasireotide LAR for the treatment of NFPAs, but due to the low efficacy and the extremely variable effects of therapy with commercially available SAs, these drugs are not currently recommended for the treatment of patients with NFPAs (25).

 

TEMOZOLOMIDE

 

Temozolomide was the first alkylating chemotherapeutic drug to show significant response rates in aggressive pituitary tumors (89). Responsiveness to temozolomide is probably dependent on the immuno-expression of O (6)-methylguanine DNA methyltransferase (MGMT), a DNA repair protein that acts by removing the alkyl group and therefore is associated with temozolomide resistance(90). Low immuno-expression of MGMT by pituitary tumors has been associated with higher response rates to temozolomide (89).

MGMT immuno-expression was assessed in a group of 45 NFPAs and the degree of expression was correlated with tumor aggressiveness (90). Low MGMT expression was observed in 50% of the aggressive NFPAs compared to 24% of the non-aggressive NFPAs. These findings suggest that aggressive NFPAs with low MGMT expression could be potential candidates for treatment with temozolomide. There have been few case reports concerning the use of temozolomide in patients who presented aggressive NFPAs or who developed pituitary carcinomas several years after the diagnosis of NFPAs (91,92). temozolomide has then been suggested as an alternative treatment for patients with aggressive NFPAs presenting tumor progression despite radiotherapy and other therapeutic measures, and for exceptional cases of pituitary carcinomas (25).

 

GnRH ANALOGUES

 

GnRH agonists and antagonists have been tested as treatment strategies for NFPAs with positivity for gonadotropins, with the expectancy that the saturation of these receptors would reduce hormone production. However, the use of GnRH agonist analogues had no effect or even exacerbated hormone hypersecretion with no change in tumor dimensions. It is therefore concluded that GnRH analogues should not be used for treating these tumors (93,94).

 

QUALITY OF LIFE AND LONG-TERM MORBIDITY AND MORTALITY

 

A substantial number of patients with NFPAs suffer from morbidities related to the tumor itself, as well as to the treatments offered. Standardized mortality ratios in these patients seem to be higher than that of the general population with deaths associated mainly with circulatory, respiratory, and infectious causes (95). Until now, there was no consensus on predictive factors of mortality but those most consistently described are older age at diagnosis (96,97)and high doses of glucocorticoid substitution therapy (98). Arecent retrospective series of 546 patients operated on for a macro NFPA between 1963 and 2011 and followed up for a median period of 8 years reported a standardized mortality ratio of 3.6 (95% CI, 2.9–4.5) (96). After adjustment for factors proven to be significant in univariate analysis - radiotherapy, tumor regrowth, and untreated growth hormone deficiency - age at diagnosis was an independent predictor of mortality, with shorter survival observed in older patients.

 

Following NFPA treatment, patient-reported health-related quality of life (HR-QoL) substantially improves, nevertheless, there are conflicting findings about HR-QoL normalization, which may also be related to the lack of a disease-specific HR-QoL questionnaire for NFPAs (99). Some studies have described a persistent decreased HR-QoL compared to healthy controls and reference data (100,101), while others have not (102). In the latest study, HR-QoL was evaluated in 193 consecutive patientswith NFPAs followed up in a tertiary endocrine referral center (102)The overall health-related quality of life and perception of subjective health in patientswith NFPAs was not compromised, however specific groups, such as females, patients with tumor recurrences, and with visual defects, were shown to be affected in various dimensions, necessitating measures to compensate for predisposing factors.

 

More recently, the use of the Wilson–Cleary model, a conceptual biopsychosocial model of HR-QoL, suggested that elements at each stage of this model could be contributing to the impairment in HR-QoL observed in patients with a NFPA (103).The authors concluded that currently available biomedical treatments, i.e., surgery, radiotherapy, and hormone replacement therapy, are clearly not sufficient for achievement of good HR-QoL in patients with a NFPA, and further improvement should be supported by a pituitary specific care trajectory, targeting not only biological and physiological variables, but also psychosocial care.

 

PROGNOSTIC FACTORS

 

There has been a search to identify reliable factors related to aggressiveness and the risk of recurrence in NFPAs. A single-center retrospective study evaluated 108 surgically-resected NFPAs followed for up to 15 years (104). Twenty two percent of the patients required further treatment, either second surgery or radiotherapy. Factors determining recurrence were the presence of residual tumor, tumor growth rate (>80 mm3/y), and suprasellar extension (104). Accordingto another retrospective case series evaluating patients with NFPAs who presented tumor regrowth after primary treatment, the NFPA subtype, categorized by anterior pituitary hormone immunostaining, was not a predictive factor for the requirement of secondary treatment or tumor regrowth.Significant risk factors were female gender and treatment approach; secondary progression was significantly higher in those patients who were followed conservatively (63%) as compared to those who received surgery (36%), radiotherapy (13%), and surgery/adjuvant radiotherapy (13%) (59).

 

The role of cellular markers, associated with cell proliferation and apoptosis, in predicting the recurrence of NFPAs has also been investigated (105). Proliferative indexes such as a high Ki-67 index, assessed by IHC, were significantly associated with a tumor size greater than 3 cm, as well as with tumor recurrence (73). Evaluation of tumor proliferation by using Ki-67 IHC is widely available and ishighly recommended as part of the assessment of NFPAs (31).

 

CONCLUSION

 

NFPAs are frequent in endocrine practice. Clinically they range from being completely asymptomatic (incidental findings on head MRI or computed tomography scans) to causing significant hypothalamic/pituitary dysfunction and visual symptoms due to mass effect. For microadenomas and asymptomatic/relatively small macroadenomas (1–2 cm), a “watch and wait” option is reasonable. If tumor growth, development of visual field defects, or progressive pituitary dysfunction is detected during follow up, then surgery is indicated. Radiotherapy is an effective treatment, although usually reserved for those patients with aggressive tumors and significant tumor remnant after pituitary surgery as considerable side effects may occur. Medical treatment with dopamine agonists stands as an alternative, despite the lack of placebo-controlled trials. As with other PitNETs, it is recommended that NFPAs be classified according to their pituitary hormone and transcription factor profiles along with proliferation markers such as Ki-67. This refined stratification may potentially aid in predicting disease course and response to adjunctive therapies and eventually result in a better personalized therapeutic strategy.

 

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Aggressive Pituitary Tumors and Pituitary Carcinomas

ABSTRACT

 

Aggressive pituitarytumors(APT) refer to pituitary adenomas exhibiting rapid growth, resistance to conventional treatments and/or early/multiple recurrences, with abandonment of the previous term ‘atypical pituitary adenoma’. Pituitary carcinomas (PC) are defined by non-contiguous craniospinal or distant metastasis. Whilst PC is exceedingly rare, comprising only 0.1-0.2% of all pituitary neoplasms, APT may account for up to 15% of all pituitary neoplasms, depending on the definition used. Typically evolving from known pituitary macroadenomas, APT/PC is most commonly diagnosed in the fifth decade of life with corticotrophand lactotroph neoplasms predominating. Diagnosis relies on MRI, hormonal studies and histological assessment including proliferative markers and immunohistochemistry for pituitary hormones and, most recently, transcription factors. Structural and molecular mechanisms have been proposed in the pathogenesis of APT/PC, although there appears to be no contribution from known familial pituitary tumorsyndrome genes such asMEN1. Treatment is multimodal, ideally delivered by an expert team with a high-volume caseload. Surgical resection may be performed with the aim of either gross total resection ortumordebulking. Radiotherapy may be administered either as fractionated external beam radiation or stereotactic radiosurgery. Standard pituitary medical therapies such as somatostatin analogues have limited efficacy in APT/PC, whereas temozolomide yields a clear survival benefit. Evidence is emerging for the use of peptide receptor radionuclide therapy, tyrosine kinase inhibitors, VEGF inhibitors, and immunotherapy. Avenues for further research in APT/PC include molecular biomarkers, nuclear imaging, establishment of an international register, and routine pituitary tumor biobanking.

 

INTRODUCTION AND DEFINITIONS

 

Pituitary adenomas (PA) are benign, typically slow-growing neoplasms originating from cells of the adenohypophysis (1). Aggressive pituitary tumors (APT) refer to PAs demonstrating rapid growth, resistance to conventional treatments and/or early/multiple recurrences (2,3).In the absence of reliable pathological predictors of tumor behavior (2), APTs lack specific diagnostic criteria and are instead best considered a clinical composite of various pituitary neoplasms exhibiting clinically aggressive behavior. Efforts should be made to be as objective as possible in diagnosing APTs (2). Postoperative recurrences should only be considered to be APTs when surgery was performed by an expert neurosurgeon with a high-volume caseload (2). Whether resection was intentionally limited because of reduced surgical fitness may also need to be considered. Intrinsic tumor resistance to medical therapy should be distinguished from inadequate dosing or poor compliance as well as drug toxicity, which is increasingly recognised in the setting of dopamine agonist (DA) treated prolactinomas (4). Progression or recurrence after radiotherapy is more compelling than resistance to only surgical and medical therapy (5-7).

 

APT is distinct from the term ‘atypical pituitary adenoma’, which was defined by the earlier 2004 World Health Organization (WHO) classification of endocrine tumors as PAs with a Ki-67 labelling index >3%, an elevated mitotic index, and extensive p53 nuclear immunostaining (8). The Ki-67 labelling index is assessed by MIB-1 immunohistochemistry (IHC). As Ki-67 is a nuclear protein with suggested roles in ribosomal RNA transcription and chromosome separation (9), MIB-1 stains cells in the S (DNA synthesis) phase of the cell cycle and thereby represents the rate of cellular proliferation (1). Immunopositivity for p53 reflects nuclear accumulation of p53 (9). In other tumors, p53 immunostaining is related to TP53mutations that prolong the half-life of p53 and result in nuclear accumulation, although TP53mutations are very rare in p53-immunopositive pituitary neoplasms (10). A threshold of >2 mitoses per 10 HPF was also included in the 2004 WHO classification as this portends a greater risk of recurrence (2). The intent of the 2004 WHO framework was to identify more aggressive tumors warranting more intensive management and follow-up. However, the term ‘atypical pituitary adenoma’ was omitted in the 2017 WHO classification of endocrine tumors as these criteria have not been clinically validated (2,3,11). Although some data show correlations between the 2004 WHO criteria and tumor behavior, the criteria do not consistently and independently predict tumor behavior in individual patients, with one study showing no difference in recurrence risk and disease-free survival in atypical PAs versus other PAs (12).

 

Although most APTs are invasive (13,14), invasiveness alone is insufficient to define APTs (2), partly because invasion is often subjective with variability between radiological, operative, and histological assessments (3). Moreover, highly invasive prolactinomas may respond well to DA therapy rather than following an aggressive clinical course. However, invasiveness is still considered a key component of aggressiveness. Compared to non-invasive, non-proliferative PA (Grade 1a), the relative risk of persistent disease is 8.0 for invasive but non-proliferative PA (Grade 2a) versus 3.1 for non-invasive but proliferative (at least two of: Ki-67 ≥3%, p53 staining with >10 strongly positive nuclei/10 HPF, mitotic count >2/10 HPF) PA (Grade 1b) (14). The impact of invasiveness and proliferation on tumor aggression is synergistic. Invasive and proliferative PAs (Grade 2b) carry a 25-fold higher risk of persistent disease and a 12-fold higher risk of tumor progression compared to non-invasive, non-proliferative PA(14). Progression-free survival is also more influenced by the effect of invasiveness than that of proliferation (15), with a relatively greater prognostic effect from invasiveness in lactotroph and corticotroph PAs than other subtypes(14).

 

At the extreme end of the spectrum, pituitary carcinoma (PC) is defined by non-contiguous craniospinalor distant metastasis. Apart from histological confirmation of the pituitary origin of suspected metastases, PC is a clinical diagnosis with no defining histopathology and remains as a distinct category in the 2017 WHO classification(8).

 

EPIDEMIOLOGY

 

Because of the lack of definitive diagnostic criteria, the prevalence of APT is unclear (2). A study incorporating radiological and histological assessments of aggressiveness found ‘grade 2b’ (invasive and proliferative) tumors in 15% of patients, although this was not a consecutive series with many patients excluded due to insufficient data and others selected to balance patients with and without persistent disease (14). Aggressiveness was also not invariable in grade 2b tumors (16). Tumor recurrence and persistence, which are generally representative of APT, are more frequently seen in younger rather than older adults (14,17,18). PAs are overall uncommon in children but tend to be more aggressive in the pediatric setting, with 26% of prolactinomas demonstrating DA resistance (19). Some (17)but not all data (15,18)show greater risks of recurrence and progression with larger PAs. As highlighted in Table 1, APT/PC development is more likely in certain tumor subtypes, namely, silent corticotroph PA, Crooke’s cell PA, plurihormonal PIT-1 positive PA (formerly ‘silent subtype 3 PA’), sparsely-granulated somatotroph PA, and lactotroph macroadenomas in men (3,20-24).

 

Table 1. PA Subtypes with Greater Tendency for APT/PC Development (3,11)
PA subtype Cell lineage Transcription factor Hormone Cytokeratin pattern Prevalence of APT/PC
Crooke’s cell PA Corticotroph T-PIT ACTH Ring-like (perinuclear hyaline bodies) Recurrence in 60%, multiple recurrence in 24%, APT/PC-related mortality in 12% (22)
Silent corticotroph PA Corticotroph T-PIT ACTH Diffuse Multiple recurrences in 57% of recurrent silent corticotroph PA vs. 3% in other non-functioning PA (P=0.001) (21)
Plurihormonal PIT-1 positive PA (previously silent subtype 3 PA) Acidophilic PIT-1 GH, PRL, beta-TSH +/- alpha-subunit Nil Postoperative residual in 65% with tumor progression in 53% of these patients (20); greater propensity for invasion and recurrence (3)
Sparsely granulated somatotroph PA Acidophilic PIT-1 GH +/- PRL Dot-like (fibrous bodies) Higher frequency of suprasellar extension/cavernous sinus invasion, larger tumors and smaller octreotide suppression test response in sparsely granulated vs. densely granulated tumors (23)
Lactotroph macroadenoma in men Acidophilic

PIT-1

ER-alpha

PRL (+GH in acidophilic stem cell subtype) Nil (or fibrous bodies in acidophilic stem cell subtype) Complete DA resistance in 8% men vs. 4% women (24); 57% of DA-resistant lactotroph PAs occur in men (24)

 

PC is rare, comprising only 0.1-0.2% of pituitary neoplasms(13,25). The incidence of PC is 4/1,000,000 person-years (26). These figures may, however, be underestimated, as up to 75% of historical PC diagnoses were only made at autopsy (27). PC typically presents in the fourth to sixth decades of life, with a mean age at diagnosis of 44 years (1), but rare pediatric cases have been reported (28,29). Whereas clinically-silent, hormone-staining tumors only account for 7% of all pituitary neoplasms (25), functioning tumors that have evolved from such tumors comprise 25% of APT/PC  (30). The commonest PC subtypes are corticotroph and lactotroph neoplasms (13). In a recent review of 72 published PC cases by Yoo et al, hormone IHC was positive for ACTH in 35%, PRL in 24%, GH in 14%, TSH in 6%, FSH in 7% and LH in 4%, and 15% were null cell (31). This rate of null cell PC was lower than other reports of 30% (13), possibly relating to the limited availability of prolactin IHC in historical studies (32). Compared to pure PA series, lactrotroph and corticotroph-derived neoplasms are overrepresented and somatotroph and null cell neoplasms are underrepresented in PC (25,30).

 

The overall epidemiology of APT/PC was recently outlined in a European Society of Endocrinology(ESE) survey of clinicians treating APT/PC patients, where APT was defined by the responding clinician. The survey cohort comprised 165 patients (40 PC, 125 APT), forming the largest APT/PC cohort to date. APT and PC were similar in age at diagnosis (43 vs. 45 yr), predominant cell subtypes (corticotroph in 45% vs. 48%, lactotroph in 20% vs. 38%), and functional status (clinically functioning in 58% vs. 63%) but initially silent and later functioning tumors were over-represented in PC (7% vs. 20%) (30). Both APT and PC demonstrated a male predilection (65% vs 63%) (30), in agreement with Yoo et al(31), but in conflict with other data showing a slight female predominance (33)or no gender predilection (13).

 

Clinically relevant germline variants in pituitary tumorigenesis genes are found in up to 20% of PA patients who are young and/or have other personal or family history of endocrine neoplasia (34). The rate of germline mutations specifically in the APT/PC setting is yet to be determined, but germlineAIPand SDHxmutations are typically associated with more aggressive tumor behavior(9,34).PC has been reported in patients with germline mutations, including SDHB(35), MSH2(36), and MEN1(37,38). However, PC appears to be no more common in patients with germline MEN1mutations than inpatients with sporadic PAs (39). To the best of our knowledge, there have been no reports of PC in patients with AIP-associated familial isolated pituitary adenoma syndrome, multiple endocrine neoplasia syndrome type 4 due toCDKN1Bmutations, Carney’s complex due to PRKAR1Amutations, McCune-Albright syndrome due to GNASmosaicism, or X-linked acrogigantism due to Xq26.3 microduplications involving GPR101. APT/PC prevalence is uncertain inUSP8, a gene where somatic mutations have been implicated in Cushing’s disease. Early data showed USP8-mutated corticotrophinomas to be smaller with lower plasma ACTH levels (40), suggesting a milder phenotype. However, subsequent data have shown higher postoperative urinary free cortisol levels in patients with USP8-mutated corticotrophinomas compared to wild-type corticotrophinomas, possibly serving as a harbinger for poorer long-term outcomes in these patients (41).

 

CLINICAL PRESENTATION

 

APT/PC nearly always evolve from pituitary macroadenomas (maximal tumor diameter ≥1cm) (13), but, conversely, many macroadenomas and even giant prolactinomas (≥4cm) respond well to standard treatments and never exhibit aggressiveness (2). Progression of a microadenoma (<1cm) to PC is exceedingly rare (42,43). The time from primary diagnosis with a pituitary neoplasm to presentation with APT/PC is highly variable (2). In APT, aggressiveness can be apparent from diagnosis, or take months to more than a decade to develop (2,44). The course of APTs may be punctuated by periods of radiological and hormonal quiescence (45). One study showed that APTs are more likely to occur following incomplete surgical resection at an odds ratio of 6.3 (17), but another study showed no relationship between APTs and the primary surgical outcome (15). These conflicting results partly reflect the difficulty in distinguishing residual tumor from normal tissue and postoperative changes (17). In PC, the mean latency from primary diagnosis is 6.5-9 years, but can range from months to 35 years (1,6,13,31,33,46,47).

 

Some symptoms, such as headache and visual field loss, overlap between PA and APT/PC, whilst cranial nerve palsies and obstructive hydrocephalus are more suspicious for APT/PC (33). Patients with Nelson’s syndrome, which is an inherently aggressive neoplasm, often present with mass effects including cranial neuropathies from the growing primary tumor as well as hyperpigmentation from proopiomelanocortin excess; distant metastasis may also occur (48). As in PA, diabetes insipidus is rare in APT/PC(49), and should raise suspicion for sella metastasis from a non-pituitary malignancy (1). Important differential diagnoses are breast and lung carcinomas, which are the commonest primary neoplasms to metastasize to the sella (46). Small cell lung cancer can produce both ectopic ACTH syndrome and sella metastasis, which may be misdiagnosed as a corticotroph PC with distant metastasis (32). PC metastases may lead to other site-specific clinical features, such as hearing loss, ataxia, motor weakness, back pain, neck masses, and liver function derangement (1,9).

 

Yoo et alshowed the site of metastases to be craniospinal in 58%, systemic in 32%, and both craniospinal and systemic in 8% of PC cases (31). This is in contrast to an earlier series of 15 PC cases reported by Pernicone et alwhere metastasis was predominantly systemic (47%), compared to craniospinal metastases in 40% and both craniospinal and distant metastases in 13% (13). Common sites of metastasis include the brain (43%), spine (38%), liver (14%) cervical lymph nodes (11%) and bone (10%)(31). Within the CNS, metastases typically involve the cortex, cerebellum and cerebellopontine angle  (49). Dural metastases may occur and can be misdiagnosed as meningiomas (33). Rare metastatic sites include the orbit, endolymphatic sac, oropharynx, heart, pancreas, kidney, skin, ovary, myometrium and pelvic lymph nodes (1,13,31,33).

 

PC subtype may influence the pattern of metastasis. In lactotroph PC compared to corticotroph PC, systemic metastases are relatively more common (71% vs. 57%), and the duration of pituitary neoplasm diagnosis to PC diagnosis is shorter (4.7 vs 9.5 years). In patients with distant metastases, the commonest site is bone in lactotroph PC and liver in corticotroph PC (13).

 

EVALUATION

 

The principles of APT/PC assessment were recently outlined in the 2018 ESE guidelines for APT/PC management (2). As in PA, the evaluation of patients with suspected or known APT/PC involves radiological, biochemical and histological investigations. Patients with APT should be followed indefinitely as recurrence and progression accumulate with time. In a study of recurrent non-functioning pituitary adenoma (NFPA), the prevalence of recurrent disease rose from 4.4% at 5 years to 10% at 10 years (6). Long-term follow-up also allows monitoring of late treatment-related complications such as radiation-induced hypopituitarism and secondary tumors, and the late development of PC which may occur decades following the initial diagnosis (2). Clinicians should be especially vigilant for metastases in patients with APTs (47), noting that metastasis often occurs insidiously and can involve various craniospinal and distant sites which may be mistakenly attributed to another primary neoplasm (2).

 

Radiological Assessment

 

The primary imaging modality in all pituitary neoplasms is MRI, ideally with thin (2-3 mm) T1- and T2-weighted slices before and after gadolinium in sagittal and coronal planes (2). T2 sequences are particularly helpful in acromegaly as T2 hyperintensity compared to normal pituitary or grey matter is often seen with sparsely granulated somatotroph PAs which tend to behave aggressively. T2 hyperintensity is also directly correlated with larger somatotroph tumors and blunted octreotide suppression test responses (50). This radiological clue is particularly helpful preoperatively, when the granulation pattern is unknown (50).

 

MRI should be performed every 3-12 months as guided by previous growth rates, proximity to vital structures and timing of interventions (2). Current images should be compared against baseline and penultimate scans (2)(1). In NFPA, volume doubling time is highly variable, ranging from one to 27 years, but tends to be stable for a given individual with an initially exponential growth pattern followed by deceleration of growth velocity (51). Deviation from this with unusually rapid growth rates are an important marker of APT (2). Rapid corticotrophinoma growth following bilateral adrenalectomy is a specific hallmark of Nelson’s syndrome, which precedes metastasis in over two-thirds of corticotroph PC cases (1).

 

Patients with APT and either discordant biochemical and radiological findings or site-specific symptoms should be screened for metastasis (2). In the absence of a formal staging system, patients with identified metastatic disease should undergo imaging by one or more modalities to define the extent of metastasis and to evaluate the possibility of a non-pituitary primary neoplasm (49). In patients with pituitary neoplasms and CNS symptoms, neck masses or back pain, pituitary MRI may be extended to include the whole brain and/or spine (1). CT imaging may be useful if bony involvement is suspected or in patients with contraindications to MRI (2). As PC is often hypermetabolic with somatostatin receptor (sst) expression including sst1, sst5 and sst2, nuclear imaging with 18FDG-PET and/or68Ga-DOTATATE-PET may be valuable in delineating the overall extent of disease (47,52). DOTATATE-PET and FDG-PET may produce discordant but useful findings. The presence of uptake on FDG-PET but not on DOTATATE PET may suggest more dedifferentiated disease. Discordant avidity may also be used to guide the selection of peptide receptor radionuclide therapy versus chemotherapy(2,52).

 

Biochemical Assessment

 

Pituitary hormones should be measured every 3-12 months, as guided by tumor subtype, clinical features and treatment interventions (2). This is imperative to identify secretory tumors responsive to medical therapies and hypopituitarism requiring hormone replacement (2). Hormone levels are also an invaluable tumor marker to guide treatment response in secretory tumors. Transition to APT/PC may be heralded by conversion of a silent PA to a clinically functioning tumor, loss of response to medical therapies, new or progressive hypopituitarism, or increasing hormonal excess despite radiological stability (1,2,9,32). In particular, an initial response to DA therapy followed by ‘escape’ was documented by Pernicone et alin 4/7 (57%) lactotroph PCs (13). Decreased hormone synthesis, reflecting tumor dedifferentiation, may also be a sign of tumor progression with declining serum levels of TSH and alpha-subunit previously reported at the time of metastasis in a thyrotroph PC (53). Another case report described a primary FSH-staining PA followed 15 years later by metastatic disease that stained negative for all pituitary hormones (54). This notion of tumor dedifferentiation may account for the increased aggressiveness of silent corticotrophinomas compared to functioning corticotrophinomas (33).

 

Histological Assessment

 

ASSESSMENT OF PROLIFERATION

 

Despite abandonment of the 2004 WHO criteria for atypical PA and the lack of a pituitary neoplasm grading system in the 2017 WHO classification, histopathology may be incorporated with clinical features to predict the trajectories of pituitary neoplasms(1,3,11). The 2018 ESE guidelines recommend performing IHC to evaluate pituitary hormones and the Ki-67 index, at a minimum, in all pituitary neoplasms, with the addition of mitotic count and p53 IHC when Ki-67 is ≥3%; however, it is ceded that the evidence basis for this is very low(2). The ESE guidelines suggest incorporating these histological markers in management decisions, such as the intensity of follow-up regimens and the use of adjuvant radiotherapy in patients with invasive and proliferative postoperative tumor remnants (2). The dominance of Ki-67 in the guidelines reiterates the finding of a Ki-67 index ≥3% being the commonest histological marker of tumor aggressiveness in the recent ESE survey, with this threshold met in 81% of APT and 85% of PC, compared to p53 positivity in 73% APT and 78% PC and mitotic count >2/10 HPF in 63% APT and 90% PC (30). Ki-67 was also the only predictive marker for tumor aggressiveness in other studies comparing various histological and clinical markers (55,56). Ki-67 thresholds of ≥3% and >10% are considered by some experts to indicate APT and PC, respectively (2). However, this is based on limited studies with variable methodologies and a lack of robust long-term data (2). Ki-67 also overlaps between indolent PA, APT and PC. Ki-67 ranges from undetectable to 80% in PC (1,30),and a Ki-67 ≥10% did not discriminate between APT and PC in the ESE survey (30). A mitotic index set at ≥2/10 HPF predicts a greater risk of recurrence (57), but there was again significant overlap between APT and PC in the ESE survey (30). Similarly, p53 immunopositivity, generally defined as >10 strongly positive nuclei per 10 HPF (2), is overrepresented in PC compared to PA, and incremental p53 staining has been observed in the progression from PA to PC (1), but p53 IHC may be negative in PC (58). Even the combination of all three histological markers of proliferation in the ESE survey did not reach statistical significance in differentiating APT versus PC (30). The unreliability of histological markers in predicting tumor behavior probably represents a combination of true biological variability between tumors given the observed variability in clinical features, as well as hampered histological assessment due to intratumoral heterogeneity, different fixation protocols, prior treatment effects, and antibody and interobserver variability (1,49).

 

MGMT IHC should be considered in suspected or known APT/PC as low expression is another potential marker of aggressive behavior and is predictive of TMZ response; however, these associations are not invariable and the decision to use TMZ should not rest on this result alone (30).

 

ASSESSMENT OF CELL LINEAGE

 

Pathological evaluation is important in identifying the more aggressive subtypes of pituitary neoplasms (Table 1). Hormone IHC is critical in identifying silent corticotroph PAs and plurihormonal PIT-1 positive PA, whilst cytokeratin staining is used to define the dot-like fibrous bodies of sparsely granulated somatotroph PAs as well as patterns specific to Crooke’s cell and silent corticotroph PAs (1). Other histological features of sparsely granulated somatotroph PAs include poorly cohesive cells with sheet-like formation and nuclear polymorphism with weak and focal GH staining (23). Although transcription factor IHC, as recommended in the 2017 WHO classification (11), may assist identification of aggressive pituitary neoplasm subtypes, it does not directly predict aggressiveness (2). Transcription factor IHC is considered most valuable in the differentiation of hormone immunonegative tumors(9). However, the clinical implications of a null cell adenoma that stains negative both for pituitary hormones and pituitary transcription factors are uncertain as the extant literature rarely defines transcription factor status. In a more recent study including 119 hormone immunonegative PAs, only 6 (5%) were also negative for cell lineage transcription factors (59). IHC for T-PIT is attractive given the greater aggressiveness of corticotrophinomas (30), but the availability of reliable T-PIT antibodies has been a concern (9). Nonetheless, the addition of transcription factor IHC is an attempt to overcome the false negative, misleadingly weak or dubious results that may be encountered with hormone IHC (3). Utrastructural analysis is not additive to the contemporary pathological assessment of pituitary neoplasms by morphology and IHC (3).

 

ASSESSMENT OF PITUITARY CARCINOMA

 

Like PAs, PCs appear microscopically as well-differentiated neuroendocrine tumors. PCs may demonstrate hypercellularity, nuclear pleomorphism, necrosis, haemorrhage and invasion, with all such features overlapping with PAs (1). Neuronal metaplasia may rarely occur in PC (1).

 

It is not possible to distinguish PC from PA on histological, immunohistochemical or ultrastructural grounds (1), and there is poor correlation between the histological and clinical features of PC metastases (47). The primary aim in the histological assessment of PC is instead to confirm a pituitary origin of metastases. Biopsy of apparent PC metastases is particularly important where another primary malignancy could explain the metastases, thereby influencing prognosis and management. Tissue diagnosis may be achieved by surgical biopsy or fine needle aspiration (FNA) biopsy of accessible sites such as the cervical lymph nodes, liver, lung or vertebrae (46,47,60). Histological diagnosis based on FNA specimens should be cautious, given its divergence from pituitary histological diagnoses which are virtually always made by craniotomy or trans-sphenoidal surgical resection (46). Key differential diagnoses based on similar cytological appearances include metastasis from renal cell carcinoma, plasmacytoma/multiple myeloma, lymphoma (46), medullary thyroid carcinoma, and other neuroendocrine tumors (47). In PC, metastatic lesions should bear cytological resemblance to the primary pituitary tumor (46,60), noting that proliferative markers, particularly Ki-67, are often higher in metastases (13,33,47). Immunohistochemical stains for neuroendocrine markers such as chromogranin A and synaptophysin aid in the differentiation of PC from non-pituitary neoplasms (1). Hormone IHC is also helpful in suspected metastases from a pituitary neoplasm that is known to be hormone producing (60). To ensure the appropriate use of these histological investigations, the reporting pathologist must be notified of the potential for metastasis from a pituitary neoplasm and aware of the frequent latency between PA onset and PC development. The small possibility of dual concurrent metastatic malignancies should be considered where there is variability in the clinical, radiological or histological features of the metastases (46).

 

Genetic Testing

 

Currently there are only weak associations between pituitary tumorigenesis genes and development of APT/PC, hence genetic testing for either germline or somatic mutations should not be performed purely on the basis of APT/PC development (2). Germline genetic testing should follow the usual indications as for non-aggressive PAs (2), including young onset and other personal or family history of related neoplasms(34).

 

PATHOGENESIS

 

As APTs represent a composite of different tumor subtypes, the contributing pathogenic mechanisms are varied. Tumor persistence, recurrence and progression after surgery at least partly relate to greater invasiveness, lowering the chance of gross total resection (15). Resistance to medical therapy in somatotroph APTs may relate to reduced sst2 expression (61)and tumor bulk (23), whilst DA resistance in lactrotroph APTs can occur in cystic tumors and tumors with decreased dopamine D2 receptor and estrogen receptor (ER) expression (2,62). Cell specific feedback sensitivity is also important. The relative indolence of somatotroph PAs with apparent insensitivity of somatotrophs to loss of negative feedback during pegvisomant treatment contrasts sharply with the typically aggressive nature of Nelson’s syndrome following bilateral adrenalectomy with loss of endogenous cortisol feedback in corticotrophs (63). A somatic inactivating mutation in the glucocorticoid receptor gene was found in one such case of Nelson’s syndrome (64). On the other hand, Cushing’s disease requiring bilateral adrenalectomy may reflect intrinsically more aggressive corticotrophinomas that drive the clinical course of disease, rather than adrenalectomy and loss of endogenous negative feedback being the underlying driver of progression (2).

 

Hypothesized mechanisms of PC dissemination include: hematogenous spread through the anterior pituitary portal system into the cavernous and petrosal sinuses and finally the jugular veins; lymphatic spread via the sphenoid sinus or in the skull base and soft tissues by connections between the intracranial perineural space and lymphatic plexus; and cerebrospinal fluid seeding along the subarachnoid space of the neuroaxis (1,13,47,49).However, there have been no studies comparing the sites of metastases in pituitary neoplasms with cavernous versus sphenoid sinus invasion. Increased matrix metalloproteinase-9 expression in PC and its association with vascular density in PC suggest that extracellular matrix degradation contributes to angiogenesis (65). Matrix metalloproteinase activity may also promote local tumor invasion, including entry into deep brain structures along the Virchow-Robin perivascular CNS spaces, resulting in non-contiguous cranial metastases (47).

 

Iatrogenesis has been purported in select PC cases with intimately located metastases following trans-sphenoidal surgery (13), craniotomy (66),  radiotherapy (47), and ventricular-peritoneal shunt placement (47). Hypotheses for the role of surgery in increasing PC risk include disruption of venous barriers intraoperatively and postoperative formation of friable new blood vessels (47). Radiotherapy has been postulated to increase tumor aggressiveness by inducing genetic mutations, in TP53for example (48). However, this theory is controversial and confounded by the fact that surgery and radiotherapy are employed in most patients with APT/PC during the typically protracted progression of PA to APT and finally to PC (1,47). Furthermore, the vast majority of operated and irradiated pituitary neoplasms never develop into PC (1), making iatrogenesis a highly unlikely cause of PC.

 

Molecular Mechanisms

 

Competing molecular models of APT/PC pathogenesis include a hyperplasia-adenoma-carcinoma sequence with accumulation of molecular alterations, versus clonal evolution of a subclone with genetic/epigenetic changes favoring cell survival, proliferation and ultimately metastasis (1,47,67). As most patients present with a long history of pituitary neoplasm (13,32), de novomalignant transformation of normal adenohypophyseal cells seems unlikely. There are, however, rare reported cases of rapid progression from pituitary neoplasm diagnosis to death (43,68). The frequent transition of PC from PA via an APT stage (1,47)suggests that pathogenic mechanisms may be shared between PAs, APTs and PCs. Although, whilst some genes like PTTGare overexpressed in PAs compared to normal pituitary tissue and in APTs compared to other PAs (69), other genes such as the RASgene only appear to be implicated in APT/PC (70,71). Whilst there is some overlap between genetic changes in APT/PC and the genes underlying more common solid organ malignancies, mutations in classic oncogenes and tumor suppressor genes are relatively uncommon (33). Certain molecular events may be specific to the different elements of APT/PC pathogenesis. A transcriptomic analysis of lactotroph pituitary neoplasms found different genetic changes in purely invasive tumors (upregulation of ADAMTS6and CRMP1; downregulation of DCAMKL3) compared to tumors that were invasive and aggressive (upregulation of ADAMTS6,CRMP1,PTTG, ASK, CCNB1, AURKBand CENPE; downregulation of PITX1). Upregulation of Pttg, Aurkb, Cenpeand Crmpand absentPitx1expression in malignant lactrotoph tumors in rats recapitulated these findings, and there is a functional basis to the involvement of these genes with ASK, PTTG, AURKB, CCNB1and CENPEinvolved in the cell cycle, ADAMTS6in the extracellular matrix, CRMPin cellular migration, and PITX1in pituitary differentiation (72).

 

Copy number variation (CNV) at the chromosomal level is the most frequent genetic aberration in pituitary neoplasms (73). CNV is particularly common in functioning neoplasms, especially prolactinomas, as well as neoplasms with high proliferative indices (73-75). The mean number of chromosomal imbalances per tumor is 1.6 in initial PAs, 3.4 in recurrent PAs and 8.3 in PC (74,75). Aneuploidy was observed in all but one of the 15 PC cases reported by Perniconeet al(13). The degree of genomic disruption is directly proportional to the Ki-67 (73). This progressive increase in CNV supports an adenoma-carcinoma sequence, as observed in other endocrine tumors such as pancreatic and adrenocortical neoplasms (75). Recurrent chromosomal aberrations in APT/PC include gains in chromosome 4q, 5, 13q and 14q and losses of chromosome 1p, 2, 8q, 10, 11, 12q, 13q and 15q (6,74,75). These chromosomes contain multiple genes implicated in APT/PC pathogenesis, as listed in Table 2, although the underlying evidence for the causal involvement of these genes is limited owing to the rarity of PC and variability in genomic technologies.

 

Table 2. Selected Genes Implicated in APT/PC Pathogenesis
Gene Locus Function Alteration in APT/PC
Oncogenes
PTTG, pituitary tumor transforming gene Chr 5q33.3* Securin protein in spindle checkpoint machinery, responsible for error-free mitosis Overexpression associated with increased risk of PA recurrence, strong correlation with Ki-67 (69)
VEGFA, vascular endothelial growth factor A (also referred to as VEGF) Chr 6p21.1 Induces angiogenesis by promoting endothelial cell survival and proliferation Increased VEGF staining in PC (76); PC stabilised by VEGF inhibition (bevacizumab) (77)
EGFR, epidermal growth factor receptor Chr 7p11.2 Receptor tyrosine kinase contributing to tumor progression by increasing proliferation, decreasing apoptosis, and inducing angiogenesis and invasion Increased EGFR expression in APT/PC (78)
HRAS, V-HA-RAS Harvey rat sarcoma viral oncogene homolog Chr 11p15.5* Promotes cellular proliferation and differentiation Rare activating mutations in APT/PC (70,71)
CCND1, cyclin D1 Chr 11q13.3* Promotes transition at the G1-S phase cell cycle checkpoint Germline CCND1 genotype associated with  locally invasive and malignant pituitary neoplasms (79);  increased CCND1 staining in APT vs other PA and normal pituitary (80)
ERBB2, V-ERB-B2 avian erythroblastic leukemia viral oncogene homolog 2 (also referred to as HER2/neu) Chr 17q12 Induces cell survival and proliferation Increased expression in PC (43)
TOP2A, topoisomerase DNA II alpha Chr 17q21.2 Enzyme modifying topological state of DNA, involved in DNA transcription and mitosis Increased topoisomerase II alpha immunostaining in invasive PA, silent type 3 PA and PC; mixed results regarding correlation with Ki-67 (81)
Tumor Suppressor Genes
MSH6, MutS E. coli homolog of 6 Chr 2p16.3* Mismatch repair protein, removes DNA base mismatches caused by errors in DNA replication or by DNA damage Loss of MSH6 in progression from atypical PA to PC,  loss of MSH6 +/- MSH2 in TMZ-resistant atypical PA/PC (44,82); inactivating MSH6mutations in PC (83)
MGMT, methylguanine-DNA methyltransferase Chr 10q26.3* DNA repair enzyme, removes alkylating adducts in DNA Decreased MGMT expression in APT/PC, correlates with activation of genes in DNA damage response and DNA repair pathways and genes involved in transcription (84)
CDKN1B, cyclin-dependent kinase inhibitor 1B (encoding p27Kip1) Chr 12p13.1 Binds cyclin/cyclin-dependent kinase complexes, regulates transition at the G1-S phase cell cycle checkpoint Loss of normal p27 expression in PC (85)
RB1, retinoblastoma 1 gene Chr 13q14.2* Regulates cellular proliferation RB1 loss of heterozygosity in highly invasive and malignant pituitary neoplasms (86)
TP53, tumor protein p53 Chr 17p13.1 Induces cellular senescence or apoptosis in response to DNA damage Increasing cellular accumulation in APT/PC, rare inactivating mutations in APT (48)
BCL2, B-cell CLL/lymphoma 2 Chr 18q21.33 Anti-apoptotic Decreased Bcl-2 expression in PC, correlates with higher rates of apoptosis in PC vs. PA (87)
Other 
PTGS2, prostaglandin-endoperoxide synthase 2 (encoding COX2) Chr 1q31.1 Cyclo-oxygenase involved in angiogenesis Increased Cox-2 expression in PC (88)
LGALS3, lectin galactoside-binding soluble 3 (encoding GAL3) Chr 14q22.3* Galactose-binding lectin regulating cyclin-E-associated kinase activity Increased Gal-3 immunopositivity in corticotroph and lactotroph PC (89)
HIF1A, hypoxia-inducible factor-1alpha Chr 14q23.2* Transcription factor mediating cellular responses to hypoxia Increased HIF1A expression in PC (90)

Abbreviations: * chromosomal loci that are frequently gained or lost in APT/PC

 

A particular gene of interest in the pathogenesis of APT/PC is MGMT, which maps to 10q26.3. Low MGMT expression is a common feature in APT/PC (30,84). It is also overrepresented in patients with plurihormonal PIT-1 positive PA, Crooke’s cell PA, Nelson’s syndrome and recurrent NFPA, all of which exhibit more aggressive behavior (84). Low MGMT expression is in turn associated with upregulation of genes involved in transcriptional activity, DNA damage response and DNA repair (84). Interestingly, in pituitary neoplasms low MGMT expression does not correlate with MGMTpromoter hypermethylation as it does in glioblastoma, suggesting that MGMTis inactivated by alternative, currently unknown mechanisms (2,7,84).

 

The conversion of PA to APT/PC does not appear to be explained by the genes causing sporadic (e.g. USP8, GNAS) and/or familial (e.g. AIP, MEN1,CDKN1B, PRKAR1A, SDHx) PAs (2). In a study of 52 patients with somatotroph PA, GNASmutations were found in 53% of tumors but there was no difference between the more common densely granulated subtype and the more aggressive sparsely granulated subtype, and Ki-67 index, invasiveness and diameter did not differ between GNASmutated and wild-type tumors (23). By contrast, a known activating GNASmutation was reported to coincide with conversion of a lactotroph PA into a somatotroph APT (91). This suggests that the conversion to hormone production in APT/PC may sometimes relate to acquired genetic mutations with a true gain of secretory function. An alternative explanation is simply increased tumor bulk with increased hormonogenesis  (30).

 

A myriad of other molecular changes has been observed in APT/PC. As in other cancers, a role for telomerase in facilitating cellular immortality has been suggested with both Ki-67 and telomerase activity shown to increase with sequential resections of a lactotroph PC, whereas telomerase activity was absent in PAs (92). Increased immune tolerance may also be contributory with reduced T-cell concentration, HLA-Bdownregulation and upregulation of genes involved in T-lymphocyte suppression shown in plurihormonal PIT-1 positive PAs (93). The role of T-lymphocytes in pituitary immune tolerance is underscored by the high rates of hypophysitis with the use of ipilimumab in other malignancies (94), and the recent successful use of combined anti-CTLA4/PD1 therapy in a corticotroph PC (83). Changes have also been observed in microRNA, which are small non-coding RNAs that bind the 3’-untranslated regions of target mRNAs, thereby regulating post-transcriptional gene expression (95). In a study of lactotroph neoplasms, miR-183 was downregulated in APTs versus non-aggressive PAs and this was associated with increased expression of PCLAF, a gene inhibiting p53 and p21 mediated cell cycle arrest. miR-183 and PCLAF also correlated with Ki-67 and p53 expression (95). In a case of a non-functioning PC with multiple intracranial metastases, miR-20a, miR-106b and miR17-5p were upregulated in the metastases compared to the primary neoplasm, in association with decreases in the tumorigenesis genes, PTENand TIMP2, which are downstream targets of these microRNAs (96). Another study showed upregulation of miRNA-122 and miRNA-493 in PC versus PA, with miRNA-493 shown to interact with the LGALS3and RUNX2genes which have been implicated in pituitary cellular proliferation (97).

 

MANAGEMENT

 

The key principle in the management of patients with APT/PC is for care to be directed by an expert multidisciplinary team. Multimodal treatment strategies are most commonly required. Surgery, radiotherapy and medical therapies all have a role in the management of APT (Figure 1). Tumor location and size, the presence of single or widespread metastatic disease (in PC), prior surgery and extent of resection(s), previous radiotherapy and cumulative doses, optimisation of standard medical therapies, past oncological treatments and patient comorbidities are all important considerations in formulating management plans.

Figure 1. Treatment options in APT/PC

Surgical Management

 

Patients with APT frequently require repeated neurosurgical procedures. In the ESE survey cohort, patients underwent a mean of 2.7 operations while 29% had four or more pituitary operations over the course of their disease (30). Multiple studies now demonstrate improved outcomes and lower complication rates when pituitary surgery is performed by high-volume neurosurgeons (98-101). The likelihood of achieving gross total resection is consistently reduced in the presence of tumor invasion, particularly of the cavernous sinus, even in the most experienced hands (102,103). Endoscopic endonasal surgical techniques utilising angled endoscopes and wide exposure may facilitate safe and more extensive surgical resection compared with transsphenoidal microsurgical approaches (104-106). In some circumstances where tumor extends to a significant degree into suprasellar or other extrasellar regions, a transcranial approach may be favored. However, the degree of resection may be limited by the risk of morbidity, depending on tumor location.

 

Surgical resection, even as a debulking procedure, should be considered in patients with APT as it may offer significant relief of compressive symptoms, particularly when there is visual disturbance (107,108). In patients with isolated metastatic deposits (either craniospinal or systemic disease) complete surgical excision may result in long-term disease-free progression particularly when followed by adjuvant radiotherapy (109-111). Repeat surgical resections of recurrent metastases may also prolong survival (13).

 

Radiotherapy

 

The use of radiotherapy should be considered in patients with APT as it may assist in long-term control of tumor growth (112). Radiotherapy is recommended in the setting of clinically significant tumor growth despite surgery, and in the case of functional tumors where standard medical therapy has been ineffective (2). In patients with PC, palliative radiotherapy may be delivered to sites of metastatic disease but there is no evidence that it prolongs survival (112). Discussion about radiotherapy should take place within a multidisciplinary setting involving an expert radiation oncologist (2). The role of further debulking surgery prior to radiotherapy should be discussed. Radiotherapy applied to a smaller tumor volume is more effective, and removing tumor in close proximity to the optic apparatus may allow safer and improved radiotherapy delivery (2,113). In previously irradiated patients, consideration must be given to the cumulative radiation dose applied to the target region. In patients with invasive tumor remnants following surgery andwhere histological markers indicate the potential for aggressive tumor behavior (high Ki-67, particularly ³10%; elevated mitotic count; increased p53 immunostaining), adjuvant radiotherapy should be considered (2). In the case of evident aggressive tumor behavior, combination radiotherapy and chemotherapy with temozolomide (TMZ) may yield improved outcomes (30).

 

Fractionated external beam radiation therapy (EBRT) and stereotactic radiosurgery (SRS, delivered as single dose or in fractions) are both highly effective in the management of PAs. In one study of NFPAs, routine use of postoperative radiotherapy was associated with a doubling of 10-year progression-free survival compared with patients who did not undergo radiotherapy (93% vs. 47% ) (114).  Success rates vary across studies because of different modalities (linear accelerators, Gamma Knife, proton beam irradiation) and variable techniques, doses and imaging protocols used between centers(115). In APT, there are limited data on the effectiveness of radiotherapy. In a series of 50 patients with persistent or recurrent adenomas despite prior radiotherapy, further focal SRS was effective in the majority of cases, although a large number of cases were treated for persistent GH excess rather than radiologically aggressive tumors (116,117). The response to radiotherapy may only be transient in more aggressive tumors or even ineffective, particularly in cases demonstrating progression despite salvage chemotherapy (30).

 

The choice of radiotherapy technique and modality is ultimately based on safety considerations (e.g. proximity to optic chiasm), volume of disease, and local center availability (2). Adverse effects of radiotherapy delivered to the pituitary, such as hypopituitarism or risk of secondary tumors, has rationalized the modern day use of radiation therapy for pituitary tumors. However, considering the morbidity and excess mortality associated with APT, these adverse effects, particularly given their significant latency, should not hinder the prompt use of radiotherapy in APT.

 

Peptide Receptor Radionuclide Therapy (PRRT)

 

Pituitary neoplasms express somatostatin receptors and have demonstrated 68Ga-DOTATATE uptake on PET/CT, stimulating interest in the use of PRRT in the management of APT (118,119). There is only limited experience with PRRT reported in the literature to date in patients with APT. A variety of radionuclides have been utilised including 111Indium-DPTA-octreotide,177Lutetium DOTATATE, 177Lutetium DOTATOC and 90Yttrium-DOTATOC(2,120). Of 14 cases, only two patients (one giant lactotroph PRL-secreting adenoma, one GH-secreting macroadenoma) have demonstrated a reduction in tumor size, one without a concomitant hormonal response (121,122). Stable disease was reported in three cases while nine patients progressed with PRRT (2).

 

Standard Medical Therapy

 

APTs typically display resistance to the standard medical therapies commonly used in the management of functional PAs. Use of maximally tolerated DA treatment should be attempted in lactrotroph APTs given occasional reported responses (123,124). Cabergoline (3.5-11mg per week) is more effective than bromocriptine or quinagolide (2). Tamoxifen has been unsuccessfully used in lactotroph PC (2). In rare cases of somatotroph PC, use of DA treatment has been associated with GH and IGF-1 reductions and symptomatic improvement, but without tumor shrinkage (27,125). Similarly, the use of first generation SSAs in somatotroph APTs is largely ineffective, whereas pasireotide may improve biochemical control although data in APT are scarce (126). Resistance to first generation SSAs (octreotide, lanreotide) due to downregulation of sst2a expression has been described among AIPmutation positive individuals with somatotroph tumors, but expression of sst5 is often preserved and thus response to second generation broader-spectrum SSAs, such as pasireotide, may be more effective (127). Temporary benefit from high dose octreotide has been described in a case of thyrotroph PC(53).

 

Aggressive corticotroph tumors represent a particular challenge, and these patients often require medical therapy to reduce hypercortisolism, a common direct cause of death (112). Adrenal glucocorticoid inhibitors, such as ketoconazole or metyrapone, are frequently used in such cases. Pasireotide has been reported in 15 cases of aggressive corticotroph tumors, including nine with Nelson’s syndrome, but with only one case exhibiting a hormonal and radiological response (2,128).

 

Chemotherapy

 

WHEN TO INITIATE CHEMOTHERAPY

 

In patients with PC, the decision to start systemic chemotherapy is clear and associated with improved survival (2,129,130). In patients with isolated metastases, loco-regional therapies such as hepatic chemoembolisation for low-bulk liver metastases may offer temporary tumor control (131). For APT, in cases of documented tumor growth, other treatment options may be explored first, such as further surgery or radiotherapy if appropriate, and histological parameters such as Ki-67 or tumor subtype, may play a role in decision making. However, it is increasingly recognised that apart from the presence of metastases, there is little that distinguishes APT from PC (16). Most importantly, time to death following diagnosis of pituitary tumor is similar between APT and PC (30). Prior to the recognition of TMZ efficacy in APT, chemotherapy was typically reserved as salvage therapy because of poor response rates. The mean survival rate in the pre-TMZ era for PC was 1.9 years (49). APT and PC treated with TMZ are now reported to have 5-year overall survival rates of 57.4% and 56.2% respectively (132). In the large French cohort, median survival was 44 months in patients who responded to TMZ compared with 16 months in non-responders (133). While TMZ is still most commonly used as a last resort therapy, it has been successfully employed during or prior to radiotherapy (30,134). In fact, the 2018 ESE guidelines suggest, in patients with rapid tumor growth where maximal doses of radiotherapy have not been reached, TMZ may be combined with radiotherapy as per the Stupp protocol used for glioblastoma (2,135). As new therapeutic modalities emerge in the coming years, clinicians will likely employ TMZ earlier in the treatment algorithm for APT. Decisions must be made within an expert multidisciplinary team setting where risk-benefit ratios are carefully deliberated, taking into account the morbidities of repeated surgery or radiotherapy as well as the potential for rare long-term consequences of chemotherapy such as hematological malignancy (2).

 

TEMOZOLOMIDE

 

TMZ is recommended as first-line chemotherapy for patients with APT and PC, with more than 200 cases now reported in the literature including the recent ESE survey (2,30). The overall response rate is 37-47% across the larger cohorts, with complete responses (both biochemical and radiological) seen in approximately 5% of cases(2,30). However, if stable disease is considered a clinically beneficial outcome, as it frequently is in oncological studies, then rates of progression-free survival are 50-87.6% in APT and PC (30,130). Clinically functioning APT are 3.35 times more likely to respond to TMZ compared with non-functioning APT (30). APT are just as likely to respond to TMZ compared with PC, although progression may be more frequent among tumors with a Ki-67 ³10% (30).

 

TMZ is a second-generation imidotetrazine alkylating agent which, when hydrolysed, forms toxic methyl adducts with DNA bases resulting in ineffective DNA repair and ultimately cellular apoptosis (136). TMZ is given as an oral outpatient-based chemotherapy, most commonly as monotherapy. Some centers advocate use of capecitabine pre-treatment (CAPTEM) because of in vitro data in neuroendocrine tumor cell lines suggesting synergistic effects with this regimen, although evidence supporting its superiority in APT is lacking (137). Similarly, it has not yet been demonstrated that TMZ in combination with any other drug(s) has enhanced efficacy (2). However, where maximal doses of radiotherapy have not been given to a patient, there is suggestion of improved response when TMZ is given concurrently with radiotherapy (2,30,138). Experimental data strongly support a radiosensistizing effect of TMZ (139,140).

 

The TMZ doing regimen given for APT is 150-200mg/m2for 5 consecutive days every 28 days. It is generally well tolerated, although mild to moderate fatigue, nausea and myelosuppression are common side effects, occurring in roughly half of patients but leading to TMZ discontinuation only in a minority (2). A dose reduction or delay in treatment cycles can allow patients to continue TMZ when myelosuppression occurs. Regular monitoring of hematological and liver function profiles is required during treatment. Hemorrhage into cerebral metastases has been described in a patient with PC who developed severe thrombocytopenia (141). Hepatoxicity has been reported when TMZ was used concurrently with ketoconazole therapy, and cholestatic hepatitis has also been reported in association with TMZ treatment in the wider literature (142,143). TMZ-induced hearing loss has been described among two pituitary cases and other rare side effects in non-pituitary literature include hypersensitivity pneumonitis, Stevens-Johnson syndrome and hematological malignancies (2). Prophylactic trimethoprim-sulfamethoxazole to protect against Pneumocystis pneumonia should be considered, particularly in hypercortisolemic patients receiving concurrent radiotherapy and TMZ and patients who develop significant lymphopenia during TMZ therapy (2).

 

WHEN TO STOP TEMOZOLOMIDE

 

Response to TMZ will be evident after 3 months of therapy(144). Treatment should cease in the event of progressive disease while receiving TMZ, or if serious adverse events occur. It is recommended to continue with treatment for at least 6 months, but therapy is often extended if there is ongoing clinical benefit (2). In the ESE survey, median treatment duration was 9 months and the longest course was 36 months (30). In this patient cohort, treatment with TMZ was initiated prior to the publication of management guidelines for APT. Hence, duration of therapy was often prescribed by oncology teams at the outset and was based on experience with TMZ clinical trials in glioblastoma (135). Following cessation of TMZ treatment in APT and PC, there is frequently a period of sustained remission. Time to tumor progression is variable, and whether longer treatment courses or degree of initial response improves progression-free survival is not currently clear. The median time to progression after cessation across patients in the ESE survey cohort was 12 months (range 1-60). Two patients exhibiting the longest time to progression were PC cases with complete response to TMZ (30). In the French multicenter cohort, patients receiving more than 12 months of TMZ achieved a median relapse-free survival of 57 months compared with 18 months in those receiving less than 12 cycles (133). However, response rates were 100% in those receiving longer treatment courses versus 75% in the shorter treatment group. Nevertheless, long-term treatment has been reported to be associated with improved progression free survival of 61% compared to 16% for short term treatment (132).

 

DETERMINANTS OF RESPONSE TO TEMOZOLOMIDE

 

The most well recognised biomarker of the likely response to TMZ is expression of O(6)-methylguanine DNA methyltransferase (MGMT). An endogenous DNA repair protein, MGMT is responsible for removal of the methyl group induced by TMZ therapy. In the absence of MGMT, unrepaired methylated guanine (O6-MeG) lesions incorrectly pair with thymine, triggering activation of the mismatch repair pathway (MMR). Intact MMR results in futile attempts at repair via incorrect reinsertion of thymine opposite the O6-MeG lesion. Cycles of ineffectual repair eventually result in DNA strand breaks which lead to cell cycle arrest followed by either apoptosis or cellular senescence. If MMR function is lost, then paradoxically cells can survive. However, even in the presence of intact MMR, MGMT can facilitate cell survival by direct repair of O6-MeG, targeting it for ubiquitination and degradation (Figure 2)(145).

 

Low expression of MGMT, as determined by IHC, is associated with a high response rate, around 75%, while tumors with high MGMT expression are unlikely to respond (30,136). Evaluation of MGMT status by IHC should be performed by a neuropathologist with expertise in APT (2). Lack of standardized IHC technique, use of different expression criteria across centers, poor fixation methods, and tumor heterogeneity are among the challenges in assessment of MGMT IHC. MGMT promoter methylation analysis has not been associated with response to TMZ in pituitary neoplasms(136,146).

 

DNA mismatch repair proteins such as MSH6, MLH1, MSH2 and PMS2 may also play a role in response to TMZ. Loss of MSH6, in the presence of low MGMT, has been described as a mechanism responsible for the development of resistance to TMZ (147). The overexpression of multidrug resistance proteins and activity of the Sonic hedgehog signalling pathway may also contribute to TMZ resistance (130).

Figure 2. Temozolomide Cytotoxicity and Mismatch Repair Pathway

TREATMENT OPTIONS BEYOND TMZ

 

There is a pressing need to identify alternative effective oncological therapies for patients progressing on TMZ or following an initial successful course of TMZ treatment. Given the paucity of treatment options, a second 3-cycle trial of TMZ treatment may be considered in patients who develop recurrence after a previous response to TMZ (2). However, a second treatment course has rarely been reported to be successful in such cases (2). If there is rapid tumor progression on TMZ treatment, a trial of other systemic cytotoxic therapy is recommended based on historical reports of transient regression and/or stabilization with some regimens (2). Lomustine (CCNU) and/or 5FU have most commonly been employed, but multiple other drugs, alone or in combination, including cyclophosphamide, doxorubicin, adriamycin, carboplatin/cisplatin, etoposide and vincristine have also been reported, with variable results (2,30).

 

Use of targeted therapies offer some promise, but data on clinical effectiveness are lacking. In vitro data demonstrating upregulation of Raf/MEK/ERK and PI3K/Akt/mTOR pathways in pituitary tumors have thus far not translated into clinical success in APT (30,148,149). There has been limited use of tyrosine kinase inhibitors (lapatinib, sunitib, erlotinib), with just one case report of a partial response with lapatanib in a lactotroph APT (150). VEGF-targeted therapy with bevacizumab, as monotherapy or in combination with TMZ, has resulted in partial response or stable disease in a few cases, although progressive disease has also been reported (30,77).

 

Finally, there is emerging interest in the potential use of immunotherapy for the treatment of APT. Pituitary neoplasms, particularly APT, have been shown to express programmed death ligand 1 (PD-L1), a T-cell immune checkpoint biomarker, along with tumor infiltrating lymphocytes  (151,152). Combination treatment with ipilimumab and nivolumab has recently been reported to result in marked tumor shrinkage and hormonal response in a patient with a hypermutated corticotroph PC (83).

 

PROGNOSIS

 

Morbidity and mortality are increased in APT even in the absence of progression to PC (2,16). This is particularly true in functioning corticotroph APTs, where morbidity and mortality are further increased in relation to cortisol excess (2).

 

In the ESE survey, mortality was higher in PC (43%) compared to APT (28%) (16), but median survival from initial diagnosis of pituitary tumor was similar (11 years in APT vs. 12 years in PC) (30). The time to death from PC diagnosis ranged from seven days to eight years in the study by Pernicone et al, with a 66% 1-year-survival (13). The mortality rate reported by Yoo et alwas 55% with an average time to death after PC diagnosis of just 10 months (31). Amongst all endocrine carcinomas, PC demonstrates the strongest decline in survival with advancing age (26). Prognosis is also poor in patients with corticotroph PC, systemic metastases, or progression during TMZ therapy (1,13,30,153). By contrast, patients who respond to TMZ experience a clear survival benefit (133). Exceedingly long-term survival over several years has been observed in selected cases (6,13), even without TMZ (153), but predictive markers for such survival remain unknown (47).

 

FUTURE DIRECTIONS

 

Comprehensive molecular studies will hopefully identify better biomarkers for PAs that are destined to become APT/PC. In addition to molecular biomarkers, the growing sphere of nuclear medicine may prove useful in the assessment of PC, which currently lacks a standard method of staging. 11C-methionine, a tracer with specific avidity for neoplastic pituitary tissue, has shown superior sensitivity to 18F-FDG-PET in localising functioning PAs (154). Though yet to be studied in PC, 11C-methionine holds promise in better delineating metastatic disease. Integration of molecular, functional and clinical data may ultimately assist clinicians in better identifying tumors with the potential for more aggressive behavior. This will allow earlier and more proactive management in affected patients with the goal of improving prognosis.

 

Because of the rarity of PC and the diverse subtypes of APT, current data are plagued by small sample size driven by case reports or series, heterogeneous case mix, short follow-up and clinical rather than histological diagnoses of PC metastases, with heavy reliance on expert opinion and local practice and a dearth of randomized controlled trials. Calls by the ESE to form an international register for APT/PC should help address the multiple evidence gaps in these rare disorders (2). As APT/PC are almost invariably diagnosed retrospectively, routine pituitary tumor biobanking with methodical storage of tissue in media that circumvent formalin-induced DNA damage will be critical in studying pathogenesis. Waiting for metastasis before labelling a pituitary neoplasm as PC is particularly problematic, given the similar time-to-death from initial pituitary tumor diagnosis between patients with APT versus PC (16). The recent suggestion to replace the term ‘pituitary adenoma’ with ‘pituitary neuroendocrinetumor’ (PitNET) is hoped to emphasise the malignant potential of a subset of these neoplasms and expand treatment intensity (9,155); however, as with all changes in nomenclature, this risks a disconnect between existing literature and contemporary clinical practice.

 

REFERENCES

 

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Calcium and Phosphate Metabolism and Related Disorders During Pregnancy and Lactation

ABSTRACT

Pregnancy and lactation require women to provide calcium to the fetus and neonate in amounts that may exceed their normal daily intake. Specific adaptations are invoked within each time period to meet the fetal, neonatal, and maternal calcium requirements. During pregnancy, intestinal calcium absorption more than doubles, and this appears to be the main adaptation to meet the fetal demand for mineral. During lactation, intestinal calcium absorption is normal. Instead, the maternal skeleton is resorbed through the processes of osteoclast-mediated bone resorption and osteocytic osteolysis, in order to provide most of the calcium content of breast milk. In women this lactational loss of bone mass and strength is not suppressed by higher dietary intakes of calcium. After weaning, the skeleton appears to be restored to its prior bone density and strength, together with concomitant increases in bone volumes and cross-sectional diameters that may offset any effect of failure to completely restore the trabecular microarchitecture. These maternal adaptations during pregnancy and lactation also influence the presentation, diagnosis, and management of disorders of calcium and bone metabolism such as primary hyperparathyroidism, hypoparathyroidism, and vitamin D deficiency. Pregnancy and lactation can also cause pseudohyperparathyroidism, a form of hypercalcemia that is mediated by parathyroid hormone-related protein, produced in the breasts or placenta during pregnancy, and by the breasts alone during lactation. Although some women may experience fragility fractures as a consequence of pregnancy or lactation, for most women parity and lactation do not affect the long-term risks of low bone density, osteoporosis, or fracture.

 

INTRODUCTION

 

During gestation the average fetus requires about 30 g of calcium, 20 g of phosphorus, and 0.8 g of magnesium to mineralize its skeleton and maintain normal physiological processes. The suckling neonate obtains more than this amount of calcium in breast milk during six months of exclusive lactation. The adaptations through which women meet these calcium demands differ between pregnancy and lactation (Figure 1). Although providing extra calcium to the offspring could conceivably jeopardize the ability of the mother to maintain calcium homeostasis and skeletal mineralization, as this review will make clear, pregnancy and lactation normally do not cause any adverse long-term consequences to the maternal skeleton. The reader is referred to several comprehensive reviews for more details and extensive reference lists for the material covered in this chapter (1-7).

 

Figure 1. Schematic illustration contrasting calcium homeostasis in human pregnancy and lactation, as compared to normal. The thickness of arrows indicates a relative increase or decrease with respect to the normal and non-pregnant state. Although not illustrated, the serum (total) calcium is decreased during pregnancy, while the ionized calcium remains normal during both pregnancy and lactation. Adapted from ref. (8), © 1997, The Endocrine Society.

 

MINERAL PHYSIOLOGY DURING PREGNANCY

 

Calcium provided from the maternal decidua aids in fertilization of the egg and implantation of the blastocyst; from that point onward the rate of transfer from mother to offspring increases substantially. About 80% of the calcium and phosphate present in the fetal skeleton at the end of gestation crossed the placenta during the third trimester and is mostly derived from the maternal diet during pregnancy. Intestinal calcium and phosphate absorption doubles during pregnancy, driven by 1,25-dihydroxyvitamin D (calcitriol) and other factors, and this appears to be the main adaptation through which women meet the mineral demands of pregnancy.

 

Mineral Ions

 

There are several characteristic changes in maternal serum chemistries and calciotropic hormones during pregnancy (Figure 2), which can easily be mistaken as indicating the presence of a disorder of calcium and bone metabolism, especially since it is not common for clinicians to measure calcium, phosphate, and calciotropic hormones during pregnancy (1). The serum albumin and hemoglobin fall during pregnancy due to hemodilution; the albumin remains low until parturition. In turn that fall in albumin causes the total serum calcium to decline to values that can be well below the normal range. The total calcium includes albumin-bound, bicarbonate-and-citrate-complexed, and ionized or free fractions of calcium. The ionized calcium, the physiologically important fraction, remains constant during pregnancy, which confirms that the fall in total calcium is but an artifact that can usually be ignored. However, that artifactual decline in total calcium means that the serum calcium cannot be relied upon to detect hypercalcemia or hypocalcemia. The ionized calcium should be measured or the albumin-corrected total calcium should be calculated to resolve any uncertainty about what the true serum calcium level is in a pregnant woman. Serum phosphate and magnesium concentrations remain normal during pregnancy.

Figure 2. Schematic illustration of the longitudinal changes in calcium, phosphate, and calciotropic hormone levels that occur during pregnancy and lactation. Normal adult ranges are indicated by the shaded areas.  PTH does not decline in women with low calcium or high phytate intakes, and may even rise above normal. Calcidiol (25OHD) values are not depicted; most longitudinal studies indicate that the levels are unchanged by lactation, but may vary due to seasonal variation in sunlight exposure and changes in vitamin D intake. PTHrP and prolactin surge with each suckling episode, and this is represented by upward spikes. FGF23 values cannot be plotted due to lack of data. Very limited data suggest that calcitriol and PTH may increase during post-weaning, and the lines are dashed to reflect the uncertainty. Adapted with permission from (1).

 

Parathyroid Hormone

 

Parathyroid hormone (PTH) was first measured with assays that reported high circulating levels during pregnancy. The finding of a low total serum calcium and an apparently elevated PTH led to the concept of “physiological secondary hyperparathyroidism in pregnancy.” This erroneous concept persists in some textbooks even today. Those early-generation PTH assays measured many biologically inactive fragments of PTH. When measured with 2-site “intact” assays or the more recent “bio-intact” PTH assays, PTH falls during pregnancy to the low-normal range (i.e. 0-30% of the mean non-pregnant value) during the first trimester, and may increase back to the mid-normal range by term. Most of these recent studies of PTH during pregnancy have examined women from North America and Europe who also consumed calcium-replete diets. In contrast, in women from Asia and Gambia who have very low dietary calcium intakes (and often high intakes of phytate that blocks dietary calcium absorption), the PTH level does not suppress during pregnancy and in some cases it has been found to increase above normal (1).

 

Vitamin D Metabolites

 

25-hydroxyvitamin D or calcifediol (25OHD) readily crosses the rodent hemochorial placenta (9) and appears to cross hemochorial human placentas just as easily because cord blood 25OHD levels generally range from 75% to near 100% of the maternal value (1,5). A common concern is that the placenta and fetus might deplete maternal 25OHD stores, but this does not appear to be the case. Even in severely vitamin D deficient women there was no significant change in maternal 25OHD levels during pregnancy (1,4,10,11).

 

Total calcitriol levels increase two to five-fold early in pregnancy and stay elevated until parturition, whereas measured free calcitriol levels were reported to be increased only in the third trimester (12). However, when the 20-40% increase in vitamin D binding protein and the decline in serum albumin during pregnancy are considered, that calculated free calcitriol should be increased in all three trimesters (11,13-16).There are several unusual aspects about this situation. PTH is normally the main stimulator of the renal 1alpha-hydroxylase; consequently, elevated calcitriol values are usually driven by high PTH concentrations. An exception to this is the ectopic expression of an autonomously functioning 1alpha-hydroxylase by such conditions as sarcoidosis and other granulomatous diseases. Another exception is pregnancy because the rise in calcitriol occurs when PTH levels are typically falling or quite low. Moreover, this increase in calcitriol occurs despite the ability of high levels of fibroblast growth factor-23 (FGF23) to suppress the synthesis and increase the catabolism of calcitriol, as shown in animal models of X-linked hypophosphatemic rickets (17-19). Evidence from additional animal models suggest that it is not PTH but other factors, such as PTH-related protein (PTHrP), estradiol, prolactin and placental lactogen, which drive the 1alpha-hydroxylase to synthesize calcitriol (1).

 

The placenta expresses 1alpha-hydroxylase and it is often assumed that autonomous placental production of calcitriol explains why the maternal calcitriol level doubles; other sources such as maternal decidua and the fetus itself could conceivably contribute to the maternal value. However, it appears that any contributions of placenta and other extra-renal sources to the maternal calcitriol level are trivial. Animal studies indicate that the maternal renal 1alpha-hydroxylase is markedly upregulated during pregnancy (20,21) and that placental expression of 1alpha-hydroxylase is many-fold less than in the maternal kidneys (17). Clinical studies have revealed that anephric women on dialysis have very low circulating calcitriol levels before and during pregnancy (1,22), confirming that maternal kidneys must be the main source of the normal 2 to 5-fold increase in calcitriol during normal pregnancy. Rodent studies, including pregnancies in mice that lack the 1alpha-hydroxylase, have confirmed that there is a small contribution of fetal or placental calcitriol to the maternal circulation (1,23,24). However, it is not enough to account for the marked increase in maternal calcitriol that normally occurs during pregnancy.

 

Calcitonin

 

Serum calcitonin levels are increased during pregnancy and may derive from maternal thyroid, breast, decidua, and placenta. The importance of these extrathyroidal sites of calcitonin synthesis has been shown by serum calcitonin levels rising from undetectable to normal values in totally thyroidectomized women who become pregnant (25). Whether calcitonin plays an important role in the physiological responses to the calcium demands of pregnancy is unknown. It has been proposed to protect the maternal skeleton against excessive resorption during times of increased calcium demand; however, there are no clinical studies that have addressed this question. Study of pregnant women who lack the gene for calcitonin or the calcitonin receptor would be informative, but no such women have been identified. On the other hand, mice that lack the gene for calcitonin have normal calcium and bone metabolism during pregnancy (26,27).

 

 

PTHrP concentrations steadily increase in the maternal circulation, reaching the highest levels in the third trimester (1,11). The assays most commonly used in these studies detected PTHrP peptides encompassing amino acids 1-86, but PTHrP is a prohormone. It is cleaved into multiple N-terminal, mid-molecule, and C-terminal peptides, which differ in their biological activities and specificities. None of these peptides have been systematically measured during pregnancy. The commonly available PTHrP1-86 assays do not measure PTHrP1-34, which is likely the most abundant of the active, PTH-like, N-terminal forms of this protein. Moreover, in many clinical studies and case reports it is evident that inappropriate blood samples were used for assaying PTHrP. Special collection and handling are required because PTHrP is rapidly cleaved and degraded in serum. Blood samples should be collected in tubes containing EDTA and aprotinin (a protease inhibitor), kept chilled, and then centrifuged, separated, and frozen within 15 minutes of sample collection. Even with these rigorous standards, PTHrP has been found to begin degrading by 15 minutes after sample collection (28). Many studies did not use this method of sample collection and preparation, but used sera that had been allowed to clot at room temperature for up to 60 minutes. This likely explains why such studies found undetectable serum concentrations of PTHrP, as compared to those that studied the plasma concentration of PTHrP during pregnancy. Individual case reports are also fraught with this problem, since standard blood collection protocols for hospital laboratories do not use the special handling described above.

 

PTHrP is produced by many tissues in the fetus and mother; consequently, it is uncertain which source(s) account for the rise in PTHrP in the maternal circulation. However, the placenta and breasts are likely the major sources of PTHrP. Whether circulating PTHrP has a role in maternal physiology during pregnancy is unclear, but its rise may stimulate the renal 1alpha-hydroxylase and contribute to the increase in calcitriol and, indirectly, the suppression of PTH. However, PTHrP appears less potent than PTH in stimulating the 1alpha-hydroxylase (29,30), which is why its contribution to the rise in calcitriol during pregnancy is uncertain. On the other hand, several case reports have clearly implicated breast- and placental-derived PTHrP as a cause of maternal hypercalcemia with elevated PTHrP and undetectable PTH, a condition called pseudohyperparathyroidism of pregnancy (see below). Since breasts and placenta were sources of excess PTHrP in these cases, those two tissues seem likely to be dominant sources of PTHrP during normal pregnancy. Moreover, since excess PTHrP impacted maternal calcium homeostasis to cause hypercalcemia in these cases, it is possible that the more modest elevations in circulating PTHrP seen during normal pregnancy also affect maternal calcium homeostasis.

 

A carboxyl-terminal form of PTHrP (so-called “osteostatin”) has been shown to inhibit osteoclastic bone resorption in vitro, and thus the notion arises that PTHrP may play a role in protecting the maternal skeleton from excessive resorption during pregnancy (31). Animal studies have shown that PTHrP has other roles during gestation such as regulating placental calcium transport in the fetus (1,32). Maternally produced PTHrP is not likely to regulate placental calcium transport since the protein should not be able to cross the placenta (1,5); instead, it is PTHrP produced within the fetus and placenta that is responsible for regulating placental calcium transport.

 

Fibroblast Growth Factor-23 (FGF23)

 

Intact FGF23 doubles its concentration in the mother’s circulation during rodent pregnancies (17-19), but whether such levels change during human pregnancy has not been reported. Within 24 hours after delivery, mean values in postpartum women were similar to non-pregnant women (33).

 

 

Other Hormones

 

This section has focused on changes in static concentrations of minerals and the known calciotropic hormones; there are no studies testing hormonal reserves or response to challenges such as hypocalcemia or hypophosphatemia. Pregnancy also induces significant changes in other hormones known to affect calcium and bone metabolism, including sex steroids, prolactin, placental lactogen, oxytocin, leptin, and IGF-1. Each of these – and possibly other hormones not normally associated with mineral and bone metabolism – may have direct or indirect effects on mineral homeostasis during pregnancy. However, this aspect of the physiology of pregnancy has been largely unexplored to date.

 

Prolactin and placental lactogen both increase during pregnancy and activate prolactin receptors. Osteoblasts express prolactin receptors, and prolactin receptor deficient mice show decreased bone formation (34). Suppressing the prolactin level with bromocriptine blunted a pregnancy-related gain in bone mineral content in rats (35). These data are consistent with the notion that prolactin or placental lactogen regulate skeletal metabolism during pregnancy. Furthermore, prolactin can indirectly affect skeletal metabolism by stimulating PTHrP synthesis and release from the breasts (36-38).

 

Circulating oxytocin levels also rise during pregnancy (39), and the oxytocin receptor is expressed by osteoclasts and osteoblasts (40). Male and female mice lacking oxytocin or its receptor have an osteoporotic phenotype with low bone formation (41). Oxytocin has been shown to stimulate osteoblast differentiation and function, stimulate osteoclast formation, but inhibit osteoclast function and skeletal resorption (41,42). Taken together, these data predict that oxytocin may regulate bone metabolism during pregnancy, but this has not been directly studied in vivo.

 

Intestinal Calcium and Phosphate Absorption

 

Intestinal absorption of calcium doubles as early as 12 weeks of human pregnancy, as shown by clinical studies that used stable isotopes of calcium, and by other calcium balance studies (1). This increase in calcium absorption appears to be the major maternal adaptation to meet the fetal need for calcium. It has been generally believed that the doubling or tripling of calcitriol levels explains the increased intestinal calcium absorption and concurrent increases in the intestinal expression of calbindin9k-D (S100G), TRPV6, Ca2+-ATPase (PMCA1), and other genes and proteins involved in calcium transport. However, intestinal calcium absorption doubles in the first trimester, well before the rise in free calcitriol levels during the third trimester. Animal studies have indicated that placental lactogen, prolactin, and other factors may stimulate intestinal calcium absorption (1) and that calcitriol or the vitamin D receptor are not required for intestinal calcium absorption to increase during pregnancy (1,23,43-45).

 

The peak fetal demand for calcium does not occur until the third trimester, and so it is unclear why intestinal calcium absorption should be upregulated in the first trimester. It may allow the maternal skeleton to store calcium in advance of the peak demands for calcium that occur later in pregnancy and lactation; some studies in rodents have shown this to be the case with the bone mineral content rising significantly before term (17,26,45). Women have also been found to be in a positive calcium balance by mid-pregnancy (46), likely due to the effect of increased intestinal calcium absorption on skeletal mineralization.

 

Intestinal phosphate absorption also undergoes a doubling during rodent and other mammalian pregnancies (1), and presumably human pregnancy as well. However, no clinical studies have studied this.

 

Renal Handling of Calcium

 

The doubling of intestinal calcium absorption in the first trimester means that the extra calcium must be passed to the fetus, deposited in the maternal skeleton, or excreted in the urine. Renal calcium excretion is increased as early as the 12th week of gestation and 24-hour urine values (corrected for creatinine excretion) often exceed the normal range. Conversely, fasting urine calcium values are normal or low, confirming that this hypercalciuria is a consequence of the enhanced intestinal calcium absorption (1). This is absorptive hypercalciuria and will not be detected by spot or fasting urine samples that have been corrected for creatinine concentration. Absorptive hypercalciuria contributes to the increased risk of kidney stones during pregnancy.

 

This absorptive hypercalciuria also renders nomograms of fractional calcium excretion invalid for the diagnosis of familial hypocalciuric hypercalcemia during pregnancy (47,48).

 

Pharmacological doses of calcitonin promote renal calcium excretion, but whether the physiologically elevated levels of calcitonin during pregnancy promote renal calcium excretion is unknown.

 

Hypocalciuria during pregnancy has been associated with pre-eclampsia, pregnancy-induced hypertension, and low (equal to non-pregnant values) serum calcitriol (49-52). These changes appear largely secondary to disturbed renal function and reduced creatinine clearance, rather than being causes of the hypertension. However, calcium supplementation reduces the risk of pre-eclampsia in women within the lowest quintile of calcium intake, and so there is a pathophysiological link between calcium metabolism and pregnancy-induced hypertension (1).

 

Skeletal Calcium Metabolism and Bone Density/Bone Marker Changes

 

As mentioned earlier, some studies in rodents indicate that bone mineral content increases during pregnancy, and other studies have shown that histomorphometric parameters of bone turnover are increased at this time. Systematic studies of bone histomorphometry from pregnant women have not been done. However, one study of 15 women who electively terminated a pregnancy at 8-10 weeks found bone biopsy evidence of increased bone resorption, including increased resorption surface and increased numbers of resorption cavities (53). These findings were not present in biopsies obtained from 13 women at term, or in the non-pregnant controls. This study bears repeating but it does suggest that early pregnancy induces skeletal resorption.

 

Bone turnover markers – by-products of bone formation and resorption that can be measured in the serum or urine – have been systematically studied during pregnancy in multiple studies (1). In the non-pregnant adult with osteoporosis these bone markers are fraught with significant intra- and inter-individual variability which limit their utility on an individual basis. There are additional problems with the use of bone markers during pregnancy, including lack of pre-pregnancy baseline values; hemodilution; increased GFR; altered creatinine excretion; placental, uterine and fetal contributions; degradation and clearance by the placenta; and lack of diurnally timed or fasted specimens. Bone resorption has been assessed using urinary (deoxypyridinoline, pyridinoline, and hydroxyproline) and serum (C-telopeptide) markers, and the consistent finding is that bone resorption appears increased from early or mid-pregnancy (1). Conversely, bone formation has been assessed by serum markers (osteocalcin, procollagen I carboxypeptides and bone specific alkaline phosphatase) that were generally not corrected for hemodilution or increased GFR. These bone formation markers are decreased in early or mid-pregnancy from pre-pregnancy or non-pregnant values and rise to normal or above before term (1). The lack of correction for hemodilution and increased GFR means that the apparent decline in bone formation markers may actually occur despite no change or even an increase in bone formation. It should be noted that total alkaline phosphatase rises early in pregnancy due to the placental fraction and is not a useful marker of bone formation during pregnancy.

 

Overall, the scant bone biopsy data and the results of bone turnover markers suggest that bone resorption is increased from as early as the 10th week of pregnancy, whereas bone formation may be suppressed (if the bone formation marker results are correct) or normal (if the bone formation markers are artifactually suppressed due to the aforementioned confounding factors) (1). Notably there is little maternal-fetal calcium transfer occurring in the first trimester, nor is there a marked increase in turnover markers during the third trimester when maternal-fetal calcium transfer is at a peak. These findings may simply underscore that resorption of the maternal skeleton is a minor contributor to calcium homeostasis during pregnancy, whereas the upregulation of intestinal calcium absorption is the main mechanism through which the fetal demand for calcium is met.

 

Another way of assessing whether the maternal skeleton contributes to calcium regulation during pregnancy is to measure bone mineral content or density. A few sequential areal bone density (aBMD) studies have been done using older techniques (single and/or dual-photon absorptiometry, i.e., SPA and DPA), and none with newer techniques (DXA or qCT) due to concerns about fetal radiation exposure. Studies of aBMD are known to be confounded by changes in body composition, weight and skeletal volumes, and all three of these factors change during normal pregnancy. The longitudinal studies used SPA or DPA and found no significant change in cortical or trabecular aBMD during pregnancy (1). Most recent studies examined 16 or fewer subjects with DXA prior to planned pregnancy (range 1-18 months prior, but not always stated) and after delivery (range 1-6 weeks postpartum) [studies reviewed in detail in (54)]. One study found no change in lumbar spine aBMD measurements obtained pre-conception and within 1-2 weeks post-delivery, whereas the other studies reported 4-5% decreases in lumbar aBMD with the postpartum measurement taken between 1-6 weeks post-delivery. A large study from Denmark obtained DXA measurements of hip, spine, and radius at baseline (up to 8 months before pregnancy) and again within 15 days of delivery in 73 women (55). DXA of the radius was also obtained once each trimester. BMD decreased between pre-pregnancy and post-pregnancy by 1.8% at the lumbar spine, 3.2% at the total hip, 2.4% at the whole body, 4% at the ultradistal forearm, and 1% at the total forearm, whereas it increased by 0.5% at the proximal 1/3 forearm (55). All women went on to breastfeed, which means that the final BMD values were confounded by lactation-induced bone loss (see lactation section). These changes in BMD were statistically significant when compared to 57 non-pregnant controls who also had serial measurements done, but the magnitudes of change were small, and would not be considered statistically significant for an individual woman.

 

Ultrasound measurements of the os calcis and fingers have been examined in other longitudinal studies, which reported a progressive decrease in indices that correlate with volumetric BMD (1,54). Whether observed changes in the os calcis accurately indicate a true or clinically meaningful decrease in volumetric BMD, or imply that losses of BMD are occurring in the spine or hip during pregnancy, is not known. The reliability or relevance of data obtained from ultrasound is questionable since this technique failed to detect any change in volumetric BMD at the os calcis during lactation (56), even though substantial bone loss occurs at the spine and hip during lactation (see lactation section).

 

Overall, the existing studies have insufficient power to allow a firm conclusion as to the extent of bone loss that might occur during pregnancy, but it seems likely (especially when data from the Danish study are considered) that modest bone loss occurs, which would be difficult to discern on an individual basis. In the long term, pregnancy does not impair skeletal strength or lead to reduced bone density. Several dozen epidemiological studies of osteoporotic and osteopenic women have failed to find a significant association of parity with bone density or fracture risk (1,57), and many have shown a protective effect of parity (58-75).

 

DISORDERS OF CALCIUM AND BONE METABOLISM DURING PREGNANCY

Osteoporosis in Pregnancy

 

The occasional woman will present with a fragility fracture during the third trimester or puerperium, and low bone mineral density may be confirmed by DXA (76). In such cases it is not possible to exclude the possibility that low bone density or skeletal fragility preceded pregnancy. In favor of a genetic predisposition is the report that among 35 women who presented with pregnancy associated osteoporosis, there was a higher than expected prevalence of fragility fractures in their mothers (77). It is conceivable that pregnancy may induce significant skeletal losses in some women and, thereby, predispose to fracture. The normal pregnancy-induced changes in mineral metabolism may cause excessive resorption of the skeleton in selected cases, and other factors such as low dietary calcium intake and vitamin D insufficiency may contribute to skeletal losses (76). A high rate of bone turnover is an independent risk factor for fragility fractures outside of pregnancy, and so the apparently increased bone resorption observed during pregnancy may increase fracture risk. In favor of pregnancy inducing fragility through excess skeletal losses is an observational study of 13 women with pregnancy-associated osteoporosis who were followed for up to eight years. Since the bone mineral density at the spine and hip increased significantly during follow-up in these women, the investigators concluded that a large part of the bone loss must have been related to the pregnancy itself (78). Taken together, fragility fractures in pregnancy or the puerperium may result from the combination of abnormal skeletal microarchitecture prior to pregnancy and increased bone resorption during pregnancy.

 

Osteoporosis in pregnancy usually presents in a first pregnancy and there is no apparent increased risk with higher parity (76,78-80). About 60% of patients present with lower thoracic or lumbar pain that may be quite debilitating due to vertebral collapse (78-80). Most cases show normal serum chemistries and calciotropic hormone levels, but in a few, secondary causes of bone loss could be identified, including anorexia nervosa, hyperparathyroidism, osteogenesis imperfecta, inactivating mutations in LRP5, premature ovarian failure, and corticosteroid or heparin therapy (76,77,79-83). Bone biopsies have confirmed osteoporosis and the absence of osteomalacia, while bone density Z-scores are often lower than expected (78-80). The pain resolves spontaneously over several weeks in most cases while the bone density has been reported to improve in most women following pregnancy. Fractures tend not to recur in subsequent pregnancies. Thus, although myriad medical treatments (bisphosphonates, estrogen, testosterone, calcitonin, teriparatide, denosumab) and surgical interventions (kyphoplasty, vertebroplasty, spinal fusion) have been used in individual cases of pregnancy-associated osteoporosis (76), the tendency for this condition to spontaneously improve may make pharmacological treatment unjustified except for the severest cases. At the least, it may be prudent to wait 12-18 months to determine the extent to which the BMD recovers on its own after a pregnancy-associated vertebral fracture (76).

 

A distinct condition is focal, transient osteoporosis of the hip (76). This is rare, self-limited, and probably not a manifestation of altered calciotropic hormone levels or mineral balance during pregnancy. Instead, it may be a consequence of local factors. A variety of theories have been offered to explain this condition, including femoral venous stasis due to pressure from the pregnant uterus, Sudeck’s atrophy or reflex sympathetic dystrophy (causalgia), ischemia, trauma, viral infections, marrow hypertrophy, immobilization, and fetal pressure on the obturator nerve. These patients present with unilateral or bilateral hip pain, limp and/or hip fracture in the third trimester or puerperium (76,84-86). Radiographs and DXA indicate radiolucency and reduced bone density of the symptomatic femoral head and neck, while MRI demonstrates increased water content of the femoral head and the marrow cavity; a joint effusion may also be present. The differential diagnosis of this condition includes inflammatory joint disorders, avascular necrosis of the hip, bone marrow edema, and reflex sympathetic dystrophy. It is a self-limiting condition with both symptoms and radiological appearance resolving within two to six months post-partum; conservative measures including bed rest are usually all that is required during the symptomatic phase (76). Of course, fractures of an involved femur require urgent arthroplasty or hip replacement. The condition recurs in about 40% of cases (not necessarily during pregnancy), unlike osteoporosis involving the spine, and this has prompted prophylactic hip arthroplasty to be done in a few cases where the opposite hip appears to be affected.

 

Primary Hyperparathyroidism

 

This is probably a rare condition but there are no firm data available on its prevalence. Two case series indicated that parathyroidectomies were done during pregnancy in about 1% of all cases (87,88). The diagnosis will be obscured by the normal pregnancy-induced changes that lower the total serum calcium and suppress PTH; however, finding the ionized or albumin-corrected calcium to be increased, and PTH to be detectable, should indicate primary hyperparathyroidism in most cases.

 

Primary hyperparathyroidism during pregnancy has been reported to cause a variety of symptoms that are not specific to hypercalcemia, and cannot be distinguished from those occurring in normal pregnancy (nausea, vomiting, renal colic, malaise, muscle aches and pains, etc.). Conversely the literature associates primary hyperparathyroidism with an alarming rate of adverse outcomes in the fetus and neonate, including a 10-30% rate for each of spontaneous abortion, stillbirth, and perinatal death, and 30-50% incidence of neonatal tetany (88-92). These high rates were reported in older literature; more recent case series suggest that the rates of stillbirth and neonatal death are each about 2%, while neonatal tetany occurred in 15% (89). The adverse postnatal outcomes are thought to result from suppression of the fetal and neonatal parathyroid glands; this suppression may be prolonged after birth for 3-5 months (89) and in some cases it has been permanent (89,91,93).

 

To prevent these adverse outcomes, surgical correction of primary hyperparathyroidism during the second trimester has been almost universally recommended. Several case series have found elective surgery to be well tolerated, and to dramatically reduce the rate of adverse events when compared to the earlier cases reported in the literature. In a series of 109 mothers with hyperparathyroidism during pregnancy who were treated medically (N=70) or surgically (N=39), there was a 53% incidence of neonatal complications and 16% incidence of neonatal deaths among medically treated mothers, as opposed to a 12.5% neonatal complications and 2.5% neonatal deaths in mothers who underwent parathyroidectomy (88). Choosing the second trimester allows organogenesis to be complete in the fetus and to avoid the poorer surgical outcomes and risk of preterm birth associated with surgery during the third-trimester (89,92,94,95).

 

Many women in the earliest published cases had a more severe form of primary hyperparathyroidism that is not often seen today (symptomatic, with nephrocalcinosis and renal insufficiency). While mild, asymptomatic primary hyperparathyroidism during pregnancy has been followed conservatively with successful outcomes, complications continue to occur, so that, in the absence of definitive data, surgery during the second trimester remains the most common recommendation (96). Milder cases diagnosed during the third trimester may be observed until delivery, although rapid and severe postpartum worsening of the hypercalcemia can occur (95,97-100). This postpartum “parathyroid crisis” occurs because the placental calcium outflow has been lost, while surging PTHrP production in the breasts means an additional factor stimulating bone resorption.

 

There are no definitive medical management guidelines for hyperparathyroidism during pregnancy apart from ensuring adequate hydration and correction of electrolyte abnormalities (96). Pharmacologic agents to treat hypercalcemia have not been adequately studied in pregnancy.  Calcitonin does not cross the placenta and has been used safely (96). Oral phosphate has also been used but is limited by diarrhea, hypokalemia, and risk of soft tissue calcifications. Bisphosphonates are relatively contra-indicated because of their potential adverse effects on fetal endochondral bone development, although a review of 78 cases of bisphosphonate use in pregnancy found no obvious problems in most cases (101). Denosumab crosses the placenta and has been shown to cause an osteopetrotic-like phenotype in fetal cynomolgus monkeys and rats (102,103), and so it should be avoided in human pregnancy. High-dose magnesium has been proposed as a therapeutic alternative which should decreases serum PTH and calcium levels by activating the calcium sensing-receptor, but it has not been adequately studied for this purpose (104,105). The calcium receptor agonist cinacalcet, which is used to suppress PTH and calcium in nonpregnant subjects with primary or secondary hyperparathyroidism and parathyroid carcinoma, has also been tried in pregnancy (106-109). However, since the calcium receptor is expressed in the placenta and regulates fetal-placental calcium transfer (110), the possibility of adverse effects of cinacalcet on the fetus and neonate remain a concern.

 

In any case that was followed medically, parathyroidectomy is recommended to be done postpartum, with monitoring in place to detect a postpartum hypercalcemic crisis.

 

Familial Hypocalciuric Hypercalcemia

 

Inactivating mutations in the calcium-sensing receptor cause this autosomal dominant condition which presents with hypercalcemia and hypocalciuria (111).  As noted above, fractional excretion of calcium is not reduced during pregnancy in this condition, because it is overridden by the physiological increase in intestinal calcium absorption that in turn causes hypercalciuria (47,48). Pregnancy in women with familial hypocalciuric hypocalcemia may be uneventful for the mother, but the maternal hypercalcemia has caused fetal and neonatal parathyroid suppression with subsequent tetany in both normal and hemizygous children (5,112,113). A hemizygous neonate will later develop benign hypercalcemia, but if the baby has two inactivating mutations of the calcium receptor (most commonly from both parents being hemizygous for FHH), then the neonate may suffer a life-threatening hypercalcemic crisis (5).

 

Hypoparathyroidism

 

Hypoparathyroidism during pregnancy usually presents as a pre-existing condition that the clinician is challenged to manage. The natural history of hypoparathyroidism during pregnancy is confusing due to conflicting case reports in the literature [reviewed in (1,3)].  Early in pregnancy, some hypoparathyroid women have fewer hypocalcemic symptoms and require less supplemental calcium. This is consistent with a limited role for PTH in the pregnant woman, and suggests that an increase in calcitriol and/or increased intestinal calcium absorption occurs in the absence of PTH. However, other case reports clearly indicate that some pregnant hypoparathyroid women required increased calcitriol replacement in order to avoid worsening hypocalcemia. Adding to the confusion is that in some case reports, it appears that the normal, artifactual decrease in total serum calcium during pregnancy was the parameter that led to treatment with increased calcium and calcitriol supplementation; fewer cases reported that dose increments in calcitriol and calcium were made because of maternal symptoms of hypocalcemia or tetany, or objective evidence of true hypocalcemia (ionized or albumin-corrected calcium). It is not possible to know in advance who will improve and who will worsen during pregnancy; the task is to maintain the albumin-corrected serum calcium or ionized calcium in the normal range for the duration of pregnancy. Maternal hypocalcemia due to hypoparathyroidism must be avoided because it has been associated with intrauterine fetal hyperparathyroidism and fetal death. Conversely, overtreatment must be avoided because maternal hypercalcemia is associated with the fetal and neonatal complications described above under Primary Hyperparathyroidism. Calcitriol and 1α-calcidiol are recommended due to their shorter half-lives, lower risk of toxicity, and the clinical experience with these agents.

 

Late in pregnancy, hypercalcemia may occur in hypoparathyroid women unless the calcitriol dosage and supplemental calcium are substantially reduced or discontinued. This effect appears to be mediated by the increasing levels of PTHrP in the maternal circulation in late pregnancy. Conversely, one case report of hypoparathyroidism in pregnancy found that there was a transient interval of increased requirement for calcitriol immediately after delivery and before lactation was fully underway (114). This may be the result of loss of placental sources of PTHrP followed by a surge in production of PTHrP by the lactating breast (see lactation section, below).

 

Pseudohypoparathyroidism

 

Pseudohypoparathyroidism is a genetic disorder causing resistance to PTH and manifest by hypocalcemia, hypophosphatemia, and high PTH levels. In two case reports of pseudohypoparathyroidism during pregnancy, the serum calcium normalized, PTH reduced by half, and calcitriol increased 2- to 3-fold (115). The mechanism by which these changes occur despite pseudohypoparathyroidism remains unclear. If maternal hypocalcemia persists during pregnancy, pseudohypoparathyroidism can lead to the same adverse fetal outcomes that have been associated with maternal hypoparathyroidism, including parathyroid hyperplasia, skeletal demineralization, and fractures (116,117). The maternal calcium concentration must be maintained in the normal range to avoid these fetal outcomes.

 

Pseudohyperparathyroidism

 

As mentioned above, pseudohyperparathyroidism is hypercalcemia that is caused by physiological release of PTHrP driving increased skeletal resorption, akin to how PTHrP also causes hypercalcemia of malignancy. In one such case the breasts were the source of PTHrP because the hypercalcemia and elevated PTHrP did not abate until a bilateral reduction mammoplasty was carried out (118,119). It has occurred in women who simply have large breasts (120,121). In another case the hypercalcemia, elevated PTHrP, and suppressed PTH reversed within a few hours of an urgent C-section, thereby confirming the placenta as the source (122). In all cases of pseudohyperparathyroidism, it should be anticipated that the cord blood calcium will also be increased, and that the baby is at risk for fetal and neonatal hypoparathyroidism with hypocalcemic tetany.

 

Vitamin D Deficiency and Insufficiency

 

There are no comprehensive studies of the effects of vitamin D deficiency or insufficiency on human pregnancy, but the available data from small clinical trials of vitamin D supplementation, observational studies, and case reports suggest that, consistent with animal studies, vitamin D insufficiency and deficiency is not associated with any worsening of maternal calcium homeostasis (this topic is reviewed in detail in (1,4,7). Maternal hypocalcemia is milder with vitamin D deficiency due to the effects of secondary hyperparathyroidism to increase skeletal resorption and renal calcium reabsorption. Consequently, hypocalcemia due to vitamin D deficiency has not been clearly associated with the same adverse fetal outcomes that maternal hypoparathyroidism causes (reviewed in detail in (5,123)). The fetal effects of vitamin D deficiency, inability to form calcitriol, and absence of the vitamin D receptor have been examined across several animal species and all have indicated that the fetus will have a normal serum calcium and fully mineralized skeleton at term (reviewed in detail in (5,123)). Neonatal hypocalcemia and rickets can occur in infants born of mothers with severe vitamin D deficiency, but it is usually in the weeks to months after birth that this presents, after intestinal calcium absorption becomes dependent on calcitriol.

 

There has been much interest in studies that have inconsistently associated third-trimester measurements of 25OHD, or estimated vitamin D intakes during pregnancy or the first year after birth, with possible extraskeletal benefits in the mother (reduced bacterial vaginosis, pre-eclampsia, pre-term delivery) or in the offspring (lower incidence of type 1 diabetes, greater skeletal mineralization, etc.). These associational studies won’t be discussed in detail (some are cited in: (1,5,124)) because they are confounded by factors which contribute to lower 25OHD levels (maternal overweight/obesity, lower socioeconomic status, poor nutrition, lack of exercise, etc.). It is necessary to test these associations in randomized clinical trials that compare higher versus lower intakes of vitamin D during pregnancy. At present the results of the associational studies are insufficient to warrant prescribing higher intakes of vitamin D during pregnancy to prevent these postulated outcomes.

 

Among many clinical trials of vitamin D supplementation that have been carried out (1), only a few have included over a 100 study participants who were vitamin D deficient at entry, while other recent studies that gained press attention did not include many vitamin D deficient subjects at all. Among the trials with over 100 participants (14,125-132), the two largest were from Bangladesh and UK with over 1,000 participants (131,132). Baseline maternal 25OHD levels were lowest (20-29 nmol/L) in trials from Bangladesh, UK, Iran, and UAE, and in the 40-60 nmol/L range in the remainder. Interventions consisted of placebo/no treatment versus low dose (400 IU/day) or high dose (1,000-5,000 IU/day) vitamin D supplementation, initiated before mid-pregnancy, and maintained until delivery.  For most trials the primary outcomes were simply maternal and neonatal-cord blood 25OHD and calcium. The most recent and largest study was from Bangladesh, and the primary outcome was pre-specified as infant length-for-age z-scores at 1 year of age (132). Offspring anthropometric parameters and/or bone mineral content were pre-specified only in a few of the remaining studies (128,130,131).

 

In all studies vitamin D supplementation increased maternal serum and cord blood 25OHD, but there was no overall effect on cord blood calcium. The largest achieved difference in a single study was 16 nmol/L (6.4 ng/mL) in the untreated and 168 nmol/L (67 ng/mL) in vitamin D-supplemented mothers at term; however, there was no obstetrical or fetal benefit (125). The incidence of neonatal hypocalcemia was reduced in offspring of vitamin-D treated mothers, reflecting the role of vitamin D/calcitriol to stimulate postnatal intestinal calcium absorption (125). In the large Bangladesh study, there were no significant differences in infant anthropometrics or any other fetal, neonatal or maternal outcomes (132). In one US-based study there was no benefit on mode of delivery, gestational age at delivery, and preterm birth (14), while in another there was no benefit on mode of delivery, C-section rates, adverse events, hypertension, infection, gestational diabetes, still birth, gestational age at delivery, or combinations of these outcomes (127). The UK MAVIDOS trial reported no obstetrical benefit, and no benefit to any of the primary (neonatal bone area, BMC, and BMD within the first 10-14 days after birth) or secondary outcomes (anthropometric and body composition parameters within 48 hours of birth). However, it received much publicity for a demonstrated increase in BMC and BMD in winter-born neonates of vitamin D-supplemented vs. placebo-treated mothers (131). Because the neonatal skeleton accretes 100 mg/day of mineral content after birth, this result may reflect improved intestinal mineral delivery over 14 days after birth, rather than a prenatal effect on skeletal mineralization (1,133,134). Curiously, autumn-born neonates of vitamin D-supplemented vs. placebo-treated mothers showed an adverse trend of similar magnitude on BMC and BMD, which suggests possible harm from vitamin D supplementation, or chance findings due to small numbers within the sub-groups (134). These sub-group analyses of treatment by season interaction were not specified outcomes in the trial registries (ISRCTN 82927713 and EUDRACT 2007-001716-23). In the UK study that achieved the greatest difference in 25OHD levels between untreated and vitamin D-treated mothers and babies, there was a trend for more small for gestational age babies born to mothers who did not receive antenatal vitamin D supplementation (28% vs. 15%, p<0.1), but the study was not powered for this outcome (125). In studies from the UAE, and Iran there was also no benefit on obstetrical outcomes (variably, mode of delivery, C-section rates, adverse events, stillbirths, gestational age at delivery) or neonatal anthropometric measurements and bone mass measurements (126,128,130).

 

The lack of any beneficial effect on maternal, immediate fetal/neonatal and neonatal outcomes (anthropometrics and cord blood calcium), even in studies that included mothers with some of the lowest 25OHD levels (125,128,130,132), suggests that vitamin D supplementation during pregnancy confers no benefit to the neonate.  The most recent study was well-powered to demonstrate a beneficial effect on infant length and other fetal/neonatal outcomes, but did not yield any significant results, despite low vitamin D levels in the mothers at study entry (132).

 

Systematic reviews have used these and the results of smaller trials to examine the effect of vitamin D supplementation during pregnancy on maternal, fetal, and neonatal extra-skeletal outcomes (135-140). Vitamin D supplementation had no significant effect on pre-eclampsia in four (136,138-140) and a positive effect in two reviews (135,137), while combined vitamin D and calcium supplementation reduced the incidence of pre-eclampsia in three systematic reviews (135-137). No consistent effect was seen on other outcomes such as preterm birth, low birth weight, small for gestational age, infections, C-section rate, and newborn anthropometrics.

 

Overall, available data are insufficient from the individual clinical trials or these systematic reviews to conclude that vitamin D supplementation during pregnancy confers any proven obstetrical benefits.

 

Genetic Vitamin D Resistance Syndromes

 

Case reports and series have provided insight into the effect of pregnancy on genetic disorders of vitamin physiology. Pregnancies have generally been unremarkable in women with vitamin D-dependent rickets type 1 (VDDR-I) which is due to absence of Cyp27b1, and in women with VDDR-II that is due to absence of functional VDRs (141-143). In one such uneventful VDR-II pregnancy, the pre-pregnancy intake of supplemental calcium (800 mg) and high-dose calcitriol were maintained until her clinicians increased the dose of calcitriol later in pregnancy “because of the knowledge that the circulating 1,25-(OH)2D concentration normally rises during pregnancy,” and not because of any change in albumin-adjusted serum calcium (142). Consequently, it’s unclear that any change was needed. However, it is reasonable to increase the dose of calcitriol to mirror the increase that happens during normal pregnancy. In women with VDDR-I, the dose of calcitriol was unchanged in one-third of pregnancies but increased 1.5 to 2-fold in others (141).

24-Hydroxylase Deficiency

 

Loss of the catabolic effects of 24-hydroxylase causes high calcitriol and mild hypercalcemia in non-pregnant adults, which may be asymptomatic (144). But during pregnancy, the physiological 2 to 5-fold increase in calcitriol is unopposed by catabolism, which causes an exaggerated increase in calcitriol, followed by symptomatic hypercalcemia. Hypercalcemia can be quite marked, with suppressed or undetectable PTH, and calcitriol concentrations that exceed what is expected for pregnancy (145-147). Pregnant patients may also present with nephrolithiasis or acute pancreatitis (147,148).

 

Treatment of the hypercalcemia is difficult because the agents that could be used are not approved for pregnancy. Increased intestinal calcium absorption is the direct cause, and so use of increased hydration and a modestly restricted calcium diet, combined with phosphate supplementation to bind dietary calcium, are relatively safe management approaches. If PTH increases above normal, then dietary calcium restriction should be lessened to prevent maternal bone resorption and fetal secondary hyperparathyroidism. Other pharmacologic therapy should be reserved for the most severe cases and used with caution. This includes oral glucocorticoids, loop diuretics, calcitonin, and bisphosphonates; denosumab should not be used because of teratogenic effects observed in cynomolgus monkeys and mice (102,103). Cinacalcet will not be useful because PTH will already be suppressed due to the combined effects of pregnancy and hypercalcemia.

 

Low or High Calcium Intake

 

Through the doubling of intestinal calcium absorption during pregnancy, women have the ability to adapt to wide ranges of calcium intakes and still meet the fetal demand for calcium. It is conceivable that extremely low maternal calcium intakes could impair maternal calcium homeostasis and fetal mineral accretion, but there are scant clinical data examining this possibility (149). Among women with low dietary calcium intake, there are differing results as to whether or not calcium supplementation during pregnancy improved maternal or neonatal bone density (150). There is short term evidence that bone turnover markers were reduced when 1.2 gm of supplemental calcium was given for 20 days to 31 Mexican woman at 25-30 weeks of gestation; their mean dietary calcium intake was 1 gm (151). In a double-blind study conducted in 256 pregnant women, 2 gm of calcium supplementation improved bone mineral content only in the infants of supplemented mothers who were in the lowest quintile of calcium intake (152). Among cases of fragility fractures presenting during pregnancy, some women had very low calcium intakes (<300 mg per day), and in such cases substantial maternal skeletal resorption must be invoked in order to meet the fetal calcium requirement and maintain the maternal serum calcium concentration (76).

 

Overall the physiological changes in calcium and bone metabolism that usually occur during pregnancy and lactation are likely to be sufficient for fetal bone growth and breast-milk production in women with reasonably sufficient calcium intake (153). However, the use of calcium supplementation for pregnant women with low calcium intake can be defended by the links between low calcium intake and both preeclampsia and hypertension in the offspring (149). Clinical trials and meta-analyses have also demonstrated that supplemental calcium will reduce the risk of preeclampsia in women with low dietary calcium intakes, but not in those with adequate intake (154-157).

 

High calcium intake, similar to primary hyperparathyroidism, can cause increased intestinal calcium absorption, maternal hypercalcemia, increased transplacental flow of calcium, and suppression of the fetal parathyroids. Cases of neonatal hypoparathyroidism have been reported wherein women consumed 3 to 6 grams of elemental calcium daily as antacids or anti-nauseates (1).

 

Hypercalcemia of Malignancy

 

Hypercalcemia of malignancy is usually a terminal condition. When it has been diagnosed during pregnancy, in some cases the baby has been spared from chemotherapy, whereas in other cases the pregnancy was terminated (or ignored) so that chemotherapy could be administered in an attempt to prolong the woman’s life. Half of published case reports haven’t even mentioned the baby’s outcome. A baby born of a mother with humoral hypercalcemia of malignancy may have a high concentration of calcium in cord blood, and is at high risk for fetal and neonatal hypoparathyroidism with hypocalcemic tetany.

 

FGF-23 Disorders

 

X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene, which lead to high circulating levels of FGF23. In turn this causes hypophosphatemia with rickets or osteomalacia. Pregnancies were normal in a mouse model of XLH. In particular, despite very high circulating levels of FGF23, which normally downregulate calcitriol synthesis and increase its catabolism, maternal serum calcitriol increased to the high levels normally seen during pregnancy (19,158). This rise in calcitriol should contribute to increased intestinal calcium and phosphate absorption. Several case reports documented persistent hypophosphatemia during pregnancy in women with XLH, but no adverse outcomes (159,160). Nevertheless, it is generally recommended to supplement with calcitriol and phosphate to keep the serum phosphate near normal during pregnancy.

 

Hyperphosphatemic disorders due to loss of FGF23 action have not been studied during human pregnancy, and animal data are also lacking because these conditions are lethal before sexual maturity. Renal insufficiency or failure causes hyperphosphatemia, and both animal and human data indicate that such renal disorders increase the risks of gestational hypertension, pre-eclampsia, eclampsia, and maternal mortality. However, the extent to which the hyperphosphatemia contributes to these risks is unknown.

 

MINERAL PHYSIOLOGY DURING LACTATION AND POST-WEANING

 

As lactation begins the mother is faced with another demand for calcium in order to make milk. The average daily loss of calcium into breast milk is 210 mg, although daily losses as great as 1000 mg calcium have been reported is some women nursing twins (1). Although women meet the calcium demands of pregnancy by upregulating intestinal calcium absorption and serum concentrations of calcitriol, a different adaptation occurs during lactation. A temporary resorption and demineralization of the maternal skeleton appears to be the main mechanism by which breastfeeding women meet these calcium requirements. This adaptation does not appear to require PTH or calcitriol, but is regulated by the combined effects of increased circulating concentrations of PTHrP and low estradiol levels.

 

Mineral Ions

 

The albumin-corrected serum calcium and ionized calcium are both normal during lactation, but longitudinal studies have shown that both are increased slightly over the non-pregnant values. Serum phosphate levels are also higher and may exceed the normal range. Since reabsorption of phosphate by the kidneys appears to be increased, the increased serum phosphate levels may, therefore, reflect the combined effects of increased flux of phosphate into the blood from diet and from skeletal resorption, in the setting of decreased renal phosphate excretion.

 

Parathyroid Hormone

 

PTH, as measured by 2-site “intact” or newer “bio-intact” assays, may be undetectable or in the lower quarter of the normal range during the first several months of lactation in women from North America and Europe who consume adequate calcium. PTH rises to normal by the time of weaning, and in two case series was found to rise above normal post-weaning. In contrast, and similar to findings during pregnancy, PTH did not suppress in several studies of women from Asia and Gambia who consumed diets that were low in calcium or high in phytate. The low PTH concentrations are an indication that PTH isn’t required for mineral homeostasis during lactation, and this is confirmed by hypoparathyroid and aparathyroid women in whom mineral and skeletal homeostasis normalize while they continue to breastfeed (see Hypoparathyroidism, below). The same is true of mice that lack the gene for parathyroid hormone. They are hypocalcemic and hyperphosphatemic when non-pregnant, but maintain normal serum calcium and phosphate concentrations while lactating and for a time during post-weaning (17).

 

Vitamin D Metabolites

 

A common concern has been that the suckling neonate will deplete maternal 25OHD stores, but this is not the case. 25OHD should not decline because it does not enter breast milk; conversely, although vitamin D can enter milk, it is present at very low concentrations because appreciable amounts exist in the maternal circulation for only a short postprandial interval. In observational studies and in the placebo arms of several clinical trials, there was either no change or at most a nonsignificant decline in maternal 25OHD levels during lactation, even in severely vitamin D deficient women (4). Calcitriol levels were twice normal during pregnancy but both free and bound calcitriol levels fall to normal within days of parturition and remain there in breastfeeding women (a single study found that women breastfeeding twins had higher calcitriol concentrations than women nursing singletons) (161). Animal studies show that severely vitamin D deficient rodents and mice lacking the vitamin D receptor are able to lactate and provide normal milk (4,45), thereby indicating that vitamin D and calcitriol are not required for lactation to proceed normally (at least in rodents). However, a more recent study found that mice lacking calcitriol produced milk with a lower calcium content (23).

 

Calcitonin

 

Calcitonin levels fall to normal during the first six weeks postpartum in women. Mice lacking the gene that encodes calcitonin lose twice the normal amount of bone mineral content during lactation, which indicates that physiological levels of calcitonin may protect the maternal skeleton from excessive resorption during this time period (26). Whether calcitonin plays a similar role in human physiology is unknown. Totally thyroidectomized women are not calcitonin deficient during lactation due to substantial production of calcitonin by the breasts, which in turn leads to systemic calcitonin concentrations that are the same as in women with intact thyroids (25). Consequently, study of totally thyroidectomized women is not the equivalent of studying a calcitonin-null state when they are breastfeeding.

 

PTHrP

 

Plasma PTHrP concentrations are significantly higher in lactating women than in non-pregnant controls. The source of PTHrP appears to be the breast, which secretes PTHrP into breast milk at concentrations that are 1,000 to 10,000 times the level found in the blood of patients with hypercalcemia of malignancy or in normal human controls. The circulating PTHrP concentration also increases after suckling (162,163). Additional evidence that the breasts are the source of PTHrP include that ablation of the PTHrP gene selectively from mammary tissue resulted in reduced circulating levels of PTHrP in lactating mice (164). PTHrP also has an intimate association with breast tissue: in animals it has been shown to regulate mammary development and blood flow, and the calcium and water content of milk in rodents, whereas in humans it is commonly expressed by breast cancers.

 

Furthermore, as described in more detail below, during lactation PTHrP reaches the maternal circulation from the lactating breast to cause resorption of calcium from the maternal skeleton, renal tubular reabsorption of calcium, and (indirectly) suppression of PTH. In support of this hypothesis, deletion of the PTHrP gene from mammary tissue at the onset of lactation resulted in more modest losses of bone mineral content during lactation in mice (164). In humans, PTHrP correlates with the amount of bone mineral density lost, negatively with serum PTH, and positively with the ionized calcium of lactating women (162,165,166). Lastly, clinical observations in hypoparathyroid and aparathyroid women demonstrate the physiological importance of PTHrP to regulate calcium and skeletal homeostasis during lactation (see Hypoparathyroidism, below).

 

Prolactin

 

Prolactin is persistently elevated during early lactation and spikes further upward with suckling. Later during lactation basal prolactin levels are normal but continue to spike with suckling. Prolactin is important for initiating and maintaining milk production (167), but it also alters bone metabolism by stimulating PTHrP production in lactating mammary tissue, inhibiting GnRH and ovarian function, and possibly (as noted earlier) through direct actions in osteoblasts that express the prolactin receptor.

 

Oxytocin

 

Oxytocin induces milk ejection by contracting myoepithelial cells within mammary tissue. If milk is not ejected, the pressure of milk stasis causes apoptosis of mammary cells, and lactation ceases. Oxytocin spikes in the maternal circulation within 10 minutes after the start of suckling (168). As noted earlier, the oxytocin receptor is expressed in osteoblasts and osteoclasts. But whether oxytocin plays a role in bone metabolism during lactation has proven difficult to determine because oxytocin null mice cannot lactate due to the lack of milk ejection (169).

 

Estradiol

 

In lactating women, estradiol levels fall and this stimulates RANKL and inhibits osteoprotegerin production by osteoblasts, thereby stimulating osteoclast proliferation, function, and bone resorption. Studies in mice have shown that increasing the serum estradiol concentration to 7 times the virgin level blunts the magnitude of bone loss during lactation (170), which confirms that estradiol deficiency plays a role in the skeletal resorption that occurs during lactation.

 

FGF23

 

FGF23 levels during lactation have not been reported. It is possible that FGF23 increases to compensate for the increased serum phosphate and low PTH that occur during lactation, but it’s also possible that FGF23 is low and contributing to the high serum phosphate.

 

Other Hormones

 

Serotonin appears to be involved in regulating PTHrP and its effect to resorb the maternal skeleton (171,172). Lactation induces changes in myriad other hormones, such as luteinizing and follicle stimulating hormone, progesterone, testosterone, inhibins, and activins. Whether these play roles in regulating skeletal metabolism during lactation has not been investigated.

 

Intestinal Absorption of Calcium and Phosphate

 

Although intestinal calcium absorption was upregulated during pregnancy, it quickly decreases post-partum to the non-pregnant rate. This also corresponds to the fall in calcitriol levels to normal. This differs from rodents which maintain increased intestinal calcium absorption during lactation; their large litters sizes mandate the need to provide some of the calcium for milk production through this route.

 

Intestinal phosphate absorption has not been measured during human lactation, whereas in rodents it remains increased.

 

Renal Handling of Calcium and Phosphate

 

Renal excretion of calcium is typically reduced to about 50 mg per 24 hours or lower, and the glomerular filtration rate is also decreased. These findings suggest that the tubular reabsorption of calcium must be increased to conserve calcium, perhaps through the actions of PTHrP.

 

Renal tubular phosphate reabsorption is increased during lactation. Despite this, urine phosphate excretion may be increased, likely due to the large efflux of phosphate from resorbed bone, which exceeds what is needed for milk production.

 

Skeletal Calcium Metabolism and Bone Density/Bone Marker Changes

 

Histomorphometric data from lactating animals have consistently shown increased bone turnover, and losses of 35% or more of bone mineral are achieved during 2-3 weeks of normal lactation in rodents [reviewed in (1)]. There are no histomorphometric data from lactating women; instead, biochemical markers of bone formation and resorption have been assessed in numerous cross-sectional and prospective studies. Confounding factors discussed earlier for pregnancy need to be considered when assessing bone turnover markers in lactating women; in particular, opposing changes from pregnancy include that the glomerular filtration rate is reduced and the intravascular volume is now contracted. Serum and urinary (24-hr collection) markers of bone resorption are elevated 2-3 fold during lactation and are higher than the levels attained in the third trimester. Serum markers of bone formation (not adjusted for hemoconcentration or reduced GFR) are generally high during lactation, and increased over the levels attained during the third trimester. The most marked increase is in the bone resorption markers, suggesting that bone turnover becomes negatively uncoupled, with bone resorption markedly exceeding bone formation, and thereby causing net bone loss. Total alkaline phosphatase falls immediately postpartum due to loss of the placental fraction, but may still remain above normal due to elevation of the bone-specific fraction. Overall, these bone marker results are compatible with significant increased bone resorption occurring during lactation.

 

Serial measurements of aBMD during lactation (by SPA, DPA or DXA) have shown that bone mineral content falls 3 to 10.0% in women after two to six months of lactation at trabecular sites (lumbar spine, hip, femur and distal radius), with smaller losses at cortical sites and whole body (1,57). These aBMD changes are in accord with studies in rats, mice, and primates in which the skeletal resorption has been shown to occur largely at trabecular surfaces and to a lesser degree in cortical bone, and as much as 25-30% of bone mass or aBMD is lost during three weeks of lactation in normal rodents. The loss in women occurs at a peak rate of 1-3% per month, far exceeding the 1-3% per year that can occur in postmenopausal women who are considered to be losing bone rapidly. This bone resorption is an obligate consequence of lactation and cannot be prevented by increasing the calcium intake in women. Several randomized trials and other studies have shown that calcium supplementation does not significantly reduce the amount of bone lost during lactation (173-176). Not surprisingly, the lactational decrease in bone mineral density correlates with the amount of calcium lost in the breast milk (177).

 

The skeletal losses are due in part to the low estradiol levels during lactation which stimulate osteoclast number and activity. However, low estradiol is not the sole cause of the accelerated bone resorption or other changes in calcium homeostasis that occur during lactation. It is worth noting what happens to reproductive-age women who have marked estrogen deficiency induced by GnRH agonist therapy in order to treat endometriosis, fibroids, or severe acne. Six months of GnRH-induced estrogen deficiency caused 1-4% losses in trabecular (but not cortical) aBMD, increased urinary calcium excretion, and suppression of calcitriol and PTH (Figure 3) [reviewed in (1,8)]. In contrast, during lactation women are not as estrogen deficient but lose more aBMD (at both trabecular and cortical sites), have normal (as opposed to low) calcitriol levels, and have reduced (as opposed to increased) urinary calcium excretion (Figure 3). The difference between isolated GnRH-induced estrogen deficiency and lactation appears to be explained by PTHrP. It stimulates osteoclast-mediated bone resorption and stimulates renal calcium reabsorption; by so doing, it complements the effects of low estradiol during lactation. Stimulated in part by suckling and high prolactin levels, PTHrP and estrogen deficiency combine to cause marked skeletal resorption during lactation (Figure 4).

Figure 3. Comparison of the effects of acute estrogen deficiency vs. lactation on calcium and bone metabolism. Acute estrogen deficiency (e.g. GnRH analog therapy) increases skeletal resorption and raises the blood calcium; in turn, PTH is suppressed and renal calcium losses are increased.  During lactation, the combined effects of PTHrP (secreted by the breast) and estrogen deficiency increase skeletal resorption, reduce renal calcium losses, and raise the blood calcium, but calcium is directed into breast milk. Reprinted from ref. (8), © 1997, The Endocrine Society.

Figure 4. Brain-Breast-Bone Circuit. The breast is a central regulator of skeletal demineralization during lactation. Suckling and prolactin both inhibit the hypothalamic gonadotropin-releasing hormone (GnRH) pulse center, which in turn suppresses the gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), leading to low levels of the ovarian sex steroids (estradiol and progesterone). PTHrP production and release from the breast is controlled by several factors, including suckling, prolactin, and the calcium receptor. PTHrP enters the bloodstream and combines with systemically low estradiol levels to markedly upregulate bone resorption. Increased bone resorption releases calcium and phosphate into the blood stream, which then reaches the breast ducts and is actively pumped into the breast milk. PTHrP also passes into milk at high concentrations, but whether swallowed PTHrP plays a role in regulating calcium physiology of the neonate is unknown. Calcitonin (CT) may inhibit skeletal responsiveness to PTHrP and low estradiol. Not depicted are that direct effects of oxytocin and prolactin on bone cells are also possible. Adapted from ref. (26) © 2006, The Endocrine Society.

 

 

The mechanism through which the skeleton is resorbed has been shown in rodents to involve two processes, both osteoclast-mediated bone resorption (1) and osteocytic osteolysis, in which osteocytes function like osteoclasts to resorb the bone matrix that surrounds them (178). Both of these processes are dependent upon PTHrP. Conditional deletion of the PTHrP gene from mammary tissue reduced the amount of bone resorbed during lactation, whereas conditional deletion of the PTH/PTHrP receptor from osteocytes appeared to eliminate osteocytic osteolysis (179). Moreover, osteocyte-specific deletion of the PTH/PTHrP receptor resulted in a 50% blunting of the amount of BMD lost during lactation (179), which may indicate that osteocytic osteolysis and osteoclast-mediated bone resorption each contribute about half of the net bone loss achieved during lactation. To date no studies have examined whether osteocytic osteolysis occurs in lactating women.

 

The lactational bone density losses in women are substantially and completely reversed during six to twelve months following weaning (1,57,174). This corresponds to a gain in bone density of 0.5 to 2% per month in a woman who has weaned her infant. The mechanism for this restoration of bone density is unknown, but studies in mice have shown that it is not dependent upon calcitriol, calcitonin, PTH, or PTHrP (17,23,26,45,180,181); nor is it fully explained by restoration of estradiol levels to normal (1). The remarkable ability of the skeleton to recover is exemplified by mice lacking the gene that encodes calcitonin. They lose up to 55% of trabecular mineral content from the spine during lactation but completely restore it within 18 days after weaning (26).

 

Although aBMD appears to be completely restored after weaning in women and all animals that have been studied, more detailed examination of microarchitecture by µCT has shown variable completeness of recovery of microarchitecture by skeletal site. In rodents, the vertebrae recover completely while persistent loss of trabeculae is evident in the long bones (182). Studies in women have similarly shown that the trabecular content of the long bones also appears to be incompletely restored (1,57,174,183,184). However, in both women (74,184,185) and rodents (26,186,187) the cross-sectional diameters and volumes of the long bones may be significantly increased after post-weaning. Such structural changes potentially compensate for any reduction in strength that loss of trabecular microarchitecture might induce, because an increased cross-sectional diameter increases the ability of a hollow shaft to resist bending (cross-sectional moment of inertia) and torsional stress (polar moment of inertia). This is supported by the finding that the breaking strength of rodent bones returns to pre-pregnant values after weaning (1,180), and limited clinical studies that correlated the increased bone volumes achieved after reproductive cycles with increased bone strength (74,185). In women, the vast majority of several dozen epidemiologic studies of pre- and postmenopausal women have found no adverse effect of a history of lactation on peak bone mass, bone density, or hip fracture risk (1,7,54,57). In fact, multiple studies have suggested a protective effect of lactation on the future risk of low BMD or fragility fractures. Consequently, although lactational bone loss can transiently increase risk of fracture (see next section), it is likely unimportant in the long run for most women, in whom the skeleton is restored to its prior mineral content and strength.

 

DISORDERS OF CALCIUM AND BONE METABOLISM DURING LACTATION

Osteoporosis of Lactation

 

On occasion a woman will suffer one or more fragility fractures during lactation, and osteoporotic bone density will be found by DXA (76). As with osteoporosis presenting during pregnancy, this may represent a coincidental, unrelated disease; the woman may have had low bone density and abnormal skeletal microarchitecture prior to pregnancy. Alternatively, it is likely that some cases represent an exacerbation of the normal degree of skeletal demineralization that occurs during lactation, and a continuum from the changes in bone density and bone turnover that occurred during pregnancy. It may be somewhat artificial, therefore, to separate “osteoporosis of lactation” from “osteoporosis of pregnancy.” But since lactation normally causes a significant net loss of bone whereas pregnancy does not, it seems more likely for lactation to cause a subset of women to develop low-trauma fractures. For example, excessive PTHrP release from the lactating breast into the maternal circulation could conceivably cause excessive bone resorption, osteoporosis, and fractures. PTHrP levels were high in one case of lactational osteoporosis, and remained elevated for months after weaning (188).

 

The diagnostic and treatment considerations described above for osteoporosis of pregnancy also apply to women who are lactating (76).

 

Primary Hyperparathyroidism

 

When surgical correctional of primary hyperparathyroidism is not possible or advisable during pregnancy, it is normally carried out in the postpartum interval. A hypercalcemic crisis is possible soon after delivery due in part to loss of the placental calcium infusion, which represented a drain on the serum calcium. If a woman with untreated primary hyperparathyroidism chooses to breastfeed, the serum calcium should be monitored closely for significant worsening due to the effects of secretion of PTHrP from the breasts being added to the high concentrations of PTH already in the circulation. The potential impact of this is even more evident in women with hypoparathyroidism, as discussed below.

 

Familial Hypocalciuric Hypercalcemia

 

The calcium-sensing receptor is expressed in mammary epithelial ducts, and it modulates the production of PTHrP and calcium transport into milk during lactation in mice (189,190). Inactivating calcium-sensing receptor mutations increased mammary tissue production of PTHrP but decreased the calcium content of milk (190). These opposing changes meant that there was a further increase in bone resorption during lactation as compared to normal mice, and the serum calcium also became higher because of reduced output of calcium into milk. Conversely, a calcimimetic drug (similar to cinacalcet) caused increased milk calcium content (190). These data predict that women with FHH will have more marked skeletal resorption during lactation, lower milk calcium content, higher serum calcium, and a greater loss of BMD during lactation as compared to normal women. However, the effect of breastfeeding on mineral and skeletal homeostasis in women with FHH has not yet been described.

 

Hypoparathyroidism

 

As noted earlier, in the first day or two after parturition the requirement for supplemental calcium and calcitriol may transiently increase in hypoparathyroid women before secretion of PTHrP surges in the breast tissue (114). The onset of lactation induces an important change in skeletal metabolism because the breasts produce PTHrP at high levels, some of which escapes into the maternal circulation to stimulate bone resorption and raise the serum calcium level. In women who lack parathyroid glands, the release of PTHrP into the circulation during lactation can temporarily restore calcium and bone homeostasis to normal. Levels of calcitriol and calcium supplementation required for treatment of hypoparathyroid women fall early and markedly after the onset of lactation, and hypercalcemia can occur if the calcitriol dosage and calcium intake are not substantially reduced (191-194). This decreased need for calcium and calcitriol occurs at a time when circulating PTHrP levels are high in the maternal circulation (191,194,195). As illustrated in one case, this is consi