Endocrine testing for the Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH)

ABSTRACT

The diagnosis of Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH) relies on an adequate assessment of a hyponatremic state (that is a serum sodium level <136 mmol/l) and on the exclusion of other causative conditions leading to an appropriate secretion of antidiuretic hormone (ADH). The understanding of mechanisms involved in pathological ADH secretion is essential for diagnosis and therapy. Although some forms are due to dysregulation in central nervous system regulation, other forms are dependent on diseases in peripheral organs and structures including ADH-producing/secreting neuroendocrine tumors, while others are induced by drugs. ADH regulation is closely linked to other systems such as the sympathetic nervous system via and baroreflex regulation. Patients with hyponatremia should be assessed carefully whether or not neurological symptoms exist. Further, assessment of volume status is needed. Based on symptoms and volume status, the need for intensive-care monitoring is determined. In parallel, laboratory findings of blood and urine must be analyzed appropriately. It is important to demonstrate true hyponatremia, which is paralleled by a decrease in serum osmolality. Mandatory laboratory diagnostic steps comprise the determination of blood and urine electrolytes and serum and urine osmolality, analysis of thyroid, adrenocortical, and kidney function as well as uric acid. Different test results such as a high fractional uric acid excretion may hint to an existing SIADH. Assessment of urine osmolality and urine sodium concentration and intravascular volume level may allow for further discrimination and may be indicative for a specific underlying disorder, causing SIADH. Brain volume changes (“hydrocephalus ex vacuo”) may depend on age rendering the elderly more tolerant to acute or chronic serum sodium changes. The course of incident hyponatremia, if documented, may affect therapy. A serum sodium drop within less than 48 hours is considered acute hyponatremia. A rapid bolus of 100 to 150 ml of intravenous 3% hypertonic saline is appropriate to avoid catastrophic outcomes in severe cases of acute symptomatic hyponatremia.

INTRODUCTION

“Antidiuretic hormone” or “arginine vasopressin” (AVP) is physiologically released into the blood stream upon increases of plasma osmolality. AVP, a nine-amino-acid peptide, originates in the supraophthalmic nucleus (SON) of the hypothalamus and is directly regulated by plasma osmolality detected via a splice variant of the capsaicin receptor, the transient receptor potential vanilloid type-1 (Trpv1) receptor (1). AVP is axonally transported to the posterior pituitary and released into the blood upon respective stimulation. AVP regulation is, however, more complex than a mere response to changes of plasma osmolality. In fact, hypovolemia enhances AVP release upon increases of plasma osmolality. Conversely, hypervolemia attenuates AVP release for given increases of plasma osmolality.

In patients with “Syndrome of Inappropriate Antidiuretic Hormone Secretion” or SIADH, the cornerstone of diagnosis is hyponatremia (Na<136 mmol/l) (2) in a state of euvolemia, i.e. absence of either over- or dehydration. SIADH-related hyponatremia is caused by excess water reabsorption due to inappropriately high levels of AVP. Specifically, AVP binds to stimulatory G-protein-coupled vasopressin V₂-receptors of the basolateral membrane of collecting-duct cells, thereby increasing the intracellular cAMP level, which, in turn, activates Aquaporin-2 channels of the brush-border membrane or urine side of the collecting-duct principal cells. AVP action results in a reduced free-water clearance resulting in a more concentrated urine and total-body water (TBW) expansion. AVP-mediated TBW expansion mediates sympathoinhibition (Figure 1).

Figure 1. Role of AVP in regulation of sympathetic tone. AVP translates into sympathoinhibition (Figure amended from (3) ).

In addition, AVP-induced TBW expansion translates into plasma-solute dilution leading to hyponatremia. Thereby, plasma osmolality (OSM) is decreased given the fact that sodium strongly determines OSM according to the following equation:

OSM (calculated) = 2 x Na (mmol/l) + Glucose (mmol/l) + Urea (mmol/l)

Besides AVP actions on OSM, AVP also enhances endothelial-cell synthesis and the release of von-Willebrand Factor, thereby affecting hemostasis (4). This AVP effect on hemostasis is therapeutically used in bleeding disorders involving Factor VIII or von-Willebrand factor deficiency (5, 6).

SIADH may be viewed as a primary central-nervous system dysregulation of OSM and/or thirst. The etiology still is incompletely understood. Alternatively, SIADH may relate to baroreceptor unloading due to clinically inapparent hypovolemia or, hypothetically, to carotid-artery atherosclerosis affecting baroreflex regulation (7). This alternative route of increased AVP release may ultimately translate into the clinical picture of SIADH. Generally, less wall distension of the carotid arterial walls and/or the aortic arch may lead to a decrease of arterial baroreceptor-related afferent autonomic nerve traffic to the rostral ventrolateral medulla and nucleus tractus solitarii (NTS) translating into less sympathoinhibition (sympathoexcitation) and to an increased release of AVP (8) (Figure 2).

Figure 2. Baroreflex regulation and SIADH: arterial hypotension lowers baroreflex-mediated afferent nerve traffic to the nucleus tractus solitarii leading to an elevated efferent sympathetic nerve activity and increased AVP release.

Lastly, hypovolemia-related cardiopulmonary (CP) reflex deactivation mediated by less wall distension of the right atrial wall and pulmonary veins may increase plasma AVP leading to SIADH (9, 10). Conversely, CP reflex activation mediated by more right-atrial wall distension e.g. after body immersion in water is able to decrease plasma AVP (11).

The term “primary SIADH” is used for all above-mentioned causes involving a known or suspected dysregulation of OSM and/or circulating-blood volume. The term “secondary SIADH” is attributed to pituitary-independent causes of AVP increases, e.g., in hormone-active neoplasms such as small-cell lung cancer. In addition, a drug-induced type of SIADH is detailed here.

CLINICAL PRESENTATION OF SIADH

Both moderate and especially severe hyponatremia (Na < 125 mmol/l) found in newly admitted hospital patients is linked with a significantly elevated in-hospital mortality of 28% compared to 9% in-hospital mortality in normonatremic, matched control patients (12). Mortality, in fact, increases when serum Na levels are below 137 mmol/L (13). While (neurological) symptoms of hyponatremia such as gait disturbances, cognitive dysfunction and dizziness may lead to falls leading to subsequent injuries requiring medical care, either preceding symptom may be subtle and difficult to diagnose. Therefore, hyponatremia often is overseen or not given full attention. Furthermore, if hyponatremia is diagnosed, it is regularly classified to be asymptomatic. Diagnostic differentiation remains absent or incomplete. Thus, underlying reasons often remain obscure (14). However, cognitive and/or geriatric functional tests regularly reveal a significant impairment in states of hyponatremia.

Clearly, symptoms of hyponatremia depend on the time elapsed since the start of hyponatremia development. Hyponatremia developing in less than 48 hours may already present with severe symptoms which are mainly caused by cerebral edema and a high intracranial pressure. These include epileptic convulsions, a pronounced somnolence or coma, vomiting and/or a compromised respiratory regulation. Symptoms like headache or modest nausea generally reflect a rather moderate severity.

In patients with acute hyponatremia, a brief patient history and a physical examination should be performed (Table 1). In cases of a rather slowly developing or chronic hyponatremia, intracellular regulations such as decreased uptake of taurin aim to adapt to the decreased extracellular osmolality. Therefore, those patients may show very subtle or even no clinical alterations. Taurin is an endogenous amino acid that mediates cellular adaptation to hyperosmotic stress (15).

Table 1. Anamnestic Factors and Conditions Responsible for the Occurrence of an Acute Hyponatremic State (<48h)

Medical interventions:

General post-operative phase

Resection of the prostate

Exercise (e.g., long distance run) with increased and rapid fluid (water) intake

Extended sauna visit

Polydipsia (transient)

Severe pain attacks (including concomitant pharmacotherapy)

Initiation of new drugs

e.g., thiazides, terlipressin, psychiatric medication

For the diagnosis of SIADH, the first diagnostic step, hyponatremia needs to be ascertained. False laboratory “measurements” of serum Na+ comprise primarily a hyperglycemic state. According to Hillier et al (16) an estimation of the true sodium concentration in serum can be drawn from the formula:

Corrected (Na+) = Measured (Na+) + 2.4 x (glucose (mg/dl) - 100 mg/dl)/100mg/dl

For example, a serum glucose level of 400 mg/dl with a measured serum sodium of 120 mmol/l corresponds to a true sodium value of 127 mmol/l (16).

Likewise, pseudohyponatremia may occur in patients with paraproteinemia, e.g. multiple-myeloma patients (17).

Another pitfall with serum sodium is the mode of its determination: the usual method involves ion-selective electrodes, not flame-photometric determination and may yield occasionally “diluted” Na⁺ levels (elevated triglyceride levels, paraproteinemia). Measured, not calculated serum osmolality may help discriminate true from pseudohyponatremia: true hyponatremia associates with a decreased serum osmolality.

True-hyponatremic patients are regularly identified at hospital admission, e.g., in the emergency room. However, a large number of hospitalized patients have or develop either mild (Na 131 – 136 mmol/l), moderate (Na 126 – 130 mmol/l) or severe (Na <125 mmol/l) hyponatremia after admission during the hospital stay. Although many hyponatremic patients will present with chronic hyponatremia, however, some patients present with a proven acute hyponatremia. Since acute hyponatremia means an incomplete adjustment of the difference in osmolality between plasma (extracellular space) and intracellular cell space of tissues and organs such as the brain, a 48-hours threshold to discern acute from chronic hyponatremia appears reasonable. However, in everyday practice, this discrimination might not be feasible due to a lack of documentation of serum sodium levels in newly hospitalized patients.

As an important step in the approach to the hyponatremic patient is to determine volume status. Accordingly, a state of overhydration characterized by the presence of peripheral edema needs to be ruled out. Hyponatremia accompanied by peripheral edema, anasarca, jugular vein distension in absence of a significant tricuspid-valve regurgitation, dyspnea, and/or signs of a lung fluid or pulmonary edema on the chest radiograph are not consistent with the diagnosis of SIADH. Here, underlying diseases such as chronic heart failure should be diagnosed and addressed. Likewise, hyponatremia in a state of hypovolemia needs to be excluded. In exsiccosis, e.g., due to diuretics, both water and solutes may be lost. Hypovolemia triggers sympathoactivation via CP and baroreflex leading to an appropriate ADH release.

In patients presenting with hyponatremia in a state of euvolemia, i.e., absence of overhydration or exsiccosis, the diagnosis of SIADH should be considered, after excluding conditions such as chronic heart failure. A known and medically treated chronic heart failure condition may present as a hypervolemic, euvolemic or – when vigorously treated – hypovolemic state. Both, chronic heart failure and liver cirrhosis are characterized by arterial underfilling and, hence, activation of both the renin-angiotensin-aldosterone system and antidiuretic hormone leading to both sodium chloride retention and water retention. The net effect may be hyponatremia, if AVP stimulation dominates. Thus, urine-sodium measurements in 24-hours urine collection may prove the presence of a sodium-sparing disorder, thereby rendering SIADH unlikely.

A useful algorithm to approach hyponatremia is depicted in Figure 3.

Figure 3. Approach to the patient with hyponatremia (adapted and modified from (18), with permission).

In the clinical assessment, euvolemic patients suspected to have primary SIADH often show a slight weight gain by 5 – 10 % of body weight and/or a worsened condition of arterial hypertension, and urine output is normal or slightly reduced. In secondary, neoplasm-associated SIADH, however, an unexplained weight loss and/or state of cachexia may prompt further diagnostics including chest radiograph and thoracic computed tomography scan, if deemed necessary.

It is crucial to assess and to treat infections effectively, since they may establish or worsen a tendency to hyponatremia.

To correctly evaluate laboratory results besides aspects of methodology, diuretics,

especially thiazides, should be discontinued, and nutritional sodium chloride intake should not exceed 5 – 6 g per day.

HYPONATREMIA IN EUVOLEMIA: ASSESSMENT FOR SIADH

The diagnostic, step-wise approach for evaluating hyponatremia in euvolemic patients is detailed below:

1^st Step

Here, a laboratory work-up (Table 2) is proposed to establish a preliminary diagnosis being consistent with SIADH.

Table 2. Laboratory Work-Up for SIADH
	Parameter	SIADH Diagnosis
Serum	Sodium Potassium Glucose Urea Creatinine Uric acid Thyroid hormones Cortisol Aldosterone Copeptin	<136 mmol/l Normal Normal Normal Normal Normal Normal Normal Normal Elevated
Urine	Osmolality Sodium Uric acid Creatinine Osmolality	>100 mosm/kg > 30 mmol/l Normal or Low Normal >12%

As a diagnostic cornerstone of SIADH diagnosis, determination of fractional uric-acid excretion has recently emerged (SIADH increased). Fenske et al. (19) confirmed earlier reports demonstrating fractional uric-acid excretion (cut-off greater than 12 %) to rule out hyponatremic states with reduced extracellular fluid volume, e.g. due to diuretics (Figure 4).

Figure 4. Calculation of fractional uric acid excretion.

There is sometimes debate on whether measurements of AVP in the plasma are helpful or not for diagnosis of SIADH or other hyponatremic circumstances. There are, however; multiple obstacles rendering AVP determination and interpretation difficult. Only a few laboratories provide tests with good sensitivity, since AVP is very unstable when isolated from plasma and binds to other structures. A potential alternative is the more stable copeptin, also called C-terminal proarginine vasopressin, which is generated by enzymatic cleavage of the vasopressin prohormone. Strikingly, SIADH patients were shown to have an elevated plasma copeptin (20). As an additional way to diagnose SIADH, correlation of plasma copeptin with changes in plasma osmolality (step 2, step 3) can be used (21). Furthermore, a hypertonic (3%) saline test has been proposed for SIADH (20). However, this test strategy still needs to be validated.

2^nd Step

Administration of 500 ml saline (NaCl 0.9 %) is regarded as an empiric, first-line therapeutic and diagnostic measure. Especially when arterial underfilling and baroreflex and/or CP reflex deactivation are thought to be relevant, i.v. saline corrects the initial disturbance that led to hyponatremia/SIADH. This relates, strictly speaking, to volume regulation and respective reflex stimulation. Isotonic saline may not resolve hyponatremia in cases of ongoing urine concentration with urine osmolality exceeding 400 mosm/kg.

In addition, a water-load test as described in www.endotext.org, NEUROENDOCRINOLOGY, HYPOTHALAMUS, AND PITUITARY may be performed the next morning under utmost scrutiny and precaution, in the hospital. The water-load test relates to the fact that normal subjects excrete 78-82% of the ingested water load within 4 hours due to AVP suppression. In patients with SIADH, the expected urine amount within 4 hours is reduced to 30-40%. However, the test involves a massive water intake in a short period of time and, therefore, is not considered a safe procedure for the majority of patients for the following three reasons:

In SIADH patients, a relative intravascular overhydration will be enhanced.
An underlying cardiac co-morbidity may be adversely affected.
The water challenge may worsen the hyponatremia increasing the

risk of symptoms such as epileptic convulsions.

In the majority of patients with hyponatremia suspected to have SIADH, this test can be replaced by the following 3^rdand 4^th diagnostic step (below). The water-load test only adds information in hospitalized individuals free of cardiac conditions presenting with rather mild hyponatremia in whom the 4^th step usually will not be performed. Again, the physician has to weigh risks and benefits of this water challenge versus alternate diagnostic steps (step 3, step 4 below).

3^rd Step

A balanced fluid-intake restriction (500 ml/day) is able to correct hyponatremia over the next 3 to 4 days with an aimed plasma-sodium increase of 0.5 mmol/l/h or less than 10 mmol/l/day. Most patient do not tolerate a very strict fluid intake reduction.

4^th Step

If step 1 – step 3 did not lead to an improvement of hyponatremia, therapy with an antagonist of the vasopressin V₂-receptors, e.g. tolvaptan, for four days should be instituted (22) with an aimed plasma-sodium increase of 0.5 mmol/l/h or less than 10 mmol/l/day.

INTERPRETATION OF CLINICAL AND LABORATORY RESULTS

SIADH leads to an increase of free-water reabsorption, thereby increasing the circulating blood volume. By virtue of dilution mediated by AVP, both hematocrit and plasma sodium are decreased. Likewise, a decrease of urine output can be found.

In cases of a prolonged, subclinical hypovolemia, baroreflex- and/or CP-reflex unloading stimulates AVP secretion leading to the clinical picture of SIADH. There, discontinuation of diuretics and/or the empirical infusion of 500 ml saline (0.9 %) as outlined above in step 2 may correct such a state of subclinical hypovolemia and lead to an improvement in hyponatremia driven by SIADH.

In assessing key laboratory results including plasma and urine sodium concentration and -osmolality, both the theoretical or calculated OSM should be compared to the actually measured OSM. That way, states of hyponatremia due to uremia or hyperglycemia can be ruled out. In such cases of hyponatremia, high plasma urea or high plasma glucose lead to a rise in OSM prompting a physiologic release of AVP, which, in turn, leads to a plasma-sodium dilution in order to maintain a normal OSM.

Urine sodium within normal range rules out a dietary sodium deficiency or states of increased tubular sodium reabsorption such as in chronic heart failure or liver cirrhosis.

After fulfilling the above-mentioned steps to diagnose SIADH, the following conditions should be separately considered as a possible differential diagnosis:

DIFFERENTIAL DIAGNOSIS OF HYPONATEMIA OTHER THAN SIADH

Sodium chloride depletion, low dietary sodium intake regularly is accompanied by hypovolemia, low urine sodium, elevated serum uric acid and serum urea.

Anterior-lobe pituitary gland insufficiency often is accompanied with signs and symptoms, and respective laboratory findings indicating hormone deficiencies such as hypothyroidism, hypocortisolism or hypogonadism. In addition, bitemporal hemianopsia and hyperprolactinemia are found in cases of anterior-lobe pituitary tumors as a cause of anterior-lobe pituitary gland insufficiency.

Adrenal-gland insufficiency including iatrogenic mineralocorticoid-receptor antagonism (spironolactone/eplerenone) regularly is accompanied by hyperkalemia and hypovolemia.

Thiazide diuretics can induce hyponatremia by an AVP-dependent mechanism and by a thiazide-induced increase of water permeability in the medullary collecting duct.

Severe hypothyroidism regularly is accompanied by dilutional hyponatremia due to a reduced free-water clearance.

Chronic kidney disease: In salt losing nephropathy, a condition that occurs in advanced kidney failure with a GFR below 15 ml/min, hyponatremia is paralleled by hypovolemia. This is a feature classically seen in interstitial kidney disease. On the other hand, many patients with near end-stage renal failure show increased Na excretion to balance body sodium content, but, due to (continuous) reduction in urine production, a diluted urine cannot be achieved, leading to hyponatremia.

Acute kidney (transplant) failure without signs of uremia, a water-excretion dysfunction may lead to dilutional hyponatremia.

Hyperglycemia or poor diabetes mellitus control may lead to a so-called translational hyponatremia due to intra- to extracellular water shift and consequent plasma sodium dilution (see above).

Cerebral salt wasting is an important differential diagnosis to SIADH occurring in cases of aneurysmal subarachnoidal hemorrhage and in other intracranial pathologies. Cerebral salt wasting still is not completely characterized and most likely involves a putative central nervous system-derived factor and/or a sudden decrease of renal sympathetic nerve activity favoring a urinary loss of sodium chloride. Cerebral-salt wasting - associated urinary sodium-chloride loss improves after successful neurosurgical care of the initial intracranial disease condition and may require temporary high amounts of sodium chloride replacement.

Overdose of antidiuretic-hormone analogs in cases of known central diabetes insipidus. If the primary physician is unaware of the underlying medical condition, SIADH may be suspected based on laboratory results described above. Patient history including the medication list clarifies the diagnosis.

THERAPY OF SIADH

If left untreated, SIADH may lead to a severe, life-threatening hyponatremia with or without clinically apparent overhydration. Acute complications of SIADH include cerebral edema and epileptic convulsions. SIADH therapy clearly depends on the specific etiology and the severity of symptoms.

Once therapy is initiated, repeat measurements of plasma sodium are mandatory to gauge the therapeutic response, and, most importantly, to ascertain a slow plasma-sodium normalization with a recommended maximum rate of 0.5 mmol/l/h plasma-sodium increase. Again, the delivery of higher concentrated sodium chloride solution is allowed strictly for symptomatic patients. A significant proportion of in-hospital mortality relating to hyponatremia likely is due to a too rapid sodium normalization in long-standing hyponatremia. The consequence of too rapid sodium normalization is the osmotic demyelination syndrome due to a rapid intra- to-extracellular water transfer and subsequent brain swelling that exceeds the percentage of cerebrospinal fluid volume capacity (usually around 8% but higher in elderly with a hydrocephalus ex vacuo).

Besides the therapeutic goal to avoid rapid changes in plasma osmolality, the underlying reason of hyponatremia in SIADH, excess total body water, should be addressed by balanced fluid-intake reduction. All therapeutic interventions discussed here target the consequences of exaggerated AVP secretion rather than sodium-chloride supplementation.

In primary SIADH, plasma-sodium dilution can be addressed by an ongoing fluid-intake restriction of 500-800 ml/day which many patients do not tolerate well.

However, subclinical hypovolemia and ensuing baroreflex and CP reflex suppression leading to AVP stimulation should be kept in mind. Addressing arterial hypotension and/or central-venous hypotension is effective in lowering AVP in plasma. At the same time, fluid-intake restriction may appear contradictory. Even though fluid-intake restriction appears to be a proven measure in terms of attenuation of AVP consequences, it is the clinician`s judgement to test both interventions and compare the best results in terms of a slow plasma-sodium increase.

In secondary SIADH, identification of the neoplasm is the goal. A thorough tumor search using positron-emission tomography and computed tomography is warranted to further determine the underlying pathology and, if applicable, consider all options of curative therapy. Chemotherapy, surgical and/or radiation therapy of malignancies with AVP activity represent definitive therapeutic approaches. On clinical grounds, neoplasm-associated, secondary SIADH often requires V2-receptor antagonism therapy until a specific oncologic care plan is employed. This might be especially true while performing cytostatic therapy cycles with increased intravenously administered fluid volumes. However, to date, no survival benefit has been demonstrated in favor of V2-receptor antagonism in oncologic care of patients with secondary, neoplasm-associated SIADH.

Besides malignancies, infections such as tuberculosis have been associated with occurrence of SIADH (24).

Only if there are severe symptoms related hyponatremia, is it advised to administer a small amount of a hypertonic NaCl-solution for a very short period of time. In practice, this must be accomplished with closely monitored sodium concentration measurements which can be accomplished on a regular ward rather than in the intensive care unit, especially when considering the urgency. 150 ml of a 3% saline solution can be infused over 20 minutes. If the symptoms do not ameliorate with this management, the infusion with 100 ml of 3% hypertonic saline can be repeated every 30 minutes until the target serum Na is reached (usually 5-8 mmol increase from baseline). Above all, a 5 mmol/l increase in serum sodium concentration should not be exceeded within the first hour. These recommendations have also been summarized in recent guideline reports (18).

The management after relief of the symptoms should be focused on a careful administration of 0.9% (only) sodium chloride solution. Independently of the initial rise in serum sodium concentration by the above measures, the maximal rise within the first 24 hours should not exceed 10 mmol/l. In some cases of overcorrection, desmopressin can be administered (13, 23). In terms of reaching a sodium goal within a short time, a rapid intermittent bolus infusion of hypertonic solution is preferred to a slow continuous infusion (25).

Drug-Induced SIADH

Drugs (see Table in Ref. 18) such as vincristine, vinblastine, cyclophosphamide, carbamazepine, tricyclic antidepressants, selective serotonin reuptake inhibitors (e.g., citalopram), oxytocin, opiates, barbiturates, and nicotine may cause SIADH. Either they enhance ADH release, are analogues of ADH, or they amplify the renal effects of ADH. However, for some drugs, the mechanism remains unclear. If applicable, a suspected drug should be discontinued with close supervision of plasma sodium levels. Once hyponatremia improves after cessation of a specific drug, drug-induced SIADH is likely. However, unless re-exposure takes place, drug- induced SIADH is not proven.

Primary and Secondary SIADH

In both primary and secondary SIADH or in euvolemic, hyponatremic patients with suspected SIADH without a therapeutic effect of fluid-intake restriction, the lowest recommended standard dose of a vasopressin V₂-receptor blocker, e.g., Tolvaptan, should be administered orally. Besides the diagnostic approach outlined above as step 4, empiric V2-receptor antagonism represents a rescue therapy and is suitable to gain time needed to perform further diagnostics and therapies in (suspected) SIADH.

Low-dose Tolvaptan therapy was shown to significantly improve hyponatremia (by 3 – 4 mmol/l) within 4 days when compared to placebo (26, 27). Alternatively, conivaptan (approved for SIADH in the United States) has a broader target than its competitors tolvaptan or mozavaptan. Conivaptan selectively targets the V1a and V2 receptors. Conivaptan can be administered intravenously in patients who are unable to take drugs orally. In addition, conivaptan has a longer bioavailability than newer vaptans including tolvaptan (28). In a large proportion of SIADH patients, whether or not the underlying circumstances leading to SIADH are known, fluid-intake restriction controls hyponatremia attributed to SIADH. It is important to note, that with the use of higher daily tolvaptan dosages, the risk of liver injury may increase, as revealed in a recent study in which vaptans were tested to treat autosomal-polycystic kidney disease (ADPKD) (29).

In essence, V₂-receptor antagonism remains both a diagnostic and a therapeutic tool for SIADH when applied under scrutiny and for a limited period of time. To date, data on long-term use of V₂-receptor blockers have not been published, especially for the use of a combined V1a-V2 receptor blockade. V2 antagonists that have been used for the treatment of SIADH are listed in Table 3. The fact that hyponatremia may reoccur shortly after V₂-receptor-blocker discontinuation emphasizes the need to identify the underlying cause of SIADH in order to devise a definitive therapy.

Table 3. V2 Receptor Antagonists
Drug	Route of administration	Receptor affinity	Literature
Tolvaptan	oral	V2	(26, 27)
(Lixivaptan)			(30)
(Mozavaptan) (only approved in Japan)			(31)
(Satavaptan)			(32)
(Conivaptan) (only approved in U.S.A.)	intravenous	V2 /V1A	(28, 33)

In parenthesis: limited or no availability.

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Cryptorchidism and Hypospadias

ABSTRACT

Undescended testis (UDT) is a common abnormality, affecting about 1/20 males at birth. Half of these have delayed testicular descent, with the testis in the scrotum by 10-12 weeks after term. Beyond this spontaneous descent is rare. Current treatment recommendations are that UDT beyond 3 months of age need surgery before 12 months of age. Some children have scrotal testes in infancy but develop UDT later in childhood because the spermatic cord does not elongate with age, leaving the testes behind as the scrotum moves further from the groin with growth of the pelvis. This is now known as ascending/acquired cryptorchidism, and orchidopexy is controversial. Many authors recommend surgery once the testes no longer reside spontaneously in the scrotum, but some groups recommend conservative treatment. The fetal testis descends in 2 separate hormonal and anatomical steps, with the first step occurring between 8-15 weeks’ gestation. Insulin-like hormone 3 (INSL3) from developing Leydig cells stimulates the genito-inquinal ligament, or gubernaculum, to swell where it ends in the inguinal area of the abdominal wall. This holds the testis near the future inguinal canal as the fetal abdomen enlarges. By contrast, in female fetuses, lack of INSL3 allows the gubernaculum to elongate into a round ligament and lets the ovary move away from the groin. The second or inguinoscrotal phase is controlled by androgen and occurs between 25-35 weeks’ gestation, where the gubernaculum and testis migrate together to the scrotum. Androgens guide this complex process, both directly and indirectly via a neurotransmitter, calcitonin gene-related peptide (CGRP), released from the genitofemoral nerve. After migration is complete the proximal processus vaginalis closes (preventing inguinal hernia) and then the fibrous remnant disappears completely, allowing the spermatic cord to elongate with age, to keep the testis scrotal. The transabdominal phase is a simple mechanical process, and abnormalities are uncommon, with intra-abdominal testes found in 5-10% of boys with UDT. Anomalies of the complex inguinoscrotal phase account for most UDT seen clinically. The undescended testis suffers heat stress when not at the lower scrotal temperature (33 degrees Celsius), interfering with testicular physiology and development of germ cells into spermatogonia. UDT interrupts transformation of neonatal gonocytes into type-A spermatogonia, the putative spermatogenic stem cells at 3-9 months of age. Recent evidence suggests orchidopexy between 6-12 months improves germ cell development, with early reports of improved fertility, but little evidence yet for changes in malignancy prognosis. Hypospadias is also a common abnormality in newborn males, affecting about 1/150 boys. Androgens control masculinization of the genital tubercle into penis between 8-12 weeks’ gestation, with tubularization of the urethra from the perineum to the tip of the glans. If this process is disrupted hypospadias occurs, with a variable proximal urethral meatus, failed ventral preputial development producing a dorsal hood, and discrepancy in the ventral versus dorsal penile length, causing a ventral bend in the penis, known as chordee. Surgery to correct hypospadias is recommended between 6-18 months, as technical advances now allow operation to be done before the infant acquires long-term memory of the surgery. Severe hypospadias overlaps with disorders of sex development (DSD), so that babies without a fused scrotum containing 2 testes and who present with ‘hypospadias’ need full DSD investigations at birth.

INTRODUCTION

Undescended testis, or "cryptorchidism", is a very common anomaly in male infants and pre-adolescent boys, with about 1 in 20 boys undergoing treatment by the time they reach puberty. Not only is it prevalent, but also there remain unresolved questions about prognosis in adult life. It is not known yet whether the dramatic changes in the recommended age for surgery (from 15 years of age in the 1950’s, to six months old now) (1-4) will decrease the risk of infertility or testicular cancer. However, current treatment is based on the assumption that early surgery will prevent germ cell degeneration during childhood, leading to improved fertility and fewer tumors (5, 6).

Our understanding of the embryology has advanced rapidly in recent years, with new theories and experimental evidence supporting a complex anatomical process controlled directly and indirectly by hormones (7, 8). The classification of cryptorchidism also is changing, with the recent recognition of acquired anomalies (9, 10). With so much change in the way we view and treat cryptorchidism, endocrinologists will need to keep checking on the evolving controversies described in this chapter.

EMBRYOLOGY

The testes descend prenatally from their initial intra-abdominal location on the urogenital ridge into the low-temperature environment of the scrotum via a complex multi-stage mechanism (11). Prior to 7-8 weeks of development, the gonadal position is similar in both sexes. With the onset of sexual differentiation, the fetal testis begins producing anti-Müllerian hormone (AMH; also called Müllerian inhibiting substance (MIS)) from the Sertoli cells, as well as androgen and insulin-like hormone 3 (INSL3) from the Leydig cells. These hormones are involved in controlling descent of the male gonad, which is held by two thickenings in the mesentery, the cranial suspensory ligament at the upper pole, and the genito-inguinal ligament, or “gubernaculum”, at the lower pole (12).

During the initial (transabdominal) phase of descent, regression of the cranial ligament and thickening of the gubernaculum allows the testis to be held near the inguinal region (13). By contrast, in the female the cranial ligament persists while the gubernaculum remains thin and elongates, which together hold the ovary higher on the posterior abdominal wall as the fetal abdomen enlarges. The inguinal canal forms by the abdominal wall muscles developing around the caudal, gelatinous end of the gubernaculum, which initially ends at the future external inguinal ring. By 15 weeks the testis is attached by a short, stout and gelatinous gubernaculum to the future internal inguinal ring, while the ovary is higher in the pelvis (14).

In mid gestation a diverticulum of the peritoneal membrane, known as the processus vaginalis, begins to elongate within the gubernaculum, which retains a central connection (known as the gubernacular cord) with the epididymal tail and the lower pole of the testis. The caudal end of the gubernaculum grows out of the abdominal wall and elongates towards the scrotum, extending the processus vaginalis eventually to the scrotum. Between 25-30 weeks’ gestation the testis descends rapidly through the inguinal canal, and then more slowly across the pubic region and into the scrotum, with descent within this peritoneal diverticulum complete by 35 weeks. After the testis reaches the scrotum, two further anatomical events complete the inguinoscrotal phase, the first of which is obliteration of the proximal processus vaginalis (15). The second event is involution of the gelatinous gubernacular bulb and its anchoring to the inside of the scrotum. The former process prevents inguinal hernia or hydrocele and the latter process prevents extravaginal or perinatal torsion of the testis (6).

Figure 1. The embryological stages of testicular descent and the postnatal growth required to keep the testis in the scrotum.

The two main phases of descent appear to be controlled independently by hormones. (Fig. 1). INSL3 is the major factor controlling gubernacular enlargement (16-18) and androgen, particularly DHT, and AMH appear to play minor roles in this "swelling reaction" of the gubernaculum (19-22). Under the influence of the hormones mentioned above, the caudal end of the gubernaculum, where it attaches to the inguinal abdominal wall, enlarges by proliferation of the embryonic mesenchyme and deposition of extracellular matrix. Androgens also are responsible for regression of the cranial suspensory ligament, but they are not sufficient alone for transabdominal descent. The phase of gubernacular migration is controlled both directly and indirectly by androgens, with the aid of the genitofemoral nerve (GFN) releasing calcitonin gene-related peptide (CGRP) (23, 24). Androgens act during a critical time window to regulate gubernacular development (25). Recent evidence suggests that the androgen receptors controlling this masculinization of the GFN may not be in the nerve itself, but in the target organ, the inguinoscrotal fat pad in the mammary line (26) . The number of sensory neurons and the amount of CGRP in the genitofemoral nerve of rats are significantly less after exposure to the anti-androgen, flutamide, consistent with androgens stimulating structural and functional changes in the nerve. The nerve is proposed to orient the direction of gubernacular migration, while the physical force needed for elongation of the processus vaginalis is probably provided by intra-abdominal pressure (27). CGRP released from the nerve stimulates mitosis and cremaster muscle development in the gubernacular tip, enabling elongation to the scrotum (16). Estrogens have a minor inhibitory role in normal gubernacular development, but estrogenic endocrine disruptors may be responsible in larger doses for cryptorchidism secondary to suppression of the "swelling reaction" by inhibition of INSL3.

The trigger that initiates active migration of the caudal tip of the gubernaculum may come from the inguinoscrotal fat pad in the mammary line (28), as the androgen receptors are present in the mammary line mesenchyme but not in the adjacent gubernaculum during the critical window of androgenic programming in rodents (25, 29). The gubernaculum has a surprisingly close link with the embryonic breast in normal marsupials as well as in eutherian animal models, such as the rat and mouse, especially after they have been exposed to the antiandrogen, flutamide (30, 31).

The primitive mammary line is in continuity with the apical ectodermal ridges of the upper and lower limb buds, and hence is likely to contain similar activated signaling systems as seen in limb bud development (32). These signals are likely to initiate outgrowth of the gubernaculum from the abdominal wall, so that it can migrate to the scrotum.

ETIOLOGY

Any anomaly in either the hormonal control or the anatomical processes in normal testicular descent will cause cryptorchidism (33). Hormonal defects in INSL3, AMH or androgenic action are identified only rarely, suggesting that mechanical anomalies may be more common. Those patients with hormonal defects may present with rare disorders of sexual development (DSD) with cryptorchidism as part of the complex genital anomaly. The first or transabdominal phase involves little movement of the testis and this may explain the low frequency (5-10%) of intra-abdominal testes. As the gubernacular swelling reaction holds the testis close to the inguinal canal while other structures grow further away, the transabdominal phase is only relative movement of the testis and hence less likely to be abnormal. By contrast, the inguinoscrotal migration phase requires very significant mechanical and anatomical re-arrangements, and consequently, anomalies are common: over 60% of testes are found just outside the external inguinal ring, consistent with anomalous or arrested gubernacular migration. Transient deficiency of androgen production in utero,perhaps related to deficiency of gonadotropin production by the fetal pituitary or the placenta (34), may account for some, particularly where there is intra-uterine growth retardation. Anomalies of the genitofemoral nerve also may cause undescended testes. For example, perineal testes may be caused by an anomalous location of the genitofemoral nerve (35).

Inherited syndromes frequently are associated with cryptorchidism. Hypothalamic dysfunction, connective tissue disorders, neurogenic (e.g., spina bifida), and mechanical anomalies (e.g., arthrogryposis multiplex congenita) may all cause disruption in testicular descent (36-38). Cryptorchidism is also common in infants with abdominal wall defects, such as exomphalos or omphalocele, gastroschisis and exstrophy of the bladder (39).

There is much current interest in the potential adverse effects of environmental estrogenic endocrine disruptors on the incidence of both cryptorchidism and hypospadias (40). In addition, there are data on the effect of diethylstilbestrol (DES) on cryptorchidism in male offspring of exposed mothers (41). In the latter case there is supporting evidence from animal models (42), although in the former, the cause-and-effect relationship is more tenuous, because the level of exposure is less clear, and the epidemiology may not have allowed for changes in diagnostic criteria over recent decades. More work is needed before we can ascertain a proven cause-and-effect link with synthetic molecules in the environment.

The body of the epididymis is hypoplastic and frequently is not tightly adherent to the cryptorchid testis (43). This is more common in high intra-abdominal testes and probably indicates significantly decreased androgen production. Whether epididymal-testicular separation is the cause or the result of cryptorchidism is not known (44). In addition, its effect on fertility is uncertain, even though the rete testis is nearly always still connected to the head of the epididymis. Recent studies show a strong link between maternal smoking and cryptorchidism in male offspring (45, 46).

CLINICAL PRESENTATION

Up to 4-5% of newborn males show cryptorchidism, but this falls to 1-2% by 12 weeks after term, following normal (but postnatal) descent in premature infants, and delayed postnatal descent in some term babies. Beyond 12 weeks, spontaneous testicular descent is rare (47). Geographic differences in prevalence of cryptorchidism have been reported, with 9% of Danish boys with undescended testes at birth, compared with only 2% of males from Finland. Some of these apparent differences, however, may be related to the definitions used for ‘cryptorchidism’ in these studies. An undescended testis is best defined as a testis that cannot be manipulated into the bottom of the scrotum (without excess tension on the spermatic cord) by 12 weeks of age. Most testes (about 85%) are near the neck of the scrotum, or just lateral to the external inguinal ring, described by Denis Browne as the "superficial inguinal pouch”(48).

A few cryptorchid testes are within the inguinal canal, making them unpalpable unless they can be squeezed out of the external inguinal ring by compression. Ten percent of testes are intra-abdominal or absent (presumed to be secondary to prenatal torsion). Ectopic cryptorchid testes are rare (< 5%), and occur in the perineum, prepubic region, thigh, or the contralateral inguinal canal (transverse testicular ectopia) (49).

ENDOCRINE EFFECTS OF CRYPTORCHIDISM

In infants with undescended testes, the testosterone and gonadotropin levels are diminished compared with normal infants between one and four months of age (50, 51), which is during the normal, transient hormonal surge, known as ‘minipuberty’ (52). Whether this is a sign of primary endocrinopathy or secondary dysfunction of the testis, caused by heat stress when the gonad is not in the low temperature environment of the scrotum, is unknown. Postnatal increase in testosterone production is also diminished in premature infants, perhaps secondary to inadequate stimulation by chorionic gonadotropin in utero (53). HCG is low compared with early pregnancy and may be of functional significance. Despite lower than normal androgen levels between 1 and 4 months of age, there is no apparent anomaly in androgen receptors from gonadal or skin biopsies collected at orchidopexy (54).

The postnatal secretion of both AMH and inhibin-B in cryptorchid infants is also deranged. Production of AMH from Sertoli cells normally increases between 4-12 months, but this surge is blunted in undescended testes (55, 56). Inhibin-B normally increases at minipuberty and remains elevated into the second year of life , but levels in infants with cryptorchidism are lower (57).

GERM CELL MATURATION IN CRYPTORCHIDISM

Germ cells mature postnatally from a primitive gonocyte through a series of steps to primary spermatocytes by 3-4 years. This process is perturbed in cryptorchid testes, with failure of transformation of gonocytes into type-A spermatogonia between 4-12 months (58-60). These observations suggest that germ cell deficiency may be at least partly secondary to early postnatal dysfunction, rather than being congenital, as proposed by some authors (61, 62).

Lack of germ cell transformation has been proposed to be secondary to postnatal androgen deficiency (60, 63) or low AMH levels (63). Recent studies, however, suggest that transformation is normal in both infants and mice with complete androgen insensitivity syndrome (CAIS), and may be mediated by activin or another TGF-family factor (64). Abnormal postnatal maturation of gonocytes could lead to both infertility and malignancy (65)), although some authors propose that there may be congenital carcinoma in-situ-cells in the cryptorchid testis (61, 66, 67).

There is now a consensus that type-A spermatogonia are likely to be the stem cells for future spermatogenesis, and that their appearance between 3 and 12 months of age, as neonatal gonocytes transform, is the key step in postnatal germ cell development (68, 69). Should this be confirmed, it implies that early surgical intervention should lead to an excellent prognosis, as long as the subsequent germ cell deficiency is secondary to postnatal heat stress of the maldescended testis, and therefore reversible. Failure of the totipotential gonocytes to transform into unipotential spermatogenic stem cells may leave some persisting gonocytes in the undescended testis, which is speculated to be the origin of subsequent tumors.

DIAGNOSIS

The aim of clinical examination is to locate the gonad, if palpable, and determine its lowest position without causing painful traction on the spermatic cord (which probably corresponds to the caudal limit of the tunica vaginalis) (70). In infants, the diagnosis is straightforward because the scrotum is thin and pendulous. Hypoplasia of the hemiscrotum indicates it does not contain a testis. The inguinal testis is within its tunica vaginalis which gives it significant mobility. Ultrasonography has become more frequently used for diagnosis of the impalpable testis, but generally is not contributory for true intra-abdominal testes. This is because absence of the testis (secondary to possible perinatal torsion) is common, and also because intra-abdominal testes are often concealed by the bowel and other viscera (71). In addition, the mobility of the undescended testis within its tunica vaginalis may make location by ultrasonography difficult. An ultrasound scan can be justified in bilateral impalpable testes, to confirm the presence of a testis. In addition AMH and inhibin-B should be measured to confirm the presence of functioning Sertoli cells (57). A simple and reliable approach is to use laparoscopy, which readily locates the testis itself (or blind-ending gonadal vessels), and allows orchidopexy in experienced hands (72).

TREATMENT

Newborn and Infant

Hormone therapy has become extremely controversial (73, 74) as it was based on the two assumptions that cryptorchidism is not only secondary to a deficiency of the hypothalamic-pituitary-gonadal axis, but also the mechanical processes were simple. Both hCG and GnRH therapy have been tried, with success rates ranging from 10-50%. Randomized, double-blind, placebo-controlled studies have not shown more than marginal benefit with either hCG or GnRH (75-77). Despite proven endocrine control of descent, the mechanical factors appear to be too complex for this simple approach to be successful except for acquired undescended testes (76). Because of its poor efficacy and possible side effects, a consensus meeting in Scandinavia several years ago recommended that hormone treatment be abandoned completely (73, 74).

Surgical treatment is based on the premise that early intervention will prevent secondary testicular degeneration caused by high temperature (35-37^oC) as the lower temperature of the scrotum (33^oC) is essential for normal postnatal germ cell maturation (78). Evidence of progressive germ cell loss in the cryptorchid testis after six months of age has accumulated over the last 50 years and now suggests that orchidopexy should be considered between 6 and 12 months of age (1-4). The first signs of abnormal germ cell development can be seen between 4-12 months of age (60), and intervention is based on the premise that these changes are secondary to high temperature and should be reversible. Certainly in animal models, early intervention prevents germ cell loss (79). A prospective study of children randomized to early (9 months) or late (3 years) surgery is showing improved testicular development with early intervention, as measured by ultrasonography at 4 years of age (80, 81). Surgery at this very early age ideally needs a trained pediatric surgeon, as the technique is quite different from that for a 5-10 year-old boy (82, 83)

All baby boys need examination at birth to document gonadal position. Those infants without two fully descended testes should be re-examined at 12 weeks of age and, if a testis is still undescended, the child should be referred to a pediatric surgeon for possible surgical treatment. Orchidopexy is done as an ambulatory procedure, with discharge home a few hours after operation. General anesthesia is supplemented with local/regional analgesia, which will provide pain relief for the first few hours postoperatively.

Prognosis

The complication rate after orchidopexy is less than 5% in experienced hands (82, 83). Wound infection is common in infants secondary to external contamination of the wound, although there is a low risk of atrophy of the testis which is greatest when intra-abdominal testes are pulled down under tension. Laparoscopy, with or without ligation of the testicular vessels (Fowler-Stephens procedure) (84), shows increasing success for high intra-abdominal gonads (72, 85, 86). The prognosis for fertility, the primary aim of orchidopexy, remains uncertain (61),(87-89). However, extensive review of the recent literature suggests improved outcomes with very early surgery (89, 90). Now that early germ cell maturation in the first year is known to be deranged, improved fertility is to be expected with very early orchidopexy (88-90). Unfortunately, it will be a few more years before the long-term outcome of this new consensus policy is known.

The risk of malignancy was previously calculated to be 5 -10 times greater than normal for a man with a history of unilateral cryptorchidism (91-95) when surgery was performed in mid-childhood. The risk in a future generation for men who underwent orchidopexy in infancy is unknown at present, but is anticipated to be much lower than in the past, as supported by preliminary evidence (90) .

Some clinical features are associated with statistically better outcomes, and include testes near the neck of the scrotum, and ascending or retractile testes (see below), where malignancy risk is now thought to be similar to men without cryptorchidism in childhood (61, 89). Poor prognostic factors are primary testicular or epididymal dysplasia, intra-abdominal or intra-canalicular position, associated strangulated inguinal hernia and (possibly) surgery late in childhood or adolescence (96).

ACQUIRED CRYPTORCHIDISM

Retractile Testes

Retraction of the testis out of the scrotum secondary to reflex contraction of the cremaster muscle is both normal and common and is involved in temperature control and protecting the testis from trauma. The reflex is absent or weak at birth and becomes more active after one year, reaching a peak in 5-10 year-old boys (97).

Many testes are erroneously described as "retractile" when they can be pulled down into the scrotum during the physical examination but retract back out of the scrotum on release. This retractability is assumed to be secondary to cremasteric activity, but an alternative explanation has been proposed recently, which is that the malposition may be caused by failure of the spermatic cord to elongate with age (98). Since the distance from external inguinal ring to the bottom of the scrotum increases from 5 cm at birth to 8-10 cm at 10 years of age, the spermatic cord must double in length to keep the testis in the scrotum during the first decade. Preliminary evidence suggests that failure of complete obliteration of the processus vaginalis may prevent normal postnatal elongation of the vas and vessels (99) (Fig. 2).

Figure 2. Acquired cryptorchidism occurs when the spermatic cord fails to elongate in proportion to growth between birth and late childhood. This figure shows what happens between birth and 5-10 years of age when the spermatic cord does not elongate with age.

Ascending Testes

The ascending testis is a special variant of acquired maldescent, in which there is delayed postnatal descent of the testis in the first three months after birth (100), (101). Follow-up studies suggest that subsequent "ascent" of the testis is common later in childhood (102-104)). The cause for ascending testes is not resolved, with the only well-documented cause being neuronal dysfunction as seen in children with cerebral palsy and spastic diplegia (105). In normal children, the explanation is likely to be persistence of the processus vaginalis, either patent or as a fibrous remnant (106).

Management of Acquired Cryptorchidism

Both “retractile” and ascending testes are likely to be different names for what is, in effect, acquired cryptorchidism caused by persistence of the processus vaginalis (107-110). The normal spermatic cord elongates gradually with growth, and hence acquired cryptorchidism develops insidiously, presenting mostly between 5 and 10 years of age (111). Orchidopexy is recommended by some authors once the testis can no longer reside spontaneously in the scrotum, and can be performed in the standard manner or by a scrotal approach (112). Once the fibrous remnant of the processus vaginalis is divided, the testis can reach the scrotum easily. In The Netherlands recently there has been a consensus to treat acquired cryptorchidism conservatively (113), with follow-up suggesting a poor outcome for fertility (114). However, whether early treatment by orchidopexy will improve the prognosis for fertility is not yet known.

The prognosis for this special group is probably much better than for congenital cryptorchidism, as the testis is normally located in the scrotum during infancy (89), (115, 116), when germ cell maturation is occurring. Unfortunately, previous studies of outcome for fertility and malignancy have not discriminated between congenital and acquired cryptorchidism, but recent studies suggest a mild suppression of fertility and little risk of malignancy (61),(89). The frequency of acquired cryptorchidism, may account for up to half of all children coming to orchidopexy (111),(117).

HYPOSPADIAS

The primitive phallus begins to enlarge at 8 weeks of development in the male, in response to fetal androgens. The inner genital folds fuse in the midline in association with elongation and canalization of the endodermal urethral plate on the penile shaft, to create the anterior urethra up to the coronal groove by about 12 weeks’ gestation, while the urethra within the glans forms in mid-gestation by canalization of the endoderm forming the distal urethral plate (118, 119). The preputial skin forms from low folds on the dorsum of the shaft at the corona, eventually covering the entire glans (118). Recent evidence suggests that some of the effects of androgen in penile development may be mediated by aromatization to estrogen, and estrogen receptors (ERα and ERβ) are located in the developing prepuce, glans and urethral plate (120).

Failure of urethral canalization and fusion leads to hypospadias (Greek for "hole underneath"), with secondary deficiency of the ventral prepuce ("dorsal hood") and relative deficiency in growth of the peri-urethral tissues compared with the corpora cavernosa, leading to "chordee", or ventral curvature of the penis (121), (Fig. 3).

Figure 3. Hypospadias is associated with a) failure of the urethral meatus to be located on the tip of the glans, and failed ventral fusion of the prepuce, causing a ‘dorsal hood’; and b) inadequate growth of the ventral shaft around the urethra, leading to bend, known as chordee.

Hypospadias occurs in one in every 100-300 boys, depending on the criteria used for diagnosis (122). About 10% of patients with hypospadias have a sibling or father with the anomaly, suggesting a polygenic inheritance pattern (123). The severity of the anomaly varies widely, from a perineal opening to an opening on the proximal glans, or even chordee with a normal urethral meatus.

Care is needed in diagnosis, as some infants with a disorder of sex development (DSD) and ambiguous genitalia may be diagnosed as "simple hypospadias” (124). Since hypospadias is an anatomical anomaly of anterior urethral development, the rest of the external (and also internal) genitalia should be normal. Patients with DSD, by contrast, have a more extensive genital anomaly, reflecting the failure of all androgen-dependent development.

A useful rule-of-thumb is to assume that any baby with "hypospadias", as well as an undescended testis and/or bifid scrotum, should be investigated for DSD, with immediate hormonal, chromosomal and anatomical studies. Immediate gender assignment as male is only safe when the scrotum is fused and both testes are descended fully (i.e., androgen-dependent genital development is normal).

Surgical treatment is required to reconstruct the penis in hypospadias (125-127). Despite numerous different operative techniques available, there are a few principles of management: a). Create an extension to the urethra to bring it to the tip of the glans, allowing normal micturition; b) Correct the chordee to create a straight shaft for normal sexual function; c) Finally, repair the dorsal hood for cosmetic reasons. In severe cases the skin is moved ventrally to create the urethra and elongate the ventral surface; in mild cases the dorsal hood can be repaired to restore the normal appearance of the foreskin. Surgery is best between 6-18 months, and this is the recommended age, as this avoids much psychological stress (128) (129) but the operation should be completed at the latest in infants or young children prior to school entry. The operation may be done as day surgery, but may need admission with urinary diversion, depending on the severity of the anomaly and the surgeon’s preference.

The prognosis for micturition and sexual function is good, with improving cosmetic appearance with newer procedures (130). However, wound infection, hematoma, urethral breakdown to create a fistula, and stricture, continue to be serious problems, as the surgery requires significant skill (131, 132).

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Androgens and Cardiovascular Disease in Men

ABSTRACT

Testosterone is the principal male sex hormone or androgen, which regulates sexual characteristics and body composition. Testosterone is converted to bioactive metabolites dihydrotestosterone and estradiol. Circulating testosterone peaks in early adulthood and declines gradually across middle and older age: older men exhibit lower testosterone and dihydrotestosterone concentrations compared to younger men. In older men, lower testosterone concentrations are associated with higher incidence of cardiovascular events. Lower testosterone and dihydrotestosterone concentrations have also been associated with higher cardiovascular mortality in older men. However, causation is unproven as a randomized placebo-controlled trial of testosterone treatment sufficiently powered to examine outcomes of cardiovascular events or mortality has yet to be reported. Potential mechanisms by which testosterone could exert beneficial actions in the vasculature include reduction in cholesterol accumulation and modulation of inflammation. Smaller randomized trials of testosterone therapy have shown improvements in surrogate endpoints related to cardiovascular risk. However, other trials of testosterone have not shown improvements in carotid atherosclerosis, as assessed by carotid intima-media thickness. One study reported an increase in coronary atheroma assessed using coronary computed tomography angiography in older men receiving testosterone therapy over 12 months. Although one randomized trial of testosterone therapy in older men with mobility limitations reported an excess of adverse events in the treatment arm, larger recent trials in middle-aged to older men did not find any excess of cardiovascular adverse events with testosterone treatment. Meta-analyses of testosterone trials generally have not shown an increase in cardiovascular adverse events. Retrospective case-control studies of health insurance databases have major methodological limitations. The results from such studies are inconsistent, associating testosterone prescriptions with either increased or decreased risk of cardiovascular events, and with lower mortality. While androgen deprivation in men with prostate cancer results in adverse metabolic effects, abuse of high dosages of androgenic steroids is associated with harm. Thus, while some epidemiological studies associate higher circulating concentrations (but within the normal range) of endogenous androgens with lower risk of cardiovascular events and mortality, the effects of exogenous androgens in the form of testosterone therapy seeking to maintain physiological circulating androgen concentrations on the cardiovascular system remain uncertain. This evidence gap has to be accommodated in the current clinical management of hypogonadal men and should be addressed by further randomized interventional studies to clarify whether testosterone treatment has beneficial, neutral or adverse effects on the cardiovascular system.

INTRODUCTION

Androgen Physiology

THE HYPOTHALAMIC-PITUITARY AXIS

Testosterone (T) is the principal male sex hormone or androgen that regulates sexual maturation and secondary sexual characteristics and body composition in adult men (1). T undergoes conversion into two major bioactive metabolites, dihydrotestosterone (DHT) a more potent ligand for the androgen receptor, and estradiol (E2), a ligand for estrogen receptors (2). T is produced primarily by the testis, under stimulation of luteinizing hormone (LH) from the pituitary gland, itself under regulation of gonadotrophin releasing hormone (GnRH) from the hypothalamus. The hypothalamic-pituitary-testicular (HPT) axis is under negative feedback regulation via T and E2, acting on the central components of the HPT axis (1,3-5).

CONSEQUENCES OF ANDROGEN DEFICIENCY

Androgens play diverse roles in the body, and androgen deficiency in men results in multiple symptoms and signs extending from loss of libido, lethargy, fatigue, poor concentration to gynecomastia, accumulation of fat, loss of muscle mass, and osteopenia or osteoporosis (6,7). Actions of T are amplified by local conversion to DHT in tissues such as the prostate and skin by the enzyme 5α-reductase (8). Of note, some of actions of T, such as in bone and adipose tissue, are mediated via conversion to E2 by the enzyme aromatase (9-11).

MEASUREMENT OF CIRCULATING ANDROGEN CONCENTRATIONS

Immunoassays have been the standard method for measurement of circulating sex hormones: however, these can exhibit non-specificity and method-dependent bias, particularly at lower hormone concentrations (12,13). Mass spectrometry is regarded as the gold standard for assay of T concentrations (14). Although mass spectrometry is preferred, it is not widely available and validated immunoassays can be informative (6,7). In the circulation, T (and DHT and E2) are bound with high affinity to sex hormone-binding globulin (SHBG), T is also bound with lower affinity to albumin with a small fraction unbound or free (15). However, whether unbound or “free” T represents a more biologically active form of the hormone in vivo is controversial (16). A major difficulty is that measurement of free T using equilibrium dialysis is technically demanding and thus rarely performed. Instead, free T is typically calculated, and this result may vary according to the equations used (15-18). Furthermore, established reference ranges for free T are lacking (7,19). Thus, analysis of actions of T in the male body and cardiovascular system involves consideration of not only its bioactive metabolites, but also the accuracy of assays used and the limitations of calculated free T as a biomarker.

Androgens and Male Ageing

DECLINE IN CIRCULATING ANDROGENS WITH MALE AGING

Circulating T peaks in early adulthood and declines gradually across middle and older age, thus older men exhibit lower T and DHT concentrations compared to younger men (20-22). Observational studies have shown longitudinal declines in T and DHT in ageing men, with parallel increases in LH and SHBG (23-25). This phenomenon suggests that in some men, there is progressive impairment of testicular endocrine function with ageing (25). However, whether older men with symptoms consistent with androgen deficiency and lower circulating concentrations of T compared to younger men, have an androgen deficiency state remains unclear (26,27). The bioactive metabolites of T, DHT and E2 measured with mass spectrometry have been associated with longer leucocyte telomere length, a measure of slower biological ageing, in middle-aged and older men (28,29). However, a study in mostly middle-aged men showed no association between T measured with immunoassay and leucocyte telomere length (30). Thus, there is considerable interest in the question of whether lower T concentrations might contribute to various manifestations of ill health in ageing men.

CONTROVERSIES OVER FUNCTIONAL HYPOGONADISM

Men with disorders of the hypothalamus, pituitary, or testes resulting in hypogonadism have symptoms and signs of androgen deficiency, and low circulating T concentrations (6). These men are classified as having pathological (or classic or organic) hypogonadism, and T treatment is routinely offered to improve symptoms and restore body composition (6,7). However, low circulating T concentrations are found in many conditions where the HPT axis is intact, including ageing, obesity, and systemic illnesses (7,31). In this context, low T concentrations result from reduction or suppression of HPT axis function, rather than an intrinsic disorder of the HPT axis, with the label of “functional hypogonadism” applied (7,31). Obesity is closely associated with reduced circulating T and SHBG, and loss of excess weight restores endogenous production of T (32-35). Thus, low T might be a biomarker for the presence of systemic illnesses, rather than a contributing or causal factor. Whether or not, and if so which, men with functional hypogonadism should receive T treatment, remains the subject of debate (7,19,20).

Androgens and Risks of Cardiovascular Disease

AGE AND OBESITY AS COMMON RISK FACTORS

Advancing age is an established risk factor for chronic diseases including cardiovascular disease (CVD) and mortality (36,37). Age is a component of all major cardiovascular risk calculators (38). Similarly, obesity, with its close associations with insulin resistance and diabetes risk, is a robust cardiovascular risk factor, and - unlike age - is a potentially modifiable one, whether via bariatric surgery or incretin-based medical therapy (41-44). This is illustrated by observational studies which show a reduction in cardiovascular events and mortality following bariatric surgery in patients who are obese (mean age 48 years) (41,42). Thus, advancing age and obesity are both associated with low circulating T concentrations, and are also risk factors for CVD.

CONFLUENCE OF AGE, OBESITY, LOW TESTOTERONE AND CARDIOVASCULAR DISEASE

Given that age and obesity are associated with both low circulating T concentrations and increased risk of CVD, understanding the relationship between these factors becomes vitally important (45). Demographic change will result in increasing numbers of older men in communities worldwide, who will have lower circulating T concentrations and who are at risk of ill health including from CVD (36). If low T contributes to CVD risk, either directly or via its association with obesity, then this represents a potential pathway for intervention to preserve health in ageing men. Conversely, if T is a biomarker for CVD, it may still have a role in risk stratification and identification of men at risk who may benefit from non-hormonal interventions directed at conventional risk factors for CVD.

EPIDEMIOLOGICAL STUDIES

Associations of Androgens with Cardiovascular Events

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

Prospective cohort studies report the association of endogenous sex hormones with incidence of cardiovascular events (Table 1). Analyses invariably adjust for age, and typically also include adjustment for body mass index (or waist circumference) and other conventional cardiovascular risk factors. From studies reporting sex hormone results based on immunoassays, some longitudinal analyses have shown no association of total T concentrations with incidence of myocardial infarction (MI) or ischemic heart disease (IHD) events (46,49,53,63,64). In an analysis of 3,443 men aged ≥70 years from the Western Australian Health In Men Study (HIMS), low total T concentrations were associated with an increased incidence of stroke (50). This finding was confirmed by later studies (57,59). In another analysis from HIMS, higher LH was associated with incident IHD (52). One smaller study reported a U-shaped association of total T with incidence of cardiovascular events (54). There were conflicting associations of total E2 with stroke (47,48), and there was no association of total E2 with the incidence of MI (49). In one study, men with lower total T had a higher risk of heart failure, but this was not confirmed in another study (65,68).

Table 1. Cohort Studies Examining Associations Between Sex Hormones with Cardiovascular Events in Middle-Aged and Older Men
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Summary of results
Smith GD, 2005 (46)	2,512	16.5	45-59	No association of total T with IHD events or deaths.
Arnlov J, 2006 (47)	2,084	10	56	Higher total E2 at baseline associated with lower incidence of CVD events, total T was not associated.
Abbott RD, 2007 (48)	2,197	≤7	71-93	Baseline total E2 in top quintile (≥125 pmol/L) associated with higher risk of stroke, total T was not associated.
Vikan T, 2009 (49)	1,568	≤13	59.6	No association of total T or E2 with incident MI, or with CVD or IHD mortality.
Yeap BB, 2009 (50)	3,443	3.5	≥70	Total and free T in the lowest quartiles (<11.7 nmol/liter and <222 pmol/liter) predicted increased incidence of stroke or TIA.
* Ohlsson C, 2011 (51)	2,416	5	69-81	Men with total T^a in highest quartile (≥19 mol/L) had lower risk of CVD events. E2 was not associated.
Hyde Z, 2011 (52)	3,637	5.1	70-88	Higher LH was associated with incident IHD.
Haring R, 2013 (53)	254	5, 10	75.5	No associations of baseline total T or total E2 with incident CVD events.
Soisson V, 2013 (54)	495; 146	4	>65	Total T in lowest and highest quintiles associated with CHD or stroke.
* Shores MM, 2014 (55)	1,032	9	76	DHT^b <1.7 or >2.6 nmol/L associated with cardiovascular events. Total T was not associated.
* Shores MM, 2014 (56)	1,032	10	76	Non-linear association of DHT^b with stroke with lowest risk in men with DHT 1.7-2.6 nmol/L. Total T^b was not associated with stroke. DHT <0.86 nmol/L associated with CVD mortality.
* Yeap BB, 2014 (57)	3,690	6.6	70-89	Higher total T^c (>12.6 nmol/L) or DHT (>1.34 nmol/L) associated with lower incidence of stroke. T^c, DHT and E2 were not associated with MI.
* Srinath R, 2015 (58)	1,558	12.8	63.1	T^d was not associated with incidence of CHD events, or cardiac-related mortality.
Holmegard HN, 2016 (59)	4,602	20	57	Total T in lowest decile (0-10^thpercentile) associated with stroke.
* Chan YX, 2016 (60)	1,804	14.9	50.3	Total T^c, DHT and E2 were not associated with CVD events.
* Srinath R, 2016 (61)	1,558	14.1	63.1	T^d was not associated with stroke.
Wang A, 2019 (62)	5,553	6	63.5	Neither total T nor free T were associated with CVD events.
* Gyawali P, 2019 (63)	1,492	4.9	54.2	Higher total T^c associated with lower risk of incident CVD events, but not with CVD mortality. E2 not associated.
Hatami H, 2020 (64)	816	12	46.1	Total T was not associated with risk of CVD events.
Zhao D, 2020 (65)	4,107	19.2	63.2	Lower total T associated with increased risk of incident heart failure.
* Collett T-H, 2020 (66)	552	7.4	72.4	Total T^d not associated with CVD events.
* Boden WE, 2020 (67)	2,118	3	≥40	643 men with total T^b <10.4 nmol/L had higher risk of combined endpoint of CHD death, MI or stroke, compared with 1,475 men with total T ≥10.4 nmol/L.
Shafer S, 2021 (68)	3,865	13.8	48.2	Lower total T not associated with incident heart failure.
Yeap BB, 2022 (69)	210,700	9	58	Total T not associated with incident MI, stroke, heart failure or MACE. Calculated free T not associated with incident MI, stroke or heart failure, but associated with incidence of MACE.

IHD=ischemic heart disease, CVD=cardiovascular disease, MI=myocardial infarction, TIA=transient ischemic attack, CHD=coronary heart disease, MACE=major cardiovascular adverse event. * denotes studies where total T, DHT and/or E2 were measured using mass spectrometry. ^aT and E2 assayed using gas chromatography-mass spectrometry (GC-MS), ^bT and DHT assayed using liquid chromatography-tandem mass spectrometry (LC-MS), ^cT, DHT and E2 assayed by LC-MS, ^dT assayed using LC-MS

Recently the association of testosterone with CVD events was examined in the largest prospective cohort study to date, the United Kingdom (UK) Biobank (69). In this study of 210,700 men aged 40-69 years at baseline, with 9 years follow-up, 8,790 had an incident CVD event. Total T was not associated with risk of incident MI, ischemic stroke, hemorrhagic stroke, heart failure, nor major cardiovascular adverse events (MACE) defined as the composite endpoint of non-fatal MI, non-fatal ischemic stroke, and CVD death. The large size of the UK Biobank, and accumulation of outcome events over the period of follow-up, provided power to examine these associations in a robust fashion. UK Biobank used an immunoassay for measurement of serum T which may underestimate results compared to mass spectrometry (70), and UK Biobank men were generally healthier than the UK male population as a whole (71). Therefore, while these results are convincing, their generalizability to different populations in other regions needs to be established.

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

Prospective cohort studies where sex hormones were measured using mass spectrometry are shown (Table 1, marked with *). In the Osteoporotic Fractures in Men Study in Sweden (MrOS), a large prospective cohort study of 2,416 men aged 69-81 years, men with higher total T had a lower incidence of CVD events (51). In HIMS, a later analysis of 3,690 men aged 70-89 years with sex hormones measured using mass spectrometry confirmed the association of low total T with higher incidence of stroke (57). In these analyses, total E2 was not associated with these outcomes (51,57). Analyses from the Cardiovascular Health Study (CHS) of 1,032 men aged 76 years suggested a U-shaped association of DHT with CVD events and stroke risk (55,56). Of note, an analysis from the Busselton Health Study (BHS) of 1,804 predominantly middle-aged men found no association of sex hormones with incidence of CVD events (60). Analyses from the Atherosclerosis Risk in Communities Study of 1,558 men aged 63 years found no association of sex hormones with CVD events (58,61), similar to the findings from a small subset of the MrOS USA study (66). By contrast, in the Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) cohort of 1,492 men followed for 4.9 years, higher total T was associated with a lower risk of CVD events (63). Furthermore, in a post-hoc analysis of 2,118 men with metabolic syndrome participating in a trial of niacin or placebo plus simvastatin, men who had a baseline total T <10.4 nmol/L had higher risk of the combined endpoint of coronary heart disease death, MI or stroke, compared with men with higher total T concentrations (65).

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR DISEASE

Taken together, these epidemiological studies suggest that there may be an association of lower endogenous T concentrations with increased risk of CVD events in middle-aged and older men. However, the studies provide a mix of positive, equivocal and negative results. Major cohort studies using mass spectrometry for assay of sex hormones associated higher total T concentrations with lower risk of CVD events in older (51,57) and middle-aged to older men (63,67). There may be a predilection for lower testosterone, measured by mass spectrometry, to be associated with stroke risk (57). In one study lower DHT was associated with CVD events including stroke, with a non-linear association (55,56). Inconsistent results in other studies may have been due to smaller cohort sizes and fewer outcome events reducing the power available to detect underlying associations. However, in the largest ever prospective cohort study, the UK Biobank, there was no association of testosterone with a range of CVD events (69). Overall, epidemiological studies would suggest a possible protective effect of endogenous androgens against CVD events in the older population of men, rather than in relatively healthy middle-aged men.

Associations of Androgens with Cardiovascular Mortality

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

Several of the studies in Table 1 reported CVD-related mortality in addition to events. Other studies where the outcome was based on CVD-related mortality are summarized in Table 2. Studies almost invariably adjusted for age, and typically adjusted for BMI and other cardiovascular risk factors. In two studies using immunoassay for assay of sex hormones, T was not associated with CVD or ischemic heart disease (IHD) mortality (46,49). However, several other studies using immunoassay for sex hormones did find associations of lower endogenous total T concentrations with increased risk of CVD-related death (72,74,77,81). Lower calculated free T was also associated with increased CVD-related mortality in some studies (76,78,81), but was associated with lower IHD mortality in one study (73). Lower total E2 was associated with CVD mortality in one study (76). In an analysis from the UK Biobank of 149,436 men followed for 11.3 years, there was no association of either total or calculated free T with risk of CVD mortality (84).

Table 2. Cohort Studies Examining Associations Between Sex Hormones and CVD-Related Mortality in Middle-Aged and Older Men
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Summary of results
Khaw K-T, 2007 (72)	825 and 1489	≤10	40-79	Total T inversely related to mortality from all causes, CVD and cancer.
Araujo AB, 2007 (73)	1,686	15.3	40-70	Lower free T associated with lower IHD mortality. Equivocal association of lower DHT with IHD mortality.
Laughlin GA, 2008 (74)	794	11.8	50-91	Total T in the lowest quartile (<8.4 nmol/L) predicted increased mortality from all causes and from CVD and respiratory causes.
* Tivesten A, 2009 (75)	3,014	4.5	75	Total T^a and E2 levels in the lowest quartiles predicted all-cause and non-CVD mortality. T and E2 were not associated with CVD mortality.
Menke A, 2010 (76)	1,114	18	≥20	Lower free T associated with overall and CVD mortality in first 9 years of follow-up. Lower total E2 associated with CVD mortality. (Difference between 90^th and 10^thpercentiles for free T and total E2)
Haring R, 2010 (77)	1,954	7.2	20-79	Total T <8.7 nmol/L associated with increased all-cause, CVD and cancer mortality.
Hyde Z, 2012 (78)	3,637	5.1	70-88	Lower free T (100 vs 280 pmol/L) predicted all-cause and CVD mortality.
* Yeap BB, 2014 (79)	3,690	7.1	70-89	Optimal total T^b (9.8-15.8 nmol/L) predicted lower all-cause mortality. Higher DHT (>1.3 nmol/L) predicted lower IHD mortality. E2 was not associated with mortality.
* Pye SR, 2014 (80)	2,599	4.3	40-79	Presence of sexual symptoms and total T^c <8 nmol/L associated with all-cause and CVD mortality, total or free T not associated.
Holmboe SA, 2015 (81)	5,323	18.5	30-70	Higher T or free T (highest vs lowest quartile) associated with lower CVD mortality.
* Hsu B, 2016 (82)	1,705, 1,367 and 958	0, 2 and 5	≥70	Decrease in total T^d over time associated with all-cause but not CVD mortality. Decrease in total E2^d was associated with all-cause and CVD mortality.
* Chasland L, 2017 (83)	1,649	20	49.8	Higher physical activity and total T^b, DHT and E2 were not associated with CVD events. Men with higher physical activity and DHT had the lowest risk of CVD death. Men with lower physical activity and higher E2 had greater risk of CVD death.
Yeap BB, 2021 (84)	149,436	11.3	58.0	Men with lower total T had higher all-cause and cancer-related mortality, no association with CVD deaths.

IHD=ischemic heart disease, CVD=cardiovascular disease, CHD=coronary heart disease. * denotes studies where total T, DHT and/or E2 were measured by mass spectrometry; free T was calculated. ^aT and E2 measured using gas chromatography-mass spectrometry (GC-MS). ^bT, DHT and E2 measured using liquid chromatography-tandem mass spectrometry (LC-MS), ^cT measured using GC-MS, ^dT and E2 measured using LC-MS.

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

Prospective cohort studies using mass spectrometry for assay of sex hormones are of interest. In an analysis from MrOS in Sweden, lower endogenous total T and E2 concentrations were associated with all-cause and non-CVD mortality, but not with CVD mortality (75). Interestingly, in an analysis from HIMS of 3,690 men aged 70-89 years at baseline, optimal endogenous total T concentrations were associated with survival, and higher DHT predicted lower IHD mortality (79). The CHS study reported consistent findings with lower DHT concentrations being associated with CVD mortality (56). An analysis from the European Male Ageing Study found that the combination of sexual symptoms and lower total T was associated with all-cause and CVD mortality, rather than total T or free T on their own (80). In an analysis from the Concord Health and Ageing in Men Project (CHAMP), longitudinal decreases in total T, DHT or E2 were associated with all-cause mortality, but only the longitudinal decrease in total E2 was predictive of CVD mortality (82). Finally, in an analysis from BHS in which physical activity and sex hormones concentrations were analyzed, men with higher levels of physical activity and higher DHT concentrations had the lowest risk of CVD death (83).

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR MORTALITY

Several cohort studies have reported an association between lower endogenous T concentrations and increased mortality related to CVD, after adjusting for age and other cardiovascular risk factors. Of large studies using mass spectrometry for assay of sex steroids, MrOS in Sweden found an association of lower total T and E2 with all-cause rather than CVD mortality, while HIMS found an optimal total T to be associated with survival (75,79). HIMS found higher DHT was associated with lower IHD mortality (79), consistent with results from CHS (56), and in BHS the combination of higher DHT and higher levels of physical activity was associated with lower risk of death from CVD (83). Declining E2 may also have a role, being associated with CVD mortality in CHAMP (82). However, in relatively healthy middle-aged men (UK Biobank), there was no evidence of an association between total T and CVD mortality risk (84).

Therefore, allowing for some heterogeneity in cohort characteristics and results, lower endogenous T concentrations, measured using mass spectrometry, may be predictors of CVD related deaths in older men, as might lower or declining concentrations of its bioactive metabolites DHT and E2. However, this may not be the case in generally healthy middle-aged men. Whether lower concentrations of endogenous sex hormones are biomarkers or possibly contributing factors to these outcomes remains unclear from these observational studies, as proof of causality ultimately requires interventional studies and randomized controlled trials (RCTs).

MECHANISTIC STUDIES

Potential Mechanisms

Knowledge of potential mechanisms by which androgens might exert protective effects against atherosclerosis and reduce the risk of cardiovascular events would bridge the findings from epidemiological studies and clinical investigation. There are a substantial number of such studies with diverse models and results, a comprehensive discussion of each being beyond the scope of this chapter (for reviews, see (85,86)). Selected studies are discussed briefly in this context.

Cholesterol Accumulation in Animal Models

Experimental studies in castrated male rabbits fed a high cholesterol diet reported effects of testosterone treatment to reduce accumulation of cholesterol in the aortic wall and to reduce atheromatous plaque area and aortic intimal thickness (87-90). Similar results have also been reported in miniature pigs (91). Castration of low-density lipoprotein receptor (LDLR)-deficient male mice results in increased fatty steak lesion formation in the aorta compared to non-castrated controls, which is attenuated with testosterone supplementation (92). At least part of this effect may be mediated via conversion of T to E2. Of note, T and DHT increased calcification of plaque in apolipoprotein E (ApoE)-null mice, even as T had a neutral effect on plaque volume and DHT decreased plaque volume (93). In testicular feminized mice with a non-functional androgen receptor (AR) and low circulating T concentrations, T supplementation to physiological levels reduced fatty streak formation (94). Similarly, AR knockout mice (ARKO) showed increased aortic atherosclerosis, and atherosclerotic lesion area that was reduced with T treatment (95). In wild-type mice, T treatment reduced the presence of necrotic cores within plaque compared with placebo. Therefore, these animal studies suggest an effect of T treatment in reducing cholesterol accumulation and the development of atheromatous plaque, while increasing calcification. However, the actions of sex hormones are complex, being mediated partly via aromatization of T to E2, and occurring at least to an extent via AR-independent mechanisms.

Neointimal Formation and Vascular Smooth Muscle Proliferation

NEOINTIMAL RESPONSES TO INJURY

In a male rabbit aorta model of neointimal plaque formation induced by endothelial denudation, T treatment in vitroinhibited plaque development (96). In a male porcine model of coronary neointimal plaque formation following moderate angioplasty-induced arterial injury, castrated males exhibited greater intimal area compared to intact males and castrated males treated with T (97). T inhibited proliferation and increased expression of the cell-cycle regulator p27^kip1 during neointimal formation. However, despite castration of wild-type mice resulting in increased neointimal formation following wire injury, selective deletion of AR from endothelial cells or smooth muscle cells did not affect lesion size (98). Therefore, effects of T on neointimal formation may be indirect, or mediated by AR-independent mechanisms.

VASCULAR SMOOTH MUSCLE

Vascular smooth muscle cells contribute to progression of atherosclerotic lesions and formation of the fibrous cap (99). T was shown to regulate expression of proliferation-associated genes in skeletal myocytes and in myofibers in different muscles (100,101). Its role in smooth muscle cells in the vasculature is not well defined. In one study, T exerted a pro-proliferative effect on vascular smooth muscle cells in vitro, with increased DNA synthesis assessed using a thymidine incorporation assay (102). In that study the effect of T was blocked by the AR antagonist flutamide. In another study, T induced apoptosis in cultured vascular smooth muscle cells, in an AR-dependent manner (103). In one study, deletion of the AR in vascular smooth muscle cells did not change atherosclerotic plaque size in LDLR knockout mice (104). However, another study demonstrated that T, acting via the AR in vascular smooth muscle cells, might be involved in promoting vascular calcification (105). Therefore, T seems to exert indirect effects on neointimal proliferation in response to injury and may play a secondary role in the development and calcification of atheromatous plaque via complex actions in vascular smooth muscle.

Inflammation

INFLAMMATION AND ATHEROTHROMBOSIS

A mechanistic link likely exists between inflammation and atherothrombosis (for reviews, see (106,107)). Statin therapy lowered both LDL cholesterol and C-reactive protein (CRP) concentrations, reducing the risk of cardiovascular events in a primary prevention setting in adults with LDL <3.4 mmol/L and high-sensitivity CRP ≥2.0 mg/L (108). Recently, anti-inflammatory intervention utilizing canakinumab, a monoclonal antibody targeting interleukin-1β, in a secondary prevention setting in adults with high-sensitivity CRP ≥2.0 mg/L demonstrated a modest reduction in major cardiovascular events (109). However, a major trial using low-dose methotrexate showed no benefit in adults with previous MI or multivessel coronary artery disease and either type 2 diabetes or metabolic syndrome (110). By contrast, colchicine has shown promise in major RCTs as an anti-inflammatory agent to reduce cardiovascular risk in both acute and chronic secondary prevention settings (111,112). These results underscore the relationship between inflammation and atherosclerosis.

TESTOSTERONE EFFECTS ON IMMUNE CELLS

Of note, in vitro studies have shown effects of T to reduce production of inflammatory cytokines from monocytes, macrophages and endothelial cells (113-115). Deletion of monocyte-macrophage AR in LDLR knockout mice resulted in reduced atherosclerosis compared to LDLR knockout mice, suggesting a role for AR-mediated actions in inflammatory cells (104). In an elegant study, pre-pubertal castration of male ApoE knockout mice increased atherosclerotic lesion area, which was abolished by an anti-CD3 antibody targeting T cells, linking hormonal and immunologic regulation of atherosclerosis (116). In that study, both castration and depletion of AR in epithelial cells resulted in increased thymus weight, and mice with depletion of AR in epithelial cells showed increased atherosclerosis and increased infiltration of T cells in the vascular adventitia (116). These findings support a mechanism by which deficiency of androgen action modulates immune/inflammatory responses to promote atherosclerosis.

TESTOSTERONE EFFECTS ON CYTOKINES

Older men treated with gonadotrophin-releasing hormone agonists to suppress HPT axis function showed increased concentrations of circulating tumor necrosis factor-α and interleukin-6 (117). In a randomized cross-over trial of 27 men ranging in age from 36-78 years, T treatment given via intramuscular injections over one month reduced circulating concentrations of tumor necrosis factor-α and interleukin-1β, and increased concentrations of the anti-inflammatory cytokine interleukin-10 (118). In another study of 20 men with type 2 diabetes, there was an inverse correlation of baseline T and interleukin-6, but T treatment over three months, while reducing waist circumference, did not alter tumor necrosis factor-α, interleukin-6, or C-reactive protein (CRP) concentrations (119). In the Testosterone Trials (T-Trials), T treatment via transdermal gel over 12 months in men aged ≥65 years did not change concentrations of high-sensitivity CRP or interleukin-6 (120). In a study of men treated with DHT or with recombinant human chorionic gonadotrophin over three months, neither intervention affected markers of endothelial cell activation or inflammation (121). By contrast, in a trial of men with metabolic syndrome, men in the T treatment arm showed a reduction in high sensitivity CRP after 12 months of treatment (122). In a trial of 76 men with newly diagnosed type 2 diabetes, T treatment over 9 months reduced markers of endothelial cell activation and inflammation, namely circulating concentrations of intracellular adhesion molecule type 1, p-selectin, and CRP (123). Therefore, the results of clinical studies are not wholly consistent. In summary, although the concept that T might exert anti-inflammatory actions protective against atherosclerosis is plausible, more evidence is needed using a direct measure of atherosclerosis.

CLINICAL TRIALS WITH SURROGATE ENDPOINTS

Testosterone Effects on Angina and Vascular Function

EFFECTS OF EXOGENOUS TESTOSTERONE ON ANGINA

Mechanistic studies in cell and animal models provide a plausible rationale for the epidemiological findings associating lower endogenous T concentrations with higher risk of CVD. However, clinical studies are necessary to clarify whether administration of T modulates clinical manifestations of CVD in vivo. Case series from the 1940s reported a beneficial effect of T therapy using intramuscular testosterone propionate to decrease the frequency and severity of angina attacks in an era where nitrate therapy was the mainstay of therapy (124-126). These early reports in men (and a small number of women) describe gradual improvements in symptoms over periods ranging from weeks to months. Conversely, a study in the 1960s found that administration of oral conjugated estrogen (that would suppress the HPT axis and serum androgen concentrations) to men resulted in adverse cardiovascular effects (127). In any case, as T is the native hormone which is metabolized in vivo to DHT and E2 (2), it is the preferred treatment for hypogonadal men (128), and represents the logical candidate for interventional studies.

More recent RCTs have revisited the issue of T treatment in men with CAD (Table 3A). In studies lasting from eight weeks to 12 months, T supplementation in men with CAD increased post-exercise ST segment depression (129), time to ischemia on exercise testing (130,132) and in a study in older men with diabetes, reduced the frequency of angina and silent myocardial ischemia during ECG Holter monitoring (133). These findings suggest either a protective effect of T on the myocardium, or an improvement in exercise capacity. A cross-over study found increased perfusion of myocardium supplied by unobstructed arteries, consistent with a vasodilatory action (131). Therefore, while existing data are limited, contemporary RCTs support historical observations suggesting a potentially beneficial effect of T supplementation in men with CAD.

Table 3. Selected Randomized Controlled Trials (RCTs) of Testosterone Supplementation in Middle-Aged and Older Men Reporting Outcomes Related to Angina (A), Artery Health (B), and Cardiovascular Adverse Events (C)
Study author and year	Population (men)	Formulation of T	N active	N placebo	Duration	Result
A
Jaffe MD, 1977 (129)	Men with ST segment depression on exercise (mean age 58 years)	T cypionate 200 mg weekly	25	25	8 weeks	Decreased postexercise ST segment depression in T-treated but not placebo group
English KM, 2000 (130)	Men with coronary artery disease (mean age 62 years)	Transdermal patch 5 mg	22	24	12 weeks	Increased time to 1-mm ST- segment depression during treadmill exercise
Webb CM, 2008 (131)	Men with angiographically proven coronary artery disease, 40-75 years	Oral T undecanoate 80 mg bd	22		8 weeks, cross-over	No difference in angina or endothelial function, decreased arterial stiffness, increased perfusion of myocardium
Mathur A, 2009 (132)	Men with chronic stable angina (men age 65 years)	Depot intramuscular T undecanoate	7	6	12 months	Increased time to ischemia, non-significant trend for decreased CIMT
Cornoldi A, 2009 (133)	Men with proven coronary artery disease and type 2 diabetes (mean age 74 years)	Oral T undecanoate 40 mg tds	45	44	12 weeks	Reduced number of angina attacks and silent ischemic episodes on ECG Holter monitoring
B
Aversa A, 2010 (122)	Men with metabolic syndrome, T ≤11 nmol/L or free T ≤250 pmol/L (mean age 57 years)	Depot intramuscular T undecanoate 1,000 mg every 12 weeks	40	10	12 months*	Decreased high sensitivity CRP, improvement in CIMT
Basaria S, 2015 (164)	≥60 years, T 3.5-13.9 nmol/L or free T <173 pmol/L	Transdermal T gel 75 mg daily	156	152	3 years	No difference in rates of change in CIMT or coronary artery calcium
Budoff MJ, 2017 (168)	Men aged ≥65 years with T <9.5 nmol/L	Transdermal T gel 50 mg daily	73	65	12 months	Greater increase in non-calcified coronary plaque volume
Hildreth KL, 2018 (141)	Mean age 66 years, T 6.9-12.1 nmol/L	Transdermal gel, titrated to 13.9-19.1 or 20.8-34.7 nmol/L	41, 43	40	12 months	No effect of T on endothelial function or on CIMT
C
Basaria S, 2010 (172)	≥65 years, T 3.5-12.1 nmol/L or free T <173 pmol/L, mobility limitation	Transdermal gel 100 mg daily	106	103	6 months	Trial stopped prematurely due to excess cardiovascular events in T arm
Srinivas-Shankar U, 2010 (173)	≥65 years, T ≤12 nmol/L or free T ≤250 pmol/L, frail or intermediate frail	Transdermal gel 50 mg daily	138	136	6 months	T improved muscle strength and physical function, no signal for cardiovascular adverse events
Snyder P, 2016 (174)	≥65 years, T <9.5 nmol/L, sexual dysfunction (A), diminished vitality (B) and/or mobility limitations (C)	Transdermal gel 50 mg daily	395 (A 230, B 236, C 193)	395 (A 229, B 238, C 197)	12 months	Modest benefit of T on sexual function, no signal for cardiovascular adverse events
Wittert GA, 2021 (175)	50-74 years, waist ≥95 cm, T ≤14 nmol/L, and impaired glucose tolerance or newly diagnosed type 2 diabetes	Depot intramuscular testosterone undecanoate 1000 mg every 3 months	504	503	24 months	All men received background lifestyle intervention (Weight Watchers). T reduced risk of type 2 diabetes at 2 years by 40%.

T=testosterone, CIMT=carotid intima media thickness, CRP=C-reactive protein. * men in placebo group switched to T after 12 months, extension study to 24 months no longer randomized.

EFFECTS OF EXOGENOUS TESTOSTERONE ON VASCULAR FUNCTION

Brachial artery endothelial function is an established measure of cardiovascular health examining both endothelial and vascular smooth muscle function, which mirrors responses in the coronary arteries (134). Assessment of arterial stiffness provides complementary insights into vascular health (135,136). In uncontrolled open-label studies in men with low baseline T concentrations, T supplementation improved both endothelial function and arterial stiffness (137,138). A study in hypogonadal older men found an improvement in arterial stiffness with transdermal T therapy (139). In a RCT of 55 obese men with type 2 diabetes, one year’s treatment with T undecanoate given as a depot intramuscular injection every 10 weeks improved endothelial function compared to placebo (140). However, other studies in middle-aged and older men did not show any effect of transdermal T treatment on endothelial function (141,142). There is a pathway by which T treatment is expected to improve endothelial function as in vitro studies demonstrate stimulation of nitric oxide synthesis in human aortic endothelial cells exposed to T (143). T might exert beneficial effects in the vasculature via actions to improve endothelial function and arterial stiffness, but additional studies are needed before a definitive conclusion can be made.

Testosterone and Atherosclerosis

CIRCULATING CHOLESTEROL CONCENTRATION

Clinical studies of T have shown consistent results for T treatment to reduce circulating concentrations of total cholesterol to a modest degree (144-146). A trend for T to lower LDL cholesterol has been noted (144). T treatment appears to lower high density lipoprotein (HDL) cholesterol again to a small degree (147). HDL cholesterol is involved in reverse cholesterol transport thus exerting anti-atherogenic activity, such that HDL function is an independent predictor of cardiovascular events (148,149). However, T may modulate HDL concentrations without a corresponding effect on HDL function (150). Of note, in observational studies, endogenous T concentrations correlated with circulating HDL and were inversely associated with total cholesterol (151,152). Thus, the prognostic significance of T-induced changes in lipid profiles, and the effect of T treatment on HDL-mediated anti-atherogenic action in men at risk for CVD remains unclear.

CAROTID ATHEROSCLEROSIS

Carotid intima-media thickness (CIMT) and the presence of carotid plaque are measures of preclinical carotid atherosclerosis, which can be assessed non-invasively using ultrasound (153). While low endogenous T concentrations are associated with increased CIMT in observational studies (154,155), it is less clear whether low endogenous T (or E2) predicts progression of CIMT (156-158). One study implicated low-grade inflammation in this process, finding an association of low non-SHBG-bound T with CIMT in older men with CRP ≥2 mg/L, but not in those with CRP <2 mg/L (159). Two cohort studies observed cross-sectional associations of low T concentrations with greater carotid plaque area (158,160). However, no association was found between baseline sex hormone concentrations and change in plaque area during follow-up, possibly due to increased use of anti-hypertensive and lipid-lowering therapy (158). One study reported an association of higher T concentrations with reduced CIMT and lower prevalence of carotid plaque in a cohort of community-dwelling men, but not in a cohort of men with angiographically proven CAD (161). In men with proven CAD, higher DHT was associated with less carotid plaque. Of note, E2 was associated with increased CIMT in community-dwelling men, but with less carotid plaque in men with CAD (161). One study found that higher E2 was associated with the presence of lipid core in carotid plaques in men, with no association of T concentrations (162). In a study of men who underwent carotid endarterectomy, the ratio of circulating T/E2 was inversely associated with plaque calcifications, macrophage staining and plaque neutrophil content, as well as plaque IL-6 protein (163). These findings associate sex hormone concentrations with CIMT and carotid plaque in men.

Several interventional studies reporting CIMT as an outcome are summarized (Table 3B). Of note, in a RCT of intramuscular T undecanoate 1,000 mg given every 12 weeks to men with metabolic syndrome, there was improvement in CIMT after 12 months of treatment (122). However, a larger RCT, Testosterone Effects on Atherosclerosis Progression in Aging Men (TEAAM), conducted over three years showed no effect of transdermal T treatment on rates of progression of CIMT (164). A smaller study, which tested both exercise training and transdermal T treatment over a period of 12 months, found no effect of either intervention on CIMT (141). Thus, while the RCT data are limited, the effects of T treatment on preclinical carotid atherosclerosis may be beneficial or neutral, but are unlikely to be adverse. See table 3, part B.

CORONARY ATHEROSCLEROSIS

Coronary computed tomography angiography (CCTA) has emerged as a non-invasive method for imaging coronary atherosclerosis (165-167). Normal findings on CCTA are associated with a low risk of cardiovascular events, while the presence and extent of CAD demonstrated on CCTA are risk predictors for future cardiovascular events in large epidemiological studies (165-167). Therefore, there is considerable interest in the Cardiovascular sub-study of the T-Trials which reported CCTA outcomes for 73 men treated with transdermal T and 65 men receiving placebo over a 12-month period (168). In this study, men in the T-treated group experienced a greater increase in non-calcified and total coronary artery plaque volume, compared to men in the placebo group (168). However, the groups were unbalanced with men in the T-treated group having considerably lower non-calcified and total plaque volumes at baseline and at the end of the study compared with placebo-treated men. The fact that the two groups of men differed substantively in a key baseline characteristic makes the result of the study challenging to interpret (169). There was no difference in the rate of change of coronary calcium score between groups, albeit men in the T-treated group had lower coronary calcium scores at baseline and at the end of the study compared with men in the placebo group (168). The results for coronary calcium scores are concordant with the TEAAM trial that also showed no difference in coronary calcium scores with T treatment (164). Men in the T-Trials Cardiovascular sub-study overall had a high burden of plaque at baseline with 32% of T-treated men and 38% of placebo recipients having baseline Agatston scores ≥300 (168).

Of note, in the T-Trials Cardiovascular sub-study, data on plaque volume were not analyzed relative to vessel lumen to address the issue of whether vascular remodeling was occurring (170). Since then, CCTA technology has progressed to allow more detailed and sophisticated analysis of plaque characteristics associated with higher risk of coronary events, which were not applied in that study (171). While these findings are important and noteworthy, a larger RCT with balanced groups using current CCTA methodology would be needed to clarify the effect of T on coronary plaque characteristics. The findings are a timely reminder that until definitive RCTs are available, the effects of T on the cardiovascular system remain uncertain and may be beneficial, neutral or adverse.

TRIALS REPORTING ADVERSE EVENTS

Testosterone RCTs and Cardiovascular Adverse Events

Selected T RCTs, which have influenced this field, are summarized (Table 3, part C). These studies were underpowered for cardiovascular events and the possibility of type 1 and type 2 errors should be considered. A key RCT randomized 209 men aged 65 years and older, with mobility limitations and low or low-normal baseline T or free T, to transdermal T gel vs placebo for six months (172). Of note the starting dose of transdermal T (100 mg daily) was greater than the usual recommended starting dose (50 mg daily). The study was discontinued after an excess of adverse events was noted in the T arm (172). A contemporaneous RCT in a comparable population of 274 older men who were frail or intermediate frail, using a 50 mg daily dose of transdermal T over 6 months, was successfully completed showing improved muscle strength and physical function, with no signal for cardiovascular adverse events (173). The Testosterone Trials (T-Trials) has reported results from the main study and component sub-studies (174,176). These have been extensively reviewed (169,176). In T-Trials, 790 men aged 65 years and older, with symptoms of sexual dysfunction, diminished vitality or mobility limitations and baseline T <9.5 nmol/L (<275 ng/dL), were randomized to transdermal T gel at a starting dose of 50 mg daily vs placebo for 12 months (174). In T-Trials, T treatment improved sexual function to a moderate degree, while the primary outcomes for physical function and vitality were not met (174). T treatment improved anemia and volumetric bone density, with a neutral effect on cognition (176). The effects on coronary artery plaque volume have been discussed (see Section 4.2.3). T-Trials had a low rate of major cardiovascular adverse events (7 in each of the T and placebo arms).

Recently, a larger Australian RCT, Testosterone for the Prevention of Type 2 Diabetes Mellitus (T4DM) has been reported (175). T4DM was a randomized, double-blind, placebo-controlled, 2-year, phase 3b trial done at six Australian tertiary care centers, which randomized 1,007 men to depot intramuscular testosterone undecanoate injections given every three months for two years, vs placebo, on a background of a lifestyle intervention (Weight Watchers) given to all participants. Inclusion criteria were age 50-74 years, waist circumference ≥95 cm and baseline T ≤14.0 nmol/L (≤403.8 ng/dL), and the presence of either impaired glucose tolerance or newly diagnosed type 2 diabetes based on oral glucose tolerance testing (175). In T4DM, testosterone treatment reduced the risk of type 2 diabetes at two years by 40% beyond the effect of the lifestyle intervention (relative risk 0·59, 95% confidence interval 0·43 to 0·80; p=0·0007). T4DM is the largest T RCT reported to date, and the incidence of cardiovascular adverse effects were similar in both T and placebo arms. In T4DM, 17 men in the placebo group and 12 in the T group had a major cardiovascular adverse event (13 men in the placebo arm and 7 in the testosterone arm had an ischemic heart disease event, 3 and 4 had cerebrovascular disease events respectively, and one in each group died from a cardiovascular-related cause) (175). Therefore, in keeping with T-Trials, the rates of major cardiovascular adverse events in T4DM were low, and comparable in testosterone and placebo-treated men.

At this point, it is worth commenting on the outcome of cardiorespiratory fitness. Low cardiorespiratory fitness, assessed as maximal oxygen consumption during exercise testing (VO₂peak), is a strong independent predictor of all-cause and CVD mortality in apparently healthy men and in men with established CVD (177-179). Cardiorespiratory fitness has been recommended as a vital sign for use in clinical assessment (180). An earlier study over 12 months did not show an effect of T treatment on fitness (141), nor did a more recent study with a 12-week intervention (181). That study used a 2x2 factorial design, while exercise training resulted in improved VO₂peak within the 12-week period of intervention, T treatment did not, and there was no evidence within this relatively short timeframe of additive benefit (181). In the TEAAM study conducted over 3 years, placebo-treated men showed a decline in VO₂peak over time, but the decline was attenuated in T-treated men (182). In TEAAM, there was no signal for cardiovascular adverse events with T (164). Part of the beneficial effect of T treatment on VO₂peak might be mediated via its effect to raise hemoglobin concentrations (147,169,176), or its action on skeletal muscle (183,184). The net effect might be to preserve (or at least attenuate the loss of) cardiorespiratory fitness in ageing men, that could translate into a reduction in cardiovascular risk. However, in keeping with the results of T4DM, an extended duration of T intervention may be required to realize these benefits.

Meta-analyses of Cardiovascular Adverse Events in Testosterone RCTs

To date, no T RCT large and long enough to be powered for the outcome of cardiovascular events has been reported. However, meta-analyses of reported T RCTs have been performed to determine whether T is associated with a difference in the rate of cardiovascular adverse events (Table 4). Earlier meta-analyses done in 2007 and 2010 had found no significant difference in risk for cardiovascular adverse events (147,185). One analysis done in 2013 claimed an association of T treatment with increased risk of cardiovascular-related adverse events (186). However, subsequent meta-analyses done from 2013 to 2020 have not supported this finding (187-193). Instead, these have found no association of T treatment with risk of cardiovascular adverse events (Table 4).

Table 4. Meta-Analyses of Cardiovascular Adverse Events in Randomized Controlled Trials (RCTs) of T Supplementation in Men
Study characteristics				Results
Study author and year	N of RCTs	N active	N placebo	Adverse signal	No adverse signal
Haddad RM, 2007 (185)	30	808	834		No significant difference in odds ratio for any cardiovascular adverse event or MI.
Fernandez-Balsells MM, 2010 (147)	51	2,716			No significant difference for all-cause mortality, coronary bypass surgery or MI.
Xu L, 2013 (186)	27	2,994		T associated with increased risk cardiovascular-related event (OR 1.54, 95% CI=1.09-2.18)*.
Ruige JB, 2013 (187)	10 (>100 participants)	1,289	848		No significant difference in cardiovascular adverse events.
Corona G, 2014 (188)	75	3,016	2,448		No association of T supplementation with cardiovascular risk. For MACE OR=1.01 (95% CI 0.57-1.77).
Borst SE, 2015 (189)	35	3,703			No significant risk for cardiovascular-related adverse events.
Alexander GC, 2017 (190)	39, cut-off for T 10.4-16.7 nmol/L	3,230	2,221		No significant increase in risk of MI OR=0.87 (95% CI 0.39-1.93)*, stroke or mortality.
Elliott J, 2017 (191)	87, cut-off for T 12 nmol/L or cFT 225 pmol/L	1,462-2088	1,372-1,851		Improved QoL, libido, depression and erectile function. No increase in risk of adverse events.
Corona G, 2018 (192)	93	4,653	3,826		No clear effect of T on incidence of CVD events. For MACE OR=0.97 (95% CI 0.64-1.46).
Diem SJ, 2020 (193)	38	N/A	N/A		Small improvement in sexual function and quality of life. Pooled risk for adverse cardiovascular outcomes did not differ between groups (OR=1.22, 95% CI 0.66-2.23).*
Hudson J, 2022 (194)	35 RCTs: 17 included in IPD meta-analysis	1,750 (IPD)	1,681 (IPD)		No significant difference between groups. For cardiovascular or cerebrovascular events OR=1·07 (95% CI 0·81–1·42).

MI=myocardial infarction, MACE=major adverse cardiovascular events, OR=odds ratio, 95% CI=confidence interval. QoL=quality of life, IPD=individual participant data. Unless otherwise specified, meta-analyses were conducted using random effects models. *fixed effects model

It is worth commenting on more recent meta-analyses that have included the results of the T-Trials. In the meta-analysis by Alexander et al. (2017), 39 RCTs were included. The meta-analysis found no significant increase in risk of MI (data from 30 RCTs utilized), stroke (9 RCTs) or mortality (20 RCTs) (190). However, caveats were noted with respect to the quality of the available evidence. In a network meta-analysis, Elliott et al. (2017) included RCTs that enrolled men with baseline T ≤12 nmol/L (≤346 ng/dl) or free T ≤225 pmol/L, including 87 RCTs overall (191). T treatment was associated with improved quality of life and libido, improvement in depression and in erectile function. There was no increase in risk of adverse events such as cardiovascular death, MI or stroke (191). Corona et al. (2018) studied 93 RCTs and found no clear effect of T on incidence of CVD events, with an odds ratio of 0.97 (95% confidence interval 0.64-1.46) for major cardiovascular adverse events (192).

Diem et al. (2020) examined 38 RCTs of at least six months duration, noting that few exceeded a 1-year duration, there was a lack of power to assess important harms, and limited data for men aged 18-50 years (193). They concluded that in older men with lower testosterone concentrations, in the absence of organic hypogonadism, T treatment resulted in small improvements in in sexual functioning and quality of life, with long-term safety and efficacy still uncertain. Recently Hudson et al. (2022) analyzed RCTs involving men with T ≤12 nmol/L and minimum duration of three months, identifying 35 studies and obtaining individual participant data from 17 of these with 3,431 participants (including T-Trials but not T4DM) (194). There was no significant difference in cardiovascular adverse events in testosterone vs placebo-treated men. The authors concluded that their results provide some reassurance about the short- to medium-term safety of T treatment for male hypogonadism (194). Clearly, the results of the TRAVERSE study, a testosterone cardiovascular safety trial, will be of considerable interest (195). For now, bearing in mind the limitations of meta-analyses of RCTs using reported adverse events as the endpoint, the weight of the currently available evidence from these sources indicates that T treatment is not associated with risk of cardiovascular adverse events.

RETROSPECTIVE CASE-CONTROL STUDIES

Retrospective Studies of Testosterone Prescriptions

Pending an adequately powered T RCT to clarify its effect on the risk of cardiovascular events, retrospective case-control studies have sought to fill this gap (Table 5). These studies are typically based on health insurance databases recording prescriptions for T and subsequent outcomes in men prescribed or not prescribed T. Limitations of these studies include lack of clinical data such as indications for prescribing, the absence of randomization and the possibility of recall and misclassification bias (196). Initial studies in male veterans and in men with type 2 diabetes associated T prescriptions with lower mortality (197,198). By contrast, two studies associated T prescriptions with increased risk of major cardiovascular events (199,200). Both have been subject of criticisms: the first over confusing statistical methodology and data inaccuracies (resulting in publication of an erratum), the second over the lack of an appropriate comparison group (209,210). Subsequent studies have associated T prescriptions with no increase in risk of MI (201), and with lower risk of death, MI and stroke (202,203). An interesting distinction was made in the studies by Sharma et al. (2015) and Andersen et al. (2016) in that men who were prescribed T who then had “normal” T concentrations, did better than men who had persistently low T concentrations or who did not receive T (202,203). However, it is important to note that these studies did not systematically assess testosterone concentrations at multiple times or multiple days. A single measurement of testosterone on a particular day, may not be an accurate reflection of testosterone concentrations achieved over sustained periods of time with different testosterone formulations (211).

Table 5. Retrospective Case-Control Studies of Men Prescribed T that Examined Associations of T Prescriptions with Cardiovascular Events and Mortality in Middle-Aged and Older Men
Study characteristics				Results
Study author and year	Size (n of men)	Follow-up (yr)	Age (yr)	Favors no T	Favors T
Shores MM, 2012 (197)	1,031	3.4	62.1		Male veterans with total T ≤8.7 nmol/L, T prescribed in 398. T supplementation associated with lower mortality.
Muraleedharan V, 2013 (198)	581	5.8	59.5		Men with Type 2 diabetes, 238 with total T ≤10.4 nmol/L. T supplementation associated with lower mortality.
Vigen R, 2013 (199)	8,709	2.3	63.4	Male veterans who had coronary angiography and total T ≤10.4 nmol/L. T prescription associated with increased risk of death, MI or stroke.
Finkle WD, 2014 (200)	55,593	90 days	54.4	Men prescribed T. Higher rate of non-fatal MI in 90 days following prescription compared to preceding 1 year.
Baillargeon J, 2014 (201)	6,355; 19,065	4.1; 3.3	≥66		6,355 men prescribed T vs 19,065 matched non-users. T prescription not associated with increased risk of MI. For men with worse prognostic scores, T associated with reduced risk of MI.
Sharma R, 2015 (202)	83,010	6.2; 4.6; 4.7	66		Male veterans with low T. TRT resulting in normalization of circulating T (n=43,931) was associated with lower risk of death, MI and stroke, compared to TRT without normalization of T (n=25,701) or no TRT (n=13,378).
Anderson J, 2016 (203)	4,736	≥3	61.2		Men with T <7.4 nmol/L. T therapy achieving normal T (n=2,241) was associated with reduced risk of MACE compared to persistent low T (n=801). T therapy achieving either normal T or high T (n=1,694) associated with lower all-cause mortality compared to persistent low T.
Wallis CJD, 2016 (204)	10,311: 28,029	5.3	≥66		Men treated with T. T treatment associated with lower mortality HR=0.88 (95% CI=0.84-0.93) and prostate cancer risk HR=0.86 (95% CI=0.75-0.99). Shorter exposure (2 months) associated with increased risk of cardiovascular events and mortality, longer exposure (35 months) with reduced risk.
Cheetham TC, 2017 (205)	8,808: 35,527	3.2	58.4		Men ≥40 years, diagnosis or T <10.4 nmol/L. T associated with reduced risk of outcome of MACE, unstable angina, coronary revascularization, TIA. HR=0.67 (95% CI=0.62-0.73).
Loo SY, 2019 (206)	15,401	4.7	≥45	Men with low T and no evidence of HPT axis disease. T associated with increased risk of composite outcome of stroke/TIA/MI (HR=1.21, 95% CI 1.00-1.46), with risk highest in first 6 months to 2 years of T use (HR 1.35, 95% CI, 1.01-1.79). Risk of all-cause mortality lower with current T use (HR=0.64, 95% CI 0.52-0.78) and higher with past T use (HR=1.72, 95% CI 1.21-2.45), compared with non-use.
Oni OA, 2019 (207)	1,470	3.2-4.0	≥50		Male veterans with low total T and history of MI. All-cause mortality lower in men treated with T who normalized total T (N=755), vs men treated with T who did not normalize total T (N=542, HR=0.76, 95% CI 0.64-0.90), or men not treated with T (N=173, HR=0.76, 95% CI 0.60-0.98). No significant difference in the risk of recurrent MI between groups.
Shores MM, 2021 (208)	204,857	4.3	60.9		Male veterans with low T. Current transdermal T use not associated with risk for incident MI/ischemic stroke/venous thromboembolism (HR=0.89, 95% CI 0.76-1.05) in men without prevalent CVD, and in those with prevalent CVD was associated with lower risk (HR=0.80; 95% CI, 0.70-0.91). Current intramuscular T use not associated with risk for composite endpoint in men without or with prevalent CVD (HR=0.91, 95% CI 0.80-1.04; HR=0.98, 95% CI 0.89-1.09, respectively).

MI=myocardial infarction, TRT=testosterone replacement therapy, MACE=major cardiovascular adverse event comprising death, non-fatal MI and non-fatal stroke, TIA=transient ischemic attack.

A study by Wallis et al. (2016) found that T treatment was associated with lower mortality overall, but men who had T for a relatively shorter duration of exposure had increased risk, while men with longer duration of exposure had reduced risk (204). In a study by Cheetham et al. (2017) of men aged ≥40 years diagnosed with low T or with T <10.4 nmol/L, T treatment was associated with a reduced risk of major cardiovascular adverse events (205). Loo et al. (2019) reported contrary findings: analyzing a cohort of men with no evidence of HPT axis disease via the UK Clinical Practice Research Datalink, they associated use of testosterone with higher risk of a composite outcome of stroke, transient ischemic attack or MI, with the risk highest in the first six to 24 months of T use (206). In that study all-cause mortality risk was lower with current T use, and higher with past T use. Those findings are not supported by two more recent analyses, involving men with prior heart disease or with multiple comorbidities (207,208). In a study of male veterans with low T concentrations and a history of MI, men receiving T treatment who had a subsequent normal testosterone concentration had a lower risk of death from any cause compared to men receiving T treatment who had a subsequent low T concentration (207). Those men also had a lower risk of death compared with men who did not receive T treatment. Finally, in a cohort of 204,857 male veterans with a mean age of 60.9 years and 4.7 chronic medical conditions who were followed for 4.3 years, current transdermal T use was not associated with risk for the composite outcome of incident MI, ischemic stroke or venous thromboembolism in men without prevalent CVD (208). On the other hand, it was associated with lower risk in men with prevalent CVD. In that study, current intramuscular T use not associated with risk for the composite endpoint in men without or with prevalent CVD (208).

In an earlier retrospective cohort study that compared the use of T gel with T injections, T injections were associated with greater risk of cardiovascular adverse events (212). Bearing in mind the limitations of non-randomized studies and the possibility of bias, and the absence of a control group not receiving T, an additional factor is that more than 90% of the T injections were T cypionate, enanthate or propionate (212), which are short acting formulations typically requiring fortnightly administration with marked fluctuations in blood concentrations. The analysis would not apply to long-acting depot injections of T undecanoate typically administered every 12 weeks, which provide more stable pharmacokinetics (128). It is worth noting in this context that a population-based case-control study (involving 19,215 patients with confirmed venous thromboembolism and 909,530 age-matched controls) found an increased risk of venous thromboembolism within the first six months of T treatment but not thereafter (213). By contrast a systematic review and meta-analysis including six RCTs (2,236 participants) and five observational studies (1,249,640 participants) found no evidence of an association between T treatment and venous thromboembolism (214). However, the authors of that study noted that the available RCT data might have had inadequate power to detect an increased risk.

In summary, earlier findings associating T prescriptions with adverse cardiovascular outcomes were echoed in a more recent study. However, most studies do not show such adverse signals, and associated T use with lower risk of adverse cardiovascular events or mortality, including several recent large studies. There is a suggestion that T treatment which achieves normal T concentrations may relate to lower risk of cardiovascular events and mortality. Bearing in mind the limitations of these retrospective, observational and non-randomized studies, which cannot prove causality, the available data provide some reassurance but are far from definitive.

Abuse of Androgenic Steroids

Androgenic steroids can serve as appearance and performance-enhancing drugs and are abused by some competitive athletes, recreational sportspersons and body builders (1,215,216). The use/abuse of androgenic steroids occurs in contravention of medical advice and applicable sporting regulations, typically without medical supervision using unapproved formulations often in excessive doses (216). The use/abuse can be interspersed with periods of non-use. Adverse effects include suppression of the endogenous HPT axis, reduced spermatogenesis and impaired fertility, decreased testicular volume, hair loss and gynecomastia and are well-recognized (1,215). There is also an appreciation that long-term abuse results in cardiovascular toxicity in the form of myocardial dysfunction and accelerated coronary atherosclerosis (217). However, this study could be confounded as men who abuse androgenic steroids may also consume many other substances and the androgen preparations might have harmful contaminants. Abuse using pharmacological dosing of various products is very distinct from medically supervised T therapy aiming to achieve physiological circulating concentrations of T (128). Nevertheless, this is a reminder that excessive exposure to androgens carries the risk of harm (1).

DISCUSSION

Lessons from the Available Evidence

Epidemiological data are consistent with a protective role for endogenous androgens against CVD. In some studies of middle-aged and older men, lower circulating concentrations of endogenous T are associated with higher incidence of cardiovascular events, particularly stroke. Lower circulating T and DHT concentrations have also been associated with higher cardiovascular mortality (discussed in sections 2.1-2.2). Potential mechanisms by which T could exert beneficial actions in the vasculature have been explored in experimental models. These include reduced cholesterol accumulation and modulation of inflammation (sections 3.1-3.4). Clinical studies have reported favorable effects of T treatment on angina symptoms and exercise tolerance, but its effect on subclinical atherosclerosis remains uncertain (sections 4.1-4.2). The T-Trials, T4DM and meta-analyses of existing T RCTs in general do not show any signal for cardiovascular adverse events (sections 5.1-5.2). Retrospective case-control studies have reported contrasting results but in general, men receiving T prescriptions appear to have lower risk of major cardiovascular events and lower mortality compared to men who did not receive T, particularly if T treatment was associated with subsequent normal concentrations of circulating T (section 6.1). It is important to bear in mind that there are contrasting findings, and beneficial associations of T with cardiovascular outcomes may be less evident in healthier middle-aged men. Therefore, epidemiological evidence and mechanistic data could be used to argue for an anti-atherogenic or a protective effect of T on the cardiovascular system, as could the majority of retrospective case-control studies. However, this remains to be proven in the context of prospective RCTs of T intervention.

Gaps in the Current Evidence Base

RCT data are lacking as to whether treatment of middle-aged and older men with T would reduce the risk of cardiovascular events. The T-Trials which used transdermal T gel over a 12-month intervention offer important evidence as to benefits of T treatment for sexual function, anemia and bone density in older men without apparent diseases of the HPT axis, who had lower circulating T concentrations compared with younger men and symptoms suggestive of (but not diagnostic for) androgen deficiency (174,176). T4DM demonstrated the benefit of T treatment to reduce the risk of type 2 diabetes in men at high risk, beyond the effects of a lifestyle intervention (175). T4DM also showed a beneficial effect of T treatment on sexual function, and on bone microarchitecture and density (175,218). The T-Trials and T4DM are also noteworthy for the absence of any adverse cardiovascular safety signal for T treatment in these populations of men (174,175). However, the findings of the T-Trials Cardiovascular sub-study regarding an increase in total coronary atheroma plaque volume, in men with substantial baseline atheromatous disease, require clarification (168).

Major evidence gaps pertain to the effects of T on the cardiovascular system, as to whether T acts to slow development or progression of coronary or carotid atheromatous plaque in middle-aged and older men, in the differing contexts of either primary or secondary prevention for CVD. If the action of T is to reduce cholesterol accumulation, and to reduce inflammation and neointimal response to injury (sections 3.1-3.4) then these actions may have more impact to prevent or reduce progression of early atherosclerosis, rather than to reverse established disease. The related questions are whether T intervention in a primary prevention setting will reduce growth of coronary or carotid atheromatous plaque, or whether in a secondary prevention setting T intervention would influence the incidence of cardiovascular events. Another important question relates whether transdermal vs depot intramuscular (T undecanoate) formulations of T have similar or differing effects on the cardiovascular system.

Neither T-Trials nor T4DM had any cardiovascular endpoints and will not answer the question as to whether T exerts beneficial, neutral or adverse effects on the cardiovascular system. The US multicenter RCT “A study to evaluate the effect of testosterone replacement therapy (TRT) on the incidence of major adverse cardiovascular events (MACE) and efficacy measures in hypogonadal men (TRAVERSE)” commenced recruitment in 2018 of men aged 45-80 years with T <10.4 nmol/L (<300 ng/dl) with evidence of CVD or at increased risk for CVD (195). TRAVERSE was designed as a cardiovascular safety study with the endpoint of myocardial infarction, stroke or death due to cardiovascular causes, aimed to enroll 6,000 men randomized to transdermal T gel or placebo, and is planned to complete in 2022. TRAVERSE will also examine outcomes of prostate cancer, sexual function, bone fractures, depression, anemia and diabetes (195). TRAVERSE will address the issue of the cardiovascular safety of T treatment in what would largely be a secondary prevention setting. This leaves unanswered the question of whether T intervention in a primary prevention setting would reduce development or progression of coronary or carotid atheroma.

Application to Clinical Practice

Current clinical practice recommendations prioritize the identification of men with classical or pathological hypogonadism who are androgen deficient due to diseases of the hypothalamus, pituitary or testes (6,7). In such men, T treatment consistently resolves symptoms and signs of androgen deficiency (6,7,19). In men with classical or pathological hypogonadism the benefits of T treatment likely outweigh possible cardiovascular risks. In any case, individualized assessment and management of cardiovascular risk factors and disease should be part of routine clinical care. Of note, the US regulatory agency required labelling to warn of a possible increased risk of cardiovascular events with T, but the European regulatory agency concluded there was no consistent evidence of increased risk of coronary heart disease with T therapy (19). Until more evidence is available, it may be prudent to adopt a degree of caution in older men who are frail or who have known CVD, and to optimize management of cardiovascular risk factors and disease before starting T treatment. Treatment should aim for physiological replacement of T using approved formulations and avoid excessive doses (128).

It is beyond the scope of this chapter to discuss controversies regarding the management of men with low endogenous T concentrations due to obesity or presence of systemic illnesses where the HPT axis is intact, but its activity may be suppressed (19,31). However, it is worth noting that in men where a clear indication for T treatment is lacking, the risks and benefits of an intervention need to be considered with special care. Further research is needed to determine whether and how T treatment might impact on the risk of CVD in men.

Conclusions

Some epidemiological studies have associated higher circulating concentrations (but within the normal range) of endogenous androgens with lower risk of cardiovascular events and mortality. In men with pathological hypogonadism, the benefits of T treatment likely outweigh putative risks of cardiovascular adverse events. However, the effects of exogenous androgens in the form of T therapy seeking to maintain physiological circulating androgen concentrations on the cardiovascular system remain uncertain. Additional information will be forthcoming once the results of the TRAVERSE trial are known. Current clinical care of hypogonadal men should recognize this evidence gap and allow for individualized assessment and management of pre-existing cardiovascular risk factors and disease in men requiring T therapy. Well-designed and adequately powered RCTs are needed to clarify whether T treatment has beneficial, neutral or adverse effects on the cardiovascular system in the general population of middle-aged and older men.

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Ovarian Reserve Testing

ABSTRACT

The ovaries affect far more than reproductive health. Estrogen affects cardiovascular, skeletal, mental health, and numerous other aspects of wellness. Additionally, ovarian dysfunction can reflect disequilibrium relating to multiple conditions. Efficient and effective ovarian testing can give women valuable answers about their fertility, time to menopause, and other conditions and symptoms they may face. Though no test is perfect, antral follicle count (AFC) and anti- Müllerian hormone (AMH) provide more sensitive and specific results that allow for the continuum of ovarian function, and have advantages over classic tests such as follicle stimulating hormone (FSH), estradiol, the clomiphene citrate challenge test (CCCT), and others. This chapter explores these and additional ovarian assays, their underlying mechanisms, and limitations that may favor one test over another depending on circumstances. Particular emphasis is given to evaluating perimenopausal status, procreation, and etiologies for amenorrhea.

INTRODUCTION

Ovarian endocrinology is dynamic. Years of quiescence are followed by oscillating secretion until near burnout, but some function remains even after menopause. “Ovarian reserve testing” assesses where the ovaries are within this spectrum. These measures seem to most clearly relate to oocyte quantity, as multiple other factors (especially age) meaningfully affect oocyte quality and fecundability. However, quantity and quality are not completely independent, as abnormal ovarian reserve testing has been linked to increased blastocyst aneuploidy (1).

This chapter will characterize the main biochemical and sonographic approaches used in both classic and modern testing. Moreover, an assay, like any tool, has value relative to the task to which it is applied. Accordingly, this chapter will also discuss application of ovarian reserve tests to several common areas: assessing perimenopausal status, evaluating ovarian reserve for fertility, and addressing primary and secondary amenorrhea. Use of these markers in assessing the male is covered elsewhere (https://www.endotext.org/chapter/laboratory-assessment-of-testicular-function).

Because consensus can be difficult, the following summaries reflect trends, though different perspectives exist and the literature continues to evolve. Existing research on ovarian reserve testing is often confusing because of heterogeneity among tested populations (the general population, infertility patients of all ages, infertility patients more than 35 years old, etc., see also data from the Society for Assisted Reproductive Technology (SART), Figure 1, (2)). Additionally, one must always keep in mind that as with all screening tests, no single result is definitive, since findings must be interpreted in context and should be repeated or supplemented as appropriate.

Figure 1. The relative effect of age on fecundity through in vitro fertilization (IVF) in 2020 according to the Society for Assisted Reproductive Technology (SART) (2).

MARKERS OF OVARIAN RESERVE

Follicle Stimulating Hormone (FSH)

MECHANISM

FSH was the first hormone directly linked to ovarian aging (3). It is secreted by the anterior pituitary and promotes the progression of antral follicles into dominant follicles. Feedback from estrogen, inhibin, and activin influence hypothalamic GnRH pulsatility, which determines pituitary FSH expression. Elevated FSH levels can be seen with dwindling reserve, where a greater FSH stimulus is required to drive folliculogenesis, but elevated levels also can be found in normal ovarian reserve if measured at the time of the LH surge. Low FSH levels are seen prior to puberty or with hypogonadotropic hypogonadism. In addition to medical conditions that shift pituitary FSH expression, exogenous hormones and their modulators (clomiphene, letrozole, etc.), cimetidine, phenothiazines, and other medications can also shift levels.

TESTING

Many non-FSH substrates can induce an FSH-like effect. Without describing in detail the spectrum of FSH assays that bypass this challenge, for which an excellent review is available (4), in the clinical setting FSH is typically measured by immunoassay. The sample is usually acquired by phlebotomy (24-hour urine collections are rarely used) on menstrual cycle day three for ovulatory patients, with day one being the first full day of flow.

Testing on cycle days two, four, or five is not unreasonable, but if a normal result would prompt retesting, a day three measurement or a different assay is preferred. Multiple cutoffs are used, with FSH levels of >16.7, >11.4, and <10 mIU/mL reflecting high, moderately high, and normal levels based on the World Health Organization (WHO) Second International Standard (5).

Because ovarian reserve is on a continuum, any cutoff selected should relate to goals of balancing positive and negative predictive values, and this is an issue that applies to other measures of ovarian reserve as well. In amenorrheic patients, a random sample is preferred to testing after hormonally induced menses. In the setting of amenorrhea, a concurrent progesterone level (<2 ng/mL) is a reasonable control to ensure that one is in the follicular phase.

LIMITATIONS

Interpersonal and intercycle variation can be meaningful in patients at risk for moderately elevated FSH, which is why it has been called “Fluctuating Severely Hormone.” The problems with FSH’s sensitivity in part stem from it being a late marker of dwindling ovarian function, as summarized in Stages of Reproductive Aging Workshop + 10 conclusions (6). This limited predictive value is reflected in the NHANES III data, which showed 75% of women aged 40 to 44 years having normal levels at less than 10 mIU/mL, even though ovarian function is typically the rate limiting step at this age, and half of women aged 45 to 49 years had levels less than 11 mIU/mL (7). Sensitivity for FSH is often worse than specificity, with findings ranging from 11-86% and 45-100%, respectively (8). With anti-muellerian hormone (AMH) and antral follicle count (AFC) demonstrating better predictive value for ovarian response than FSH, these are more likely to be the tests of choice (9). Accordingly, relative to emerging alternatives, FSH testing increasingly is seen as less valuable than it used to be for procreative testing and more useful for evaluating perimenopausal status, hypergonadotropic and hypogonadotropic hypogonadism, and central precocious puberty.

Estradiol

MECHANISM

As with FSH, estradiol levels vacillate over the course of a menstrual cycle, peaking in both the late follicular and mid luteal phases. As ovarian reserve declines, the follicular phase shortens because of decreasing feedback inhibition by follicles recruited during the previous cycle. (This is why the first clinical sign of decreasing ovarian reserve is shortening menstrual cycle length.) With the follicular phase starting earlier, estradiol levels start rising closer to menses (and the classic day three FSH peak actually can occur prior to menses). As a result, an elevated day three estradiol level could reflect diminishing ovarian reserve.

Elevated estradiol (>60-80 pg/mL) may also lead to an artificially normal FSH, where higher estradiol levels lead to feedback suppression of FSH. Conversely, estradiol levels <20 pg/mL on day three depending on the circumstances can be consistent with normal ovarian function, hypogonadotropic hypogonadism, or ovarian failure.

TESTING

Estradiol is also typically measured by immunoassay after phlebotomy. The sample is usually drawn at the same time as FSH levels or randomly when assessing amenorrhea. Estrone, the primary postmenopausal estrogen, and estriol, the primary pregnancy estrogen, are not typically tested when evaluating ovarian function. Also, because oral estrogens are typically metabolized into many byproducts (with varying activity), serum estradiol levels often won’t reflect exogenous exposure. (However, transdermal estrogen administration can be monitored through serum levels.) Medical conditions, glucocorticoids, sex steroids, clomiphene, letrozole, GnRH agonists and antagonists, and other medications can alter estradiol levels, just as they could shift FSH levels.

LIMITATIONS

For many conditions, an estradiol level is a reasonable proxy for ovarian inactivity. However, for assessing decreasing ovarian reserve, estradiol is neither a sensitive nor specific assay (9). Accordingly, when used for measuring ovarian reserve, estradiol has its greatest value as an internal control to ensure that one is testing at the expected portion of the menstrual cycle (Figure 2).

Figure 2. Ovarian, hormonal, and endometrial changes over the menstrual cycle. Adapted from Hall, et al., Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal follicular transition (10).

Clomiphene Citrate Challenge Test (CCCT)

MECHANISM

The clomiphene citrate challenge test combines measurement of FSH and estradiol levels prior to clomiphene exposure and FSH levels after clomiphene exposure. Clomiphene is a selective estrogen receptor modulator (SERM) that inhibits negative feedback inhibition by estradiol on the hypothalamus. Normally, increased estrogen levels decrease GnRH pulsatility, resulting in lower FSH levels through negative feedback. By using clomiphene to block feedback inhibition by estradiol, there is an increase in FSH, which enhances follicular recruitment, and which is why clomiphene can be used for ovulation induction and superovulation.

TESTING

FSH and estradiol levels are assessed through immunoassay, as previously described. The CCCT is performed by having an FSH level drawn on the third day of the menstrual cycle, taking 100 mg of clomiphene orally cycle days five to nine, and then repeating the FSH level on cycle day number ten (11, 12). An estradiol level is also frequently drawn on the third day and sometimes on the tenth day as well.

LIMITATIONS

When assessing ovarian reserve for fertility, FSH is a limited measure of ovarian response and a poor predictor of pregnancy and estradiol is predictive of neither (9). When combining the two through the CCCT, it is difficult to assess the degree of benefit through receiver operator curves (9). If benefit is unclear, cost-effectiveness is even less so. Accordingly, other measures of ovarian reserve are increasingly used instead of the CCCT, although this assay is still more commonly used than other provocative tests, such as the exogenous FSH ovarian reserve test (EFORT) and the GnRH agonist stimulation test (GAST). The CCCT has particularly suboptimal value in anovulatory patients. The reason is that the CCCT is primarily used to help discriminate normal ovarian reserve from poor reserve in patients with potentially borderline function. However, the typical anovulatory patient tends to have robust reserve (PCOS, hypogonadotropic hypogonadism) or poor reserve (primary ovarian insufficiency), so relative to alternative assays, a test designed to elicit subtleties is typically less important in this population.

Antral Follicle Count (AFC)

MECHANISM

Follicular recruitment is in constant flux during the reproductive years, with less than 0.1% of oogonia present at birth ever making it to ovulation. Fluid surrounding numerous oocytes not selected to be the dominant follicle can be seen sonographically prior to regression. The more follicles visualized within the ovary, the greater the probable ovarian reserve, and AFC has been shown to correlate closely with the primordial follicular pool on histologic analysis. (13, 14). Though it remains for debate as to how much a dwindling follicular pool reflects oocyte quality as well as quantity, women with infertility are more likely to have lower antral follicle counts than those without infertility (15). Similarly, women with low antral follicle counts are much more likely to have cancellation for under response with IVF than those with normal counts (16). However, though low quantity in younger women may reflect fewer oocytes with which blastocysts can form, it does not clearly seem associated with higher rates of aneuploidy or miscarriage (17).

TESTING

Antral follicle count can be measured at any time during the menstrual cycle, as well as when a woman is on hormonal contraceptives or is pregnant. Classically, a woman’s AFC is the total number of ovarian follicles measuring between two and nine millimeters, though many studies count follicles up to and including ten millimeters in size (Figure 3).

Figure 3. Ovarian sonographic imaging of women in their mid-30’s. Figure 3A is from a woman with premature ovarian failure and there are no visualized antral follicles (the sonographically anechoic regions measuring approximately two to nine millimeters within the ovary). Figure 3B is from a woman with tubal factor infertility, and for whom seeing a few follicles within a single plane of the ovary would be normal. Figure 3C is from a woman with polycystic ovarian syndrome. Though her ovary is arguably more multicystic than polycystic (which would typically have follicles concentrated on the periphery of the ovary), she met the criteria for PCOS and her ovary is clearly distinct from those shown in 3A and 3B. Of note, all three ultimately conceived with their own oocytes, so it should be remembered that the absence of visualized antral follicles makes conception far less probable, but not impossible.

Multiple cutoffs are used for what constitutes normal and poor ovarian reserve. Given that antral follicle count varies among cycles, it is reasonable to view the AFC as a continuum, with four total antral follicles reflecting limited reserve, but five antral follicles not being entirely reassuring. Additionally, what constitutes normal is age dependent, where ten total antral follicles may be common for women in their 30’s, but not their teens. Though many measures have been used to define polycystic ovarian morphology, the most accepted standard is that used in the Rotterdam criteria of, “12 or more follicles in each ovary measuring 2 to 9 mm in diameter, and/or increased ovarian volume (>10 ml).” This cutoff was chosen, as it was associated with 75% sensitivity and 98% specificity for distinguishing polycystic ovarian morphology (PCOM) from normal ovaries (18). Another frequently used definition comes from Adams, who considered an ovary polycystic if there were ≥ 10 follicles measuring <9 mm (19). Of note, in the development of guidelines for the WHO on PCOS, sonography was deemed preferable to AMH levels from a pragmatic standpoint. (20)

LIMITATIONS

There is debate as to how much moving outside of the early follicular phase or hormonal modulation such as pregnancy and oral contraceptives will shift the measurement of antral follicle count. Both central and paracrine effects can occur and these are more likely to be meaningful in patients with suboptimal ovarian reserve. However, patients with reassuring ovarian reserve are unlikely to move into a non-reassuring category through these conditions if the ultrasound resolution allows for early antral follicle visualization and measurement.

Patient dependent and observer dependent limitations should also be considered. Patients with elevated BMI (particularly with increased vaginal adiposity) and/or scarring of the pelvis may be more likely to have ovaries with limited resolution for assessment, which could potentially underestimate ovarian reserve. Similarly, large cysts or endometriomas could exert a temporary paracrine effect underestimating reserve. Patients with previous ovarian surgery could also have inclusion cysts appearing similar to antral follicles, but these won’t develop with stimulation or have oocytes at follicular aspiration for IVF. For observer dependent limitations, it should be noted that in some multi-center studies where anti-Müllerian hormone (AMH) is found superior to antral follicle count, one can find most institutions having AMH and AFC equally predictive, but one site has an observer where there is a meaningful difference. This has led some to conclude AMH superior to AFC, but failure to properly train observers prior to research is a limitation to study design and may not necessarily reflect true diminished value in utilizing AFC for assessing ovarian reserve. ASRM 2022 Practice Committee Guidelines note, “When performed in an experienced center, AFC is a reasonable alternative to AMH” (29).

Anti-Müllerian Hormone (AMH, Müllerian Inhibiting Substance, MIS)

MECHANISM

AMH is a homodimeric glycopeptide that in reproductive aged women is predominantly granulosa cell derived. The role of systemic AMH is not clear, but at the level of the ovary, it is believed to downregulate FSH mediated folliculogenesis. AMH expression is highest in secondary, preantral, and small antral follicles up until approximately 4 mm in size, and it stops being expressed by granulosa cells when the follicle measures in the 4 to 8 mm range (Figure 4).

Figure 4. The interplay of follicular development and hormonal secretion and responsiveness.

AMH seems to have a role in selecting the dominant follicle in addition to generally mediating preantral follicular recruitment. AMH levels start undergoing a log-linear decline approximately fifteen years prior to menopause and drop to very low levels approximately five years before menopause (21).

The AMH level associated with diminished ovarian reserve is assay specific and depends on the desired balance of sensitivity and specificity, but is typically below 1 ng/mL. The threshold for menopause is typically lower than the lower detectable limit for many assays, being slightly below 0.1 ng/mL (22). AMH <0.5 ng/mL seems associated with fewer than three follicles available at retrieval, 0.5-1 ng/mL with reduced response, 1-3.5 ng/mL with normal response, and >3.5 ng/mL with overresponse, reflecting greater risk for ovarian hyperstimulation syndrome (23). Normal AMH values often exceed 2 ng/mL at 30, 1.5 ng/mL at 35, and 1 ng/mL at 40 as a quick reference for expected reserve at a given age.

The role of weight loss on AMH levels is open for debate, but should be substratified in women with and without PCOS. (51) Though a lack of association cannot be excluded due to limitations in sample size, there does not seem to be a clear shift in AMH with weight loss for non-PCOS patients. However, for those with elevated AMH from PCOS that has been effectively treated through diet, exercise, and/or bariatric surgery, there is improved fertility, even as AMH lowers to more normal levels. (51) It is unclear which signal transduction pathways drive these lower levels, as one would expect weight loss-associated shifts in adiponectin, leptin, and insulin to actually increase AMH through recognized mechanisms. Lifestyle may have a broader impact beyond weight on ovarian reserve, as both AMH and AFC are statistically lower in women with lower socioeconomic status (56).

TESTING

AMH levels are measured through immunoassay on a sample obtained through phlebotomy. Values obtained have the distinct advantage of being equally valid at any point in the menstrual cycle. Because AMH is expressed primarily before FSH responsiveness occurs, it is believed that AMH remains a valid assay even when ovarian suppression occurs through smoking, oral contraceptives, GnRH agonists, and pregnancy (24). Though these factors can lead to transient ovarian suppression, they are unlikely to change levels so much as to meaningfully underestimate true reserve. The magnitude of effect through these reversible factors seems to be low, with age-specific AMH percentiles decreasing by 11% with oral contraceptives and 17% with pregnancy (25). Additionally, AMH levels drawn on day seven of the pill free interval seem to closely correlate with levels seen after oral contraceptive discontinuation (26).

A popular misconception is that just because it is valid to assess AMH throughout the

menstrual cycle and under a variety of inhibitory conditions, this should not be mistaken as meaning that AMH levels are static. Though levels of AMH tend to be steady state in perimenopausal patients, for those with higher ovarian reserve, AMH levels fluctuate significantly. This fluctuation, however, is not to the point where a person with robust ovarian reserve is likely to be categorized as having limited reserve (21).

LIMITATIONS

AMH seems to have fewer limitations than most other assays. In fact, it seems to have the advantage that it not only is useful in predicting ovarian response to gonadotropin stimulation, but may even have limited value in predicting pregnancy rates (27). However, like other ovarian reserve assays, it does not appear particularly valuable in predicting viability once pregnancy has already been established. When there is discordance between AFC and AMH levels (e.g., low AFC but normal AMH or vice versa), ovarian response is often a hybrid of the two findings (above those with diminished reserve but less than that of those with normal reserve) (28).

Inhibin B

MECHANISM

Inhibin B is similar to AMH in that it is a glycoprotein secreted by preantral follicles, with levels declining with age. Both inhibin A and B downregulate pituitary FSH secretion. However, Inhibin A levels are not used to predict ovarian reserve because they arise primarily from the dominant follicle rather than an earlier follicular cohort and therefore are less predictive. Inhibin B levels are relatively more useful, but overall remain suboptimally predictive, as they are a late finding for diminished ovarian reserve and typically start falling around four years prior to menopause (21).

TESTING

Inhibin levels are measured by immunoassay after phlebotomy. Inhibin B levels fluctuate over the menstrual cycle, with peaks in the early to mid-follicular phase, as well as during ovulation. Accordingly, inhibin B is typically measured on the third day of the menstrual cycle in ovulatory women. Outside of ovarian reserve testing, in postmenopausal women, where inhibin B levels should be consistently low, a random level is particularly good for following granulosa cell tumors (>89% have elevated inhibin B) and also can be useful for following some epithelial cell ovarian tumors.

LIMITATIONS

In addition to significant variation within the cycle, there is also meaningful variation among cycles. Because of limited sensitivity and specificity, this assay has greater value in those far more likely to have diminished reserve. Some have proposed using inhibin B in combination with other assays, but it is the opinion of the American Society for Reproductive Medicine that “combined ovarian reserve tests models do not consistently improve predictive ability over that of single ovarian reserve tests.” (29).

Ovarian Volume

MECHANISM

Follicles, stroma, and vasculature all contribute to ovarian volume. The percentage that each contributes depends on the individual, her age, underlying gynecologic conditions, and where she is during the menstrual cycle.

TESTING

Typically, ultrasound is used to measure the ovary in all 3 dimensions. These measurements are then applied in the formula for calculating the volume of an ellipse (D1 x D2 x D3 x 0.523). An ovarian volume of >10 cm^3 is considered consistent with PCOS. Although increased ovarian stromal volume distinguishes polycystic ovarian morphology from the multicystic ovary, stromal volume is not routinely measured. Alternative approaches that may improve the effectiveness of ovarian volume include the use of trapezoidal volume (30), 3D ultrasound (31), and color Doppler (32).

LIMITATIONS

Ovarian volume shifts in response to normal physiologic changes (such as the presence of a dominant follicle) and coexisting medical conditions (such as endometriomas). Exogenous hormones can decrease ovarian volume (33), even though ovarian reserve itself has not changed. For these reasons, if evaluating the ovaries by ultrasound, antral follicle count is believed to be a better proxy for ovarian reserve.

APPLICATION OF OVARIAN RESERVE TESTS

Assessing Perimenopausal Status

Classically, ovarian insufficiency and failure have been defined as present when persistent FSH levels >40 µIU/mL are found with at least two radioimmunoassays more than a month apart. No detectable antral follicles in a patient without ovarian suppression is consistent with a perimenopausal state and fewer than two antral follicles has been deemed a more sensitive cutoff (29). The reason to not require the complete absence of follicles is that minimal follicular development is not unusual in postmenopausal women, as there can be a 14% prevalence and an 8% incidence of simple cysts in a given year (34). Similarly, though an undetectable AMH level would be consistent with menopause, in women with primary ovarian insufficiency, approximately a quarter of them will have below normal but detectable AMH levels and a sixth will have normal AMH levels (35). Though women with advancing age will have higher FSH levels, it remains unclear if women with elevated FSH earlier in their reproductive life will go through menopause earlier (36). Finally, it should be remembered that confirmation of primary ovarian insufficiency does not automatically mean completion of testing, as fragile X carrier screening and other evaluations may be appropriate.

Evaluating Ovarian Reserve for Fertility in Ovulatory Patients

For ovarian reserve testing prior to fertility therapy, there is more data on FSH than other measures. Generally, women of the same age with higher FSH levels seem to have lower fecundability (37). However, younger women with elevated FSH levels often have much better fecundability than older women with comparably elevated FSH (38) and age can be a better predictor of outcome than FSH (39). Though differences in pregnancy rates can be shown between those with high and low FSH, the assay in general has suboptimal sensitivity for both ovarian response and pregnancy rates, as reflected by receiver-operator curves (9).

A rarely addressed caveat is that though it is true that multiple studies are showing AMH and AFC to have a better balance of sensitivity and specificity than FSH, meta-analyses regarding the predictive value of FSH run the risk of being biased towards the null. The reason is that the earliest ovarian reserve testing research (using FSH) was done at a time when IVF success rates were lower. This caveat won’t apply to modern studies were FSH is directly compared with AMH or AFC, but one should account for temporal bias in meta-analyses if studies from the 1990s are included. One should also note, that 20% of the time there will be AMH and FSH discordance, particularly in older women where this can be as high as 33% (55).

Anti-Müllerian hormone levels and antral follicle count seem to be emerging as the best approaches to procreative testing. A survey of 796 centers noted 51% thought AMH the best measure for ovarian reserve, while 40% selected AFC, though ultimately 80% felt age was the best predictor for pregnancy (50). After accounting for age, AFC and AMH seem highly accurate in predicting poor response with IVF, while FSH does so only moderately (40). Not only are these measures commonly used for predicting under response, but they can also be used to predict hyperstimulation (41). Ovarian reserve assessment for reproductive purposes is fraught with controversy because different practitioners prefer different balances of sensitivity and specificity. At the minimum it should be recognized that this type of testing is meant to be screening for women who are more likely to have a poor response to ovarian stimulation, and findings are not necessarily diagnostic of ovarian failure or the degree of risk for premature menopause. However, results consistent with perimenopausal findings should be confirmed and appropriate counseling given. As stated by ASRM, “Extremely low AMH levels should not be used to refuse treatment in IVF” (29). The American College of Obstetricians and Gynecologists (ACOG) draws similar conclusions (42).

Since combined tests do not consistently improve the ability to predict ovarian response, many clinicians are simply using either AMH or AFC in the context of the patient’s age and reserve additional testing for atypical clinical pictures or to confirm significant ovarian insufficiency. In spite of this ASRM recommended approach, some argue that combined testing improves sensitivity in detecting suboptimal ovarian reserve. Whether or not the literature ultimately demonstrates this, a way of side-stepping this debate is by noting that combined testing is unlikely to be cost-effective. The reason is that if additional testing is unlikely to change management (especially when the vast majority of patients have normal results), it is very hard to show cost-effectiveness when doubling or tripling costs without clear benefit. Accordingly, if using combined testing, it should be selective rather than universal.

An additional note on ovarian reserve tests in procreation relates to their limitations. Though some appear better than others in predicting ovarian response to stimulation, most are limited at best in predicting pregnancy, and this predictive value is highly dependent on patient demographics within a study. This is not inherently a flaw in the assays; rather, infertility is often multifactorial, so when ovarian reserve testing is a subset of factors, this tends to bias its relevance towards the null. Studies showing an ovarian reserve test to be predictive of pregnancy in general tend to have older populations. (It has been argued as to whether this constitutes enrollment bias or limits external validity; however, it is reasonable to find a test having greater value when applied to a population at risk.)

Another limitation is that abnormal ovarian reserve testing does not always increase miscarriage rates (43), despite an association between abnormal testing and blastocyst aneuploidy (1). Studies show roughly a 25% increase in the probability per embryo of being aneuploid in the setting of diminished ovarian reserve (52, 53). This may underestimate the magnitude of effect, as many aneuploid embryos may not survive to biopsy and evaluation. This is why one can see the bottom quartile having higher rates of all embryos being aneuploid (19.3% vs 10.3%) and of having only one embryo to biopsy (31% vs 11%) (52). Further muddying the waters, for a particular age, though women with diminished ovarian reserve may have quantity issues without this always translating to quality (euploidy), looking at the 5%-10% of patients with lowest reserve may give a different answer than looking at the bottom quartile. The better performers among the population with the lowest reserve may bias the data towards the null. This is why when comparing the cited Jaswa (53) and Fouks (52) articles, one sees a higher rate of aneuploidy in the Jaswa study (71% DOR vs 55% controls) relative to Fouks (50% bottom quartile vs 60% middle quartiles), as the Jaswa DOR group arguably had more pronounced diminished ovarian reserve. A study by Morin focused on those with the bottom 10% for ovarian reserve and noted a decline in quantity didn’t shift quality (54). However, the bottom decile had triple the rate of no usable blastocysts (17% vs 5.3%) and lower live birth rates (41.2% vs 53.1% per cycle start), where arguably successful pregnancy is the ultimate metric of quality. The Morin DOR population was meaningfully younger than the Jaswa population, so DOR associated with aging is likely to be more concerning than comparable ovarian reserve in younger women (53, 54).

To further complicate the quantity vs. quality debate, there can be heterogeneity, where some women with DOR have far lower quality than others. Finally, follicular quantity is more valuable in identifying ovarian factors for subfertility patients but does not seem predictive of outcomes in patients who have suboptimal reserve, but have never tried to conceive (44).

Evaluating Amenorrhea in the Post-menarche, Pre-menopausal Patient

Numerous conditions can cause amenorrhea in reproductive aged women. Testing falls into two categories: diagnosing etiology and reassuring the patient that she is not in ovarian failure. For a more comprehensive discussion of how to evaluate etiology, please see the Endotext section on the Endocrinology of Female Reproduction. In general, and in addition to remembering to exclude pregnancy as a cause for amenorrhea, ASRM Practice Committee guidelines recommend FSH, TSH, and prolactin levels in addition to the usual history and physical exam (45) (An important contextual caveat is that AMH and AFC were not as well established when these guidelines came out in 2008). Though increasingly AFC is used in place of FSH, especially for evaluating hyperandrogenic women since AFC is part of the Rotterdam criteria (46), FSH still has a role in differentiating PCOS and forms of functional hypothalamic amenorrhea (47). See figure 5 for interpretation of test results.

Figure 5. Laboratory parameters in the setting of amenorrhea (43). Reproduced with permission of the author.

Regarding reassuring the patient, for gynecologists a transvaginal ultrasound to assess antral follicle count is relatively easy to perform, can often be performed promptly at the initial office visit, and can have a reassuring tangibility to patients when antral follicles are identified and their importance is explained. When sonographic evaluation of ovarian reserve is less available, a normal AMH level should be reassuring. Additionally, for patients who have been placed on oral contraceptives or other hormonal therapy without a diagnosis of etiology for amenorrhea, AFC and AMH levels may be lowered, but are still likely to remain within the normal range if the patient truly has normal reserve. Figure 6 provides information on the relative strengths of assays.

Figure 6. Relative strengths of assays for determining ovarian reserve (balancing positive and negative predictive values, typical costs, and available alternatives). AFC= antral follicle count, AMH= anti-mullerian hormone, CCCT= clomiphene citrate challenge test. * When combined with LH

LOOKING TO THE FUTURE OF OVARIAN RESERVE TESTING

Advancing technologies and improving cost has made direct to consumer (DTC) fertility testing a reality. DTC testing enhances patient access care, but can do so at the expense of oversight, insight, and broader perspective. Moreover, though abnormal results may encourage a patient to seek out a physician for counseling, normal results may provide false security for fertility, which is multifactorial and more than ovarian reserve. Additionally, ovarian reserve testing in patients with untested fertility may provide limited predictive value for fecundability and ultimately fecundity (57). Ultimately, how do physicians interpret results in patients who may not warrant assessment? If DTC ovarian reserve testing is typically performed by fingerstick blood sampling, relative to a larger, better-preserved sample through traditional phlebotomy, sample accuracy at times can be suboptimal. Additionally, lack of assay standardization among labs can also hinder counseling patients on their results (58). If more information is available, the true questions are if we can use and trust the results?

When (and if) ovarian reserve testing is predictive, therapy can be focally applied early. Current testing methods are often more reliable after DOR has already occurred and better predict response to therapies than pregnancy itself. The known causative association between Fragile X premutation in women and risk of decreased ovarian reserve leads to interest in genetic causes of decreased ovarian reserve. Studies have linked target genes and epigenetic changes to patients with diagnosed DOR (59, 60). If testing Fragile X, as well as testing for blepharophimosis, ptosis, and epicanthus inversus (BPES), can anticipate DOR, the challenge is not just in making the most of ovarian reserve where possible, but there is also a role for potentially reducing transmissible morbidity.

CONCLUDING REMARKS

It has been said that a Rolex keeps time well, but makes for a lousy hammer. All ovarian reserve tests are merely tools and their value relates to the task to which they are applied. Even as we see increased use of AFC and AMH (48), we have to remember that ideal testing is “systematic, expeditious, and cost-effective” (49). In other words, when evaluating ovarian reserve, one should account for not only the symptoms and probable diagnosis, but also the turnaround time for results, and how to maximize value in testing. These latter two factors vary by site, so clinicians will have to find the right balance for their practice. Finally, one of the most important and cost-effective predictors is age (see Figure 1). In the procreative setting, after age is combined with another ovarian reserve test, the marginal benefit from further assays tends to be less (8). Accordingly, and with the exception of premature ovarian failure where independent confirmation is appropriate (due to discordance between age and the assay), until further studies justify effectiveness and cost-effectiveness, simultaneously using multiple ovarian reserve tests should be for selected patients rather than universal.

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Male Contraception

ABSTRACT

Men continue to have a strong interest and commitment to effective family planning. Traditional methods of male contraception have long included periodic abstinence, non-vaginal ejaculation, condoms, and vasectomy, the latter two representing physical methods to prevent sperm from reaching the site of fertilization. However, for male contraception the reversible methods are not reliable, and the only reliable method, vasectomy, is not intended as reversible. During the 20^th century, a wide array of reversible and highly reliable female hormonal contraceptive methods was marketed; however, no new methods for male fertility regulation have been introduced for centuries. For men to share more equally the burdens as well as the benefits of family planning, more effective reversible male contraceptive methods need to be available. Most studies into male contraception have been conducted with hormonal methods, analogous to well-known female hormonal contraceptives, making them the closest to introducing a reliable, reversible male contraceptive method. The most promising hormonal approach is the combination of an androgen (usually testosterone) with a progestin and multiple studies have shown such combinations of depot steroids displays high contraceptive efficacy, based on reliable and reversible suppression of sperm output, with few side effects. While research into novel methods for male fertility regulation has continued in the public sector, private sector research into male contraception, essential for effective commercial product development, has stalled in recent decades.

BACKGROUND

A male contraceptive must reduce the number of fertile sperm ejaculated into the vagina to levels that reliably prevent fertilization (1). Conception can be prevented by diverting or suppressing sperm output and/or inhibiting sperm fertilizing capacity. So far, all male methods depend on mechanical means to reduce female exposure to sperm using either traditional drug and device-free methods (abstinence, withdrawal, non-vaginal intercourse) or contemporary male methods comprising condoms and vasectomy. Unfortunately, among current male contraceptive options, the reversible methods are not reliable, and the reliable method is not intended as reversible. No new male contraceptive methods were introduced during the 20^th century contrasting with highly reliable, reversible hormonal contraceptives developed for women over the last half century. Yet male involvement in family planning remains extensive. Globally, one third of couples using contraception rely on methods requiring active male participation (2) and current male methods remain (together with medicated intrauterine devices) the most cost-effective contraceptive methods in the USA (3). While reflecting traditional and ongoing reliance of family planning on male involvement, greater participation by men in sharing the burdens as well as the benefits of effective family planning require developing more effective, reversible male methods. Such greater participation could ameliorate the still large unmet need for contraception globally as 40% of pregnancies are still unintended with half of unintended pregnancies ending in abortion and those rates are higher among developed countries (4). Family planning must always remain a matter of personal choice by couples. This is highlighted by the human rights abuses of coercive national family planning programs enforcing family limitation (“one-child family”), including through forced sterilization and abortion, in India and China (5, 6). Such coercive national programs originated in the 1970’s prompted by uncritical belief in the “population explosion”, notably from influential publications such as the Club of Rome’s “Limits to Growth” and Ehrlich’s “Population Bomb”, now discredited by their failed, alarmist predictions. India’s forced sterilization program was overturned by popular democratic action within a few years of its imposition whereas China’s one-child family policy operated for nearly 4 decades before its ending in 2016. These issues echo in medical mistrust as a barrier to acceptance of male contraception among the Black American minority (7).

Extensive public sector clinical research over the last 4 decades (8), despite minimal pharmaceutical industry involvement, has proven in principle that hormonal male contraception is feasible. Proof-of-principle is established for androgen-based hormonal suppression of spermatogenesis (9) achieving full reversibility (10), sufficient short-term safety (11) and acceptability to men and women (12, 13) culminating in the crucial proof of reliable contraceptive efficacy using androgen alone (14-17) or depot (18) or daily use (19) androgen-progestin combinations. Demographic modelling shows that introducing new male contraceptive methods, even if adopted by only a minority of men, would significantly reduce unintended pregnancies in Nigeria, South Africa, and the USA (20). Surveys of participants in clinical male contraceptive studies suggest that men may prefer a male contraceptive to be provided by their family doctor rather than specialists (21); however, the practical knowledge of non-specialists remains unsatisfactory and would require specific training (22). In addition to boutique contraception for adventurous early adopters, practical niches for male contraception include contraception after birth, during breast-feeding, or after abortion (23). Nevertheless, despite the proof-of-principle, strong community interest, and medical priority on the need for new, reversible male contraceptives, pharmaceutical industry development has ceased. This market failure reflects pharmaceutical industry perceptions of low profitability coupled with high risk in the context where a better return on investment is expected for developing drugs in more lucrative, less risky areas of chronic disease compared with the higher product liability risk of developing novel contraceptives for healthy men to compete with existing low-cost female methods. The possibility that Western market failure might be overcome if the growing perception of needs for voluntary family planning as a national priority in rapidly industrializing countries such as China, India and Brazil (24), has proved a failed hope. Novel approaches to finance further research such as philanthropic private sector funding (25) and public-private partnerships (26) have been developed. The 2022 overturning by the US Supreme Court of what was long accepted as a constitutional right to abortion has refreshed interest in developing wider coverage of effective contraception, including for men, to reduce the demand for abortion.

NON-HORMONAL METHODS

Traditional Methods

PERIODIC ABSTINENCE

Although theoretically effective, neither celibacy nor castration is an acceptable or practical contraceptive method. Periodic abstinence, the limiting of sexual intercourse to "safe" days (27), has high contraceptive efficacy if the rules are followed perfectly but the failure rates rise steeply with rule breaking (28). This cost- and device-free method is used by >40 million couples world-wide for family planning (2). The typical use 1^st year failure rate is ~25% with ~47% of couples continuing use at 1 year in USA (29) but 8% and 52%, respectively, in France (30, 31). While inherently safe, it has limited acceptability due to low reliability, inflexibility, and interference in the spontaneity of sex.

EX-VAGINAL EJACULATION

Withdrawal is a traditional male method of contraception whereby intercourse culminates in extra-vaginal ejaculation (32). Often overlooked as a contraceptive method, withdrawal, together with abortion, was the major pre-industrial method of family planning largely responsible for the demographic transition from high to relatively low birth rates in industrial nation states and continues to be used by 40 million couples (2). This cost- and device-free method has limited reliability in its demanding requirement for skill and self-control. The typical use 1^st year failure rate is 18-22% with 46% continuing use at 1 year in the USA (29, 33) and 10% and 55%, respectively, in France (30, 31). While safe and reasonably effective for experienced users, interfering with the pleasure of coitus leads to a correspondingly high failure rate in practice. Other sexual practices that avoid intravaginal ejaculation have also been used traditionally to avoid conception. These include masturbation, oral and anal intercourse, deliberate anejaculation and retrograde ejaculation (34).

CONDOM

After centuries of use in preventing sexually transmitted infections and pregnancy, now over 50 million couples rely on condoms for contraception (2). Condoms provide safe, cheap, widely available, user-controlled, and reversible contraception with few side-effects. In case of latex allergy, non-rubber (polyurethane, natural membrane) condoms can be substituted. Latex condoms are moderately effective at preventing pregnancy with a typical 1^st year failure rate of 18% with 43% continuing use at 1 year in the USA (29, 33) and 3.3% and 47%, respectively, in France (30, 31). Recent US data suggest a reduction in failure rate to 13% (35). The discrepancy from the estimated 2% perfect-use failure rate (29) is mainly to human error, notably rushed use (36), misuse, or non-use rather than mechanical failure (breakage or slippage) (37). Differences in contraceptive use behaviors may explain lower reported 1^st year failure rates for condoms in France (31). The major limitations of condoms for contraception are relatively high failure rates and interference with sexuality. The requirement for regular and correct application during foreplay disturbs the spontaneity of sex and dulls erotic sensation. These aesthetic drawbacks limit the popularity of condoms especially among stable couples (38). Latex condoms are perishable through tears or snagging on fingernails, clothing, or jewelry as well as deterioration from exposure to light, heat, humidity, or organic oils.

Polyurethane condoms with improved tactile sensitivity were developed in the 1990’s to enhance acceptability (39), but they have shown reduced efficacy and mechanical performance compared with latex condoms in prospective randomized controlled clinical trials (40). Although the theoretical requirements for condom use to protect against sexually transmitted infections differ from those to prevent pregnancy, in practice the protections are similar (41). Laboratory testing of condoms standardizes integrity and durability for strength and leakage. Although viral penetration is not routinely tested, synthetic (latex or polyurethane) but not natural membrane condoms are not perfect at preventing passage of prototype human pathogenic viruses (42). Using a sensitive, objective biochemical marker (PSA) for seminal plasma exposure in vaginal swabs, vaginal exposure to semen was reduced by 50-80% even after mechanical condom failure (breakage, slippage) (43). Novel spermicides with virucidal properties that are being developed to provide dual antimicrobial and contraceptive protection (44) might not enhance condom efficacy for either function because non-compliance is the major cause of failure for both (37).

VASECTOMY

Vasectomy, used by over 40 million couples for family planning (2), varies widely in prevalence between countries depending upon cultural factors, public education, and availability of male-oriented facilities (45). Recent data suggests a steady decline in vasectomy in the US over the first two decades of the 21^st century (46). Having overcome a sordid history of applications to eugenics and other historical misadventures into the 20^th century (47), vasectomy is now more widely used than female sterilization in some economically developed countries although female sterilization remain ~4 times more frequent worldwide (48). For men having completed their family and fit for minor surgery, vasectomy is a very safe and highly effective office procedure (49, 50). Relative contraindications include risks from office-type surgery (bleeding disorders, allergy to local anesthetic) or scrotal pathology (post-inguinal surgery scarring, keloid-proneness, active genitourinary or groin infections). Vasectomy is performed under local anesthesia via scrotal incisions and usually involves excising a segment of both vasa deferentia. Interposing a fascial barrier between the occluded cut ends significantly reduces the risk of failure due to recanalization (51, 52). The Chinese-developed "no-scalpel" technique (53)minimizes skin incision and reduces immediate side-effects (bleeding, infection) 10-fold to 0.3% compared with conventional vasectomy (54, 55). Chronic post-vasectomy pain (56) occurs with an estimated prevalence of ~15% at 7 months after vasectomy (57). It may be reported as groin or testicular pain precipitated or aggravated by intercourse, ejaculation, or exertion and accompanied by tender, distended epididymides (56, 57). Fascial interposition significantly reduces failure rate (52, 58) so that, in the hands of an experienced practitioner, no-scalpel vasectomy with fascial interposition is now the method of choice (55, 59). Additional studies suggest that cautery may further enhances reliability (60, 61) and that leaving an open testicular end reduces retrograde pressure-related complications (pain, sperm granuloma, epididymal and testicular damage) thereby better preserving reversibility (62-64).

Vasectomy is highly effective once sperm are cleared from the distal vasa deferentia. However, flushing with saline or water (65-69) or spermicides (nitrofurazone (70), euflavine (71, 72) or chlorhexidine (73)) during surgery does not accelerate sperm clearance, but the evidence remains weak (74). Non-irritant spermicides that inhibit sperm function without chemical sclerosis of the vasa that would impair potential reversibility, might have promise (75). Immediately following vasectomy, additional contraception must continue until azoospermia or near azoospermia (<0.1 million non-motile sperm per mL) is demonstrated usually at 3 months post-vasectomy when at least 95% of men reach this clearance criterion (76-79). Although azoospermia might occur sooner (80), reliable evidence is lacking to support the reliability of earlier time points as recanalization, where motile sperm persist in the ejaculate, may occur within the first few weeks after vasectomy (81) and persistence of motile sperm in the ejaculate indicates technical failure. Although detailed information on the rate of sperm clearance from the ejaculate after vasectomy remains sparse, time since vasectomy rather than number of ejaculations is more predictive of sperm clearance (80). Contraceptive failures are rare; early failures are most often due to not awaiting sperm clearance (82) or occasionally misidentification or duplication of the vasa deferentia whereas late failures are due to spontaneous vas(a) recanalization (~0.1%) (83). Complications of vasectomy include post-surgical bleeding, wound or genito-urinary infections and fistulae, and chronic scrotal pain (50, 84) with risk of death estimated at ~1 per million vasectomies in developed countries although higher in developing countries (85). Vasectomy causes no consistent changes in circulating hormones (86), sexual function, or risk of cardiovascular or other diseases (49, 87, 88) including testicular cancer (89-91). A small increased risk of prostate cancer after vasectomy has been observed in case-control studies (92-94), but the increased risk is unaffected by vasectomy reversal (95, 96). This risk seems to be attributable to surveillance and detection bias, rather than a biological effect as indicated by consistent evidence from numerous cohort studies (96-102). Sperm antibodies develop in most vasectomized men but have no known deleterious health effects apart from a possible role in reducing fertility after vasectomy reversal (103) when sperm antibody titers are very high (>512) (104).

Vasectomy is a quick, simple, highly effective, and convenient method of permanent sterilization; its major drawback as a male contraceptive is its limited reversibility. Elective sperm cryostorage is occasionally useful but may reflect ambivalence about the irreversible intent of vasectomy. Cumulative rate of requests for reversal, mostly prompted by remarriage, are 2.4% at 10 year post-vasectomy but exceed 10% for young men (aged <25 years at vasectomy) (105). Failed vasectomy reversal is now a significant cause of male infertility. Following microsurgical vasovasostomy, 80-100% have any sperm in the ejaculate ("patency") but normal sperm concentration is less common, and cumulative conception rate at 12 months is only ~50% compared to 80-85% at 12 months in healthy young couples not using contraception (105). This discrepancy is most probably attributable to technical limitations of microsurgery as even the lowest reported rates of azoospermia (bilateral non-patency) after microsurgical vasovasostomy indicate that nearly half such men have at least one non-patent vas deferens (106) so that re-operation should be a prominent consideration if pregnancy does not ensue. After technical failure, the wife’s age and time since vasectomy appear to be the dominant predictors of successful reversal (107, 108) Whether robotic microsurgery can improve the technical success of vasovasostomy to become a cost-effective and widely available alternative to human microsurgery remains to be established (109-111). Reversibility is better with microsurgery (106), presence of sperm in the vasal fluids from testicular end of stump (112), in younger men with shorter duration since vasectomy (105) and possibly with longer testicular vasal stump (113, 114); unfavorable predictors include non-microsurgical techniques, older age of wife (especially after 40 yr) (106, 115), high titers of sperm antibodies (104), and long duration (>10 years) since vasectomy (116-118) due to long-term epididymal (119), vasal (120) and testicular damage (116, 117, 121, 122). Experimentally in a variety of mammalian species, open-ended vasectomy is preferable to complete vas occlusion in preventing the back pressure-induced damage to spermatogenesis and seminiferous tubular integrity which are important contributors to failure of vasectomy reversal (123, 124). An alternative to surgical vasectomy reversal either instead of, or after failed vaso-vasostomy, is sperm harvesting from epididymis or testis in conjunction with intracytoplasmic sperm injection (ICSI)/in-vitro fertilization. Currently cost-benefit analyses suggest that microsurgical vaso-vasostomy is more cost-effective and safer in both North America (125) and Europe (126), with the wife’s age being a key determinant (107). However, optimal management depends on local clinical expertise as well as access to microsurgery and reproductive technologies. The feasibility of successful vasectomy reversal has led to the proposal that, although vasectomy is intended as permanent, some individuals may consider it a reversible contraceptive method (127). USA national economic indicators strikingly influence rates of vasectomy and of vasectomy reversal, which are increased and decreased, respectively, according to the unemployment rate and personal income (128); whether this applies in countries with national health schemes that reduce financial limitations on access to elective health care remains unknown.

Modern Methods

VAS OCCLUSION

The efficacy, safety, simplicity, and acceptability of vasectomy suggest that a reversible mechanical method of vasa occlusion would be an attractive male contraceptive option. Since vasectomy reversal is neither cheap nor widely available, more reversible vasa occlusion methods are needed (129). A nonsurgical, potentially reversible technique involving percutaneous injections of polymers that harden in-situ to form occluding plugs which might be later removed to restore fertility was reported (130) but, despite preliminary positive findings (131), formal evaluation showed vasa occlusion had lower efficacy (inducing azoospermia) than vasectomy (132). In a phase II randomized clinical trial, urethane-coated nylon thread intra-vasa devices were more acceptable with fewer complications but were less effective in producing azoospermia, compared with no-scalpel vasectomy (133). A hydrophilic gel, composed of co-polymer of styrene and maleic anhydride delivered in dimethyl sulfoxide, forms a charged spermicidal biopolymer when injected into the vas deferens that is stable but potentially removable. Experiments in rats favor sodium bicarbonate over dimethyl sulphoxide for intravasal injection for effective reversal of occlusion (134). Preliminary non-comparative clinical evaluation showed azoospermia in 12 men with no pregnancies in their wives for 12 months following intravasal injection suggesting effective vasal occlusion (135); however, some morphologically damaged and non-functional sperm persist in the ejaculate (136) but the mechanisms of the deleterious effects on sperm structure and function remain unexplained(137). In an extended multi-center phase III study of 139 fertile married men who had a single, bilateral intravasal injection of a styrene maleic anhydride copolymer in 120 µl of dimethyl sulphoxide solution (Reversible Inhibition of Sperm under Guidance, RISUG) were followed for six months after injection. In six men, the injections failed to be delivered, but in the remainder, 110 achieved azoospermia within 1 month and 23 achieved azoospermia 3 to 6 months after the procedure. After ceasing condom use in the first 2 months after injection, there were no pregnancies in the 133 men with successful occlusion over the next 4 months whereas pregnancies were reported in two of six men who had failed injections; however, reversibility was not reported (138). Another phase III randomized controlled clinical trial has shown that a prototype implantable non-occlusive intra-vasal device, comprising a nylon thread encased in barium-impregnated polyurethane, is as effective for sterilization as standard no-scalpel sterilization with no more adverse effects (139); however the reversibility of this device remains to be demonstrated. A promising development for a reversible vas occlusion is a solution for transcutaneous intravasal injection which forms a hydrogel in the vas to reversibly occlude it. The injection solution, comprising sodium alginate conjugated with thioketal and including titanium dioxide and calcium chloride, transforms after injection into the vas deferens into an occluding hydrogel. Subsequently, the occluding hydrogel can be cleared in vivo by non-invasive application of ultrasound that generates reactive oxygen species in the hydrogel causing cleavage of the thioketal and recanalizing the vas deferens. Experimental studies in rats demonstrated fully effective contraception and reversibility (140). Other technical developments including percutaneous injection of sclerosants and transcutaneous delivery of physical agents (ultrasound, lasers) continue to be developed slowly (141).

HEATING

It has long been known (142) that even brief elevations of testicular temperature can profoundly suppress spermatogenesis (143) while sustained elevation may contribute to testicular pathology in cryptorchidism, varicocele, and occupational male infertility (144). A highly novel experimental approach has been reported using intravenously injected iron oxide nanoparticles that accumulate in the testis where they can be activated by an external alternating magnet field to generate testicular heating and damage to spermatogenesis (145); however, safe clinical application, and notably reversibility, remain to be demonstrated. Clinical studies evaluating the potential for tight scrotal supports as a practical male contraceptive method (146, 147) showed a reversible decrease in sperm output but of inadequate magnitude for reliable contraception. Experience of small numbers of men utilizing thermal male contraception has been favorable, apart from discomfort (148) but wider uptake would require substantial population education (149). Given the dubious acceptability and safety (150) of heat-induced suppression of sperm output, the feasibility of a male contraceptive method based on testicular heating remains to be established.

IMMUNOCONTRACEPTION

Sperm vaccines to interrupt fertility have long been of interest (151) with a 1937 patent issued for vaccination with semen (152). Sperm express unique epitopes within the immunologically protected adluminal compartment of the seminiferous tubules at puberty, long after the definition of immune self-tolerance hence explaining their potential autoimmunogenicity. Sperm autoimmunity may contribute to subfertility after vasectomy reversal and in ~7% of infertile otherwise healthy men (other than orchitis). Experimental models for an effective multivalent chimeric protein sperm vaccine targeting surface-expressed antigens involved in fertilization have been reported (153) but remain untested in men. Practical application of this method requires resolving problems of the millions of sperm ejaculated, representing a large antigenic load requiring virtually complete functional blockade, variability of individual immune responses, restricted access of antibodies into the seminiferous tubules and epididymis and the risks of autoimmune orchitis or immune-complex disease. Passive immunization may overcome the present limited predictability of active immunization with sperm antigens to reach quickly and maintain, as well as allowing for volitionally controllable offset, of effective immunocontraceptive titers (154). The smaller antigenic burden in the female reproductive tract requiring complete neutralization suggests that a sperm vaccine, using modern genetic engineering of sperm epitopes (155), may be better targeted for administration to women. However, the most suitable targets for contraceptive vaccines might be feral and wild animals (156, 157).

CHEMICAL (NON-HORMONAL) METHODS

The lack of commercial product development for hormonal male contraceptives has made attractive the alternative option of innovative non-hormonal mechanisms to inhibit sperm production and/or function. These approaches have focused on developing feasible, druggable targets lacking off-target adverse effects for novel product development using either opportunistic or planned approaches to identify novel leads.

Opportunistic approaches include the identification through fortuitous pharmacological observation of male reproductive effects of drugs or natural products. Among older drugs, an orally active spermicide concentrated in semen (158), drugs inhibiting spermatogenesis (159), ejaculation (160) or epididymal sperm function (161) have been identified. Further, among numerous plant products and natural medicines reputed to inhibit male fertility, the most widely tested was gossypol, a polyphenolic yellow pigment identified in China as causing epidemic infertility among workers ingesting raw cottonseed oil. In over 10,000 men purified gossypol reduced sperm output to <4 million/ml in >98% within 75 days with suppression maintained by a lower weekly maintenance dose (162). Although an effective male contraceptive, the systemic toxicity of gossypol, due to mitochondrial apoptosis (163), and irreversibility of sperm suppression precluded further clinical development (164) although it has potential anti-cancer applications (163). Subsequently, extracts of Tripterygium wilfordii, a traditional Chinese herbal medicine for rheumatoid arthritis and skin disorders, decrease sperm output and function and inhibit fertility in rodents and men. Studies aiming to characterize triptolide, an active alkaloid as a potential lead for an orally effective sperm function inhibitor, reveal prominent induction of germ cell apoptosis (165) at testicular (166) in addition to a post-testicular site of action (167). Additional opportunistic approaches include recognizing that the rapidly proliferating germinal epithelium is highly susceptible to cytotoxins such as drugs, heat, or ionizing irradiation which damage germ cell replication, resulting in inhibition of spermatogenesis. However, complete elimination of sperm by non-specific toxicity compromises full reversibility and the accompanying mutagenic risk from direct interference with DNA replication precludes safe use for reversible male contraception.

Alternatively, potential non-hormonal approaches to developing chemical male contraception focus on sperm development maturation and function (168) ensuring that targets are specific for sperm (169). Such approaches may exploit the numerous biological processes required for developing functionally mature, fertile sperm that create abundant targets for reversible inhibition of male fertility (170) . Prominent targets include either reducing sperm output via non-hormonal mechanisms regulating spermatogenesis or post-testicular inhibition of sperm functions. One approach to reduce sperm output is by exploiting the essential requirement for retinoic acid in spermatogenesis (171). Experimental genetic or pharmacological inhibition of vitamin A action inhibits the generation of mature sperm and male fertility (172, 173); however, the ubiquitous roles of vitamin A in cellular replication and differentiation requires thorough safety evaluation for off-target effects in clinical trials. More specific focus on the retinoic acid alpha receptor by developing alpha-specific inhibitors continue in pre-clinical development (174). Another series of orally active indazole carboxylic acid analogs, adjudin, gamendazole and indenopyridine derivatives (175-178), have been developed from the compound lonidamine but aiming to eliminating its non-specific muscle and liver toxicity while retaining its mechanism of action in causing reversible male infertility. The indazole carboxylic acid analogs cause reversible subfertility by disrupting highly specialized intercellular junctions between elongating spermatids and Sertoli cells leading to precocious detachment of immature, elongating spermatids that are then shed prematurely from the germinal epithelium. Clinical evaluation of such drugs remains at an early pre-clinical stage. Other discoveries of molecules with specific expression in sperm and contributions to sperm development and/or fertilizing ability may provide clues to novel leads for chemical non-hormonal male contraceptive drug development. These include protein phosphatase complex (179), cyclin dependent kinase 2 (180), testis-specific bromodomain (181), homeodomain-interacting protein kinase 4 (182) and histone demethylase KDM5B (183).

The seclusion of functionally immature post-meiotic, haploid sperm during their transit through seminiferous tubules and epididymis offers targets for chemical methods to regulate male fertility as sperm are stored and mature functionally. Post-testicular targets offer the advantages of fast onset and offset of action compared with hormonal methods; however, specific target identification, selective drug targeting to the epididymis or testis and human dose optimization remain challenging problems. A model, rapid-onset oral spermicide was first provided by the chlorosugars that showed rapid, irreversible effect on rodent epididymal sperm (184) but proved too toxic for clinical development. A recent promising drug lead was the recognition that an alkylated iminosugar drugs that inhibit glucosyltransferase, used therapeutically to reduce lysosomal glycosphingolipid accumulation in storage disorder type 1 (Gaucher's disease), miglustat, was a potent and reversible oral inhibitor of male mouse fertility but free from apparent systemic toxicity (161, 185). Miglustat treatment produced structural malformation of sperm acrosome, head and mid-piece with consequential impaired motility although sperm retain the ability to fertilize oocytes in-vitro and produce normal offspring. However, miglustat effects were species- and mouse strain-dependent and were not effective in rabbits (186) or men (187). Numerous proteins identified as specifically or uniquely expressed in the epididymis provide additional opportunities for development of novel non-hormonal male contraceptive targets (188-192). Signaling enzymes involved in the epididymal acquisition of sperm motility and capacity for hyperactivation and fertilization such as sperm-specific protein phosphatase PPIγ2, glycogen synthase kinase 3 (GSK3), and calcium regulated phosphatase calcineurin (PP2B) and protein kinase A (PKA) have key role in sperm maturation, activation and fertilization that might provide clues to novel chemical, non-hormonal male contraceptive drugs (193). Post-testicular inhibition of purinergic and adrenergic receptors in the vas deferens to inhibit sperm movement through the ejaculatory ducts are feasible (194, 195), and a series of these receptor antagonists have been developed (196). However, interference with ejaculation is unlikely to be an acceptable form of male contraception. An opportunistic approach based on a FDA-approved excipient, N,N dimethylacetamide that produced a reversible reduction in fertility of rats without hormonal effects has been reported (197).

The most rapidly growing area of opportunity arises from serendipitous discoveries of genes found to be necessary for normal fertility, often from gene knock-out mouse models displaying unexpected male sterility or subfertility (170). This rapidly expanding list includes distinctive steps in spermatogenesis involving either biological processes unique to spermatogenesis, notably meiosis (two-stage reductive division of diploid germinal cells into haploid gametes) and spermiogenesis (metamorphosis of haploid round spermatids into spermatozoa) or inhibiting functions essential to sperm fertilizing ability such as flagellar motility and sperm motion (including hyperactivation (198)), excurrent ductular transport, acrosome reaction (199), chemoattraction to and fertilization of oocytes (194, 200), acrosomal function (201)and testis-specific serine/threonine kinases involved in development of normal sperm morphology and function (202, 203). The most frequent mechanisms involves inhibition of ion channels in sperm (200, 204-211) or vas deferens smooth muscle (194, 195) leading to inhibition of sperm motility, hyperactivation and/or transport required for fertilization. One leading candidate is CatSper, the principal calcium channel activated by progesterone (212-216) and essential for the sperm hyperactivation required for fertilization (217). Experimental vaccines against CatSper epitopes inhibit murine fertility (218, 219).

In addition, drug screening programs using extensive high-throughput chemical libraries can be employed to identify suitable lead compounds using selective sperm function targets for further development including target optimization (220). For example, the inhibitor analogs of CatSper using patch clamping to identify channel blockade have identified probes suitable for drug discovery development (221). Similarly, screening of a chemical library identified phosducin-like 2, a testis specific phosphoprotein, which when knocked-out in mice, induced sterility due to globozoospermia with impaired sperm head formation (222) as well as screening of natural plant (223-225) and microbial (226) products continue as a source for lead compounds continues. Other products of drug screening that may interfere with DNA replication by targeting meiosis, incur additional theoretical safety risks of mutagenesis if intended for male contraception (227).

HORMONAL METHODS

Hormonal methods are the closest to meeting the requirement for a reliable, reversible, safe, and acceptable male contraceptive. Although reliability is judged by the efficacy in preventing pregnancy in fertile female partners, as a hormonal male contraceptive aims to prevent pregnancy by reversible inhibition of sperm output, the suppression of spermatogenesis constitutes a useful surrogate marker for development and evaluation of prototype male contraceptive regimens. This makes defining the degree of suppression of sperm output required a key strategic issue in developing a hormonal male contraceptive (228).

Two landmark WHO studies, the first ever male contraceptive efficacy studies, involving 671 men from 16 centers in 10 countries established the proof of principle that hormonally-induced azoospermia provides highly reliable, reversible contraception (14, 15). Among the minority (~25%) of men who remained severe oligozoospermic (0.1-3 million sperm/ml) using weekly testosterone enanthate injections, contraceptive failure rate (~8% per annum) was directly proportional to their sperm output. Hence to achieve highly effective contraception, azoospermia is analogous to anovulation as a sufficient, but not necessary, requirement. Nevertheless, reliable contraception by modern standards (29) requires uniform azoospermia as the desirable target for male contraceptive regimens (229). No regimen yet achieves this consistently in all men, although in some Asian countries (e.g. China (15), Indonesia (230, 231)) an approximation to uniform azoospermia can be achieved by a variety of regimens. A study involving 308 Chinese men in 6 centers has shown that monthly injections of testosterone undecanoate provides highly effective and reversible contraception (17). No pregnancies were recorded among men who were azoospermic or severely oligozoospermic (≤3 million sperm per mL) providing a 95% upper confidence limit of pregnancy (contraceptive failure) rate of 2.5% per annum. The overall failure rate based on suppression of spermatogenesis was <4%. The prototype regimen was well tolerated apart from injection site discomfort due to large oil injection volume (4 mL) and reversible androgenic effects (acne, weight gain, hemoglobin, lipids). Nevertheless, despite these promising findings, non-Chinese men require combination hormonal regimens involving a 2^nd gonadotropin suppressing agent, notably progestins, together with testosterone to ensure uniformly adequate spermatogenic suppression. Proof-of-principle for this combination approach was provided by a depot androgen/progestin regimen that observed no pregnancies among 55 couples during 35.5 person-years of exposure (95% upper limit of failure rate ~8%) in a study with satisfactory tolerability and reversibility for a prototype regimen (18). Hence, contraceptive efficacy studies show that highly effective contraception can be achieved with suppression of sperm output to near azoospermia (≤1 million per mL) (229). Ultimately, the efficacy of male contraception must be established by enumerating pregnancies prevented, and not counting sperm. The present paucity of male contraceptive efficacy studies, for which placebo controls are ethically impossible (232), makes systematic evaluations comparing practical clinical regimens a task for the future (233).

The reversibility of hormonal male contraceptive regimens is clearly established by an integrated re-analysis that pooled primary data from over 90% of all hormonal male contraceptive studies reported to show that all regimens show full reversibility within a predictable time course (234). This comprehensive review of the recovery of 1549 healthy eugonadal men, aged 18-51 years, who underwent 1283.5 man-years of treatment and 705 man-years of post-treatment recovery, showed the median times for recover to sperm densities of 10 and 20 million per mL were 2.5 months (2.4-2.7) and 3.0 months (2.9-3.1), respectively. Covariables such as age, ethnicity and hormonal or sperm output kinetics had significant but minor influence on the rate, but not the extent, of recovery.

Acceptability of a hormonal male contraceptive is high across a wide range of countries and cultures in potential male as well as female users (12). Willingness to use a hypothetical hormonal male contraceptive averaged 55% (range 29-71%) in an extensive, population representative survey of 9342 men aged 18-50 yr from 9 countries (4 Europe, 3 South America, Indonesia and USA) with consistency across a wide range of socio-economic and cultural settings (235, 236). Similar findings are reported in a 4-country study (UK, South Africa, Hong Kong, Shanghai) with 44-83% in each center (237) as well as 75% in Australia willing to try a hormonal male contraceptive (238). A majority of men in a US study were satisfied with and would recommend a transdermal gel-based hormonal contraceptive (239) and a majority of Chinese men were satisfied with monthly injections (240). Female partners from a variety of cultures also indicate high acceptability in a survey of 1894 women in 4 countries, among whom 40-78% would support and trust their male partners in stable relationships to use a hormonal male contraceptive (241). Corroborating the acceptability of hormonal male contraception are findings from experimental studies of prototype regimens for up to 12 months usage in which most participants confirm high levels of satisfaction and willingness to try a commercial product (239, 240, 242, 243).

Modern safety evaluation for hormonal contraceptive methods requires long-term, large scale studies of marketed products (244). Consequently, in the absence of any marketed male contraceptive, no long-term safety profile can be discerned. Nevertheless, 4 decades of clinical trials have consistently identified mainly minor adverse effects in short- or medium-term studies of prototype male hormonal contraception regimens (1, 8, 228). This was verified in an unique, placebo-controlled study of a combined androgen-progestin regimen which again proved highly effective at reversible suppression of sperm output although the inclusion of a placebo arm rendered it ethically impossible to evaluate contraceptive efficacy but provided insight into drug-related side effects (11). The study confirmed the benign medium-term safety profile for this prototype regimen in observing few serious adverse effects, none attributable to the steroid regimen, and, among the non-serious adverse effects reported, expected androgenic effects (acne, weight gain, sweating, changes in mood or libido) were more frequent than placebo but mild and rarely led to discontinuation (11). Nevertheless, the long-term safety evaluation of novel contraceptives requires large scale observational studies of extensively used drugs to define low frequency risks but can only occur after the marketing of suitable products.

Hence, prototype hormonal methods have proven reliability and reversibility and reasonable prospects for being well accepted and safe. Although they are the most likely opportunity in the foreseeable future to develop a practical contraceptive method for men, progress depends on pharmaceutical industry development. However, leadership in male contraceptive development has come almost exclusively from university-based investigators working with public sector organizations (8), notably WHO (245), CONRAD (246), and the Population Council (247). By contrast, commitment from pharmaceutical companies, including those that flourished in the post-war decades through developing female hormonal contraception, continued to languish over recent decades (24, 248) and is effectively ceased (249).

Steroidal Methods

ANDROGEN ALONE

Testosterone provides both gonadotropin suppression and androgen replacement making it an obvious first choice as a single agent for a reversible hormonal male contraceptive. Although androgen-induced, reversible suppression of human spermatogenesis has long been known (250-253), systematic studies of androgens for male contraception began in the 1970's (254, 255). Feasibility and dose-finding studies (256), mostly using testosterone enanthate (TE) in an oil vehicle as a prototype, showed that weekly im injections of 100-200 mg TE induce azoospermia in most Caucasian men (257)but less frequent or lower doses fail to sustain suppression (258-261). The largest experience with an androgen alone regimen arises from the two WHO studies in which over 670 men from 16 centers in 10 countries received weekly injections of 200 mg TE. In these studies ~60% of non-Chinese and >90% of Chinese men became azoospermic and the remainder were severely oligozoospermic (14, 15) (Figure 1). The high efficacy among Chinese men has also been replicated using monthly TU injections (16, 17). Effective gonadotropin suppression is a prerequisite for effective testosterone-induced spermatogenic suppression in human (18, 262-266) and non-human primates (267, 268). However, the reasons for within and between population differences in susceptibility to hormonally-induced azoospermia remain largely unexplained (269). Possible factors include population differences in reproductive physiology of environmental (270, 271), genetic (272-274) or uncertain (275, 276) origin that may lead to differences in rate and extent of suppression of circulating gonadotropins and/or depletion of intratesticular androgens. Limited invasive studies measuring intratesticular testosterone (and DHT) suggest that the degree of depletion may not predict reliably complete suppression of sperm output (277-279) but other more widely applicable, non-invasive markers of endogenous Leydig cell function such as circulating epitestosterone (262) or 17-hydroxyprogesterone (280) or non-steroidal testicular products such as INSL3 (281) may be more analytically informative as to the relative roles of gonadotropin suppression and intratesticular androgen depletion. Exogenous testosterone causes suppression of sperm output with an average of 13 weeks to reach severe oligozoospermia (<1 million per mL) or azoospermia with suppression being maintained consistently during ongoing treatment (282) (Figure 2). Following cessation of treatment, sperm reappear within 3 months to reach sperm densities of 10 and 20 million per mL at an average of 11.5 and 13.6 weeks, respectively (282)with ultimately full recovery (10) (Figure 3). Apart from intolerance of weekly injections, there were few discontinuations due to acne, weight gain, polycythemia, or behavioral effects and these were reversible as were changes in hemoglobin, testis size, and plasma urea. There was no evidence of liver, prostate, or cardiovascular disorders (14, 15, 283).

Figure 1. Pooled summary of contraceptive efficacy from two WHO male contraceptive efficacy studies (14, 15) where contraceptive failure rate (pregnancy rate) is plotted against the current sperm concentration in the ejaculate. This illustrates a summation of all data pooled from both studies. Data comprise monthly observations of the mean sperm concentration (averaging monthly sperm counts) and whether a pregnancy occurred in that month or not. Pregnancy rate (per 100 person-years, Pearl index) on the y-axis is plotted against the cumulative sperm density (in million sperm per ml) indicating that contraceptive failure rates are proportional to sperm output. The inset is the same data re-plotted in discrete sperm concentration bands rather than cumulatively. For comparison, the average contraceptive failure rates in the first year of use (33, 407) of modern reliable contraceptive methods are indicated by diamond symbols.

Figure 2. Plot of suppression (left panel) and recovery (right panel) of sperm output from the WHO Studies 85921 (15) and 89903 (14) involved pooling ~14,000 semen samples from 16 centers in 10 countries (282). Data-points represent mean and SE (error bar) of semen samples grouped within weeks with between six and 383 samples per time-point. Note cube-root scale on y-axis. For suppression, the smooth line is the two-parameter, single term exponential decay function plotted to fit the data by non-linear regression. For recovery, the smooth line is the three-parameter sigmoidal curve plotted to fit the data by non-linear regression.

Figure 3. Recovery of spermatogenesis after cessation of treatment with hormonal male contraceptive regimens modified after an integrated re-analysis of over 90% of all reported studies (234). Data is plotted as a Kaplan-Meier survival plot of the increasing proportion of men recovering to various thresholds over time since last treatment. The data of last treatment is defined as the time elapsed from the end of the last treatment cycle, that is the latest date of the first missed treatment dose. The thresholds are a sperm concentration of 3, 10 or 20 million sperm per mL in the ejaculate or a return to their own pre-treatment baseline sperm concentration. The median time to achieving each threshold is tabulated together with its 95% confidence interval.

The pharmacokinetics of testosterone products are crucial for suppressing sperm output. Oral androgens have major first-pass hepatic effects producing prominent route-dependent effects on hepatic protein secretion (e.g., SHBG, HDL cholesterol) and inconsistent bioavailability. Short-acting testosterone products requiring daily or more frequent administration (oral, transdermal patches or gels) which may be acceptable for androgen replacement therapy present serious feasibility challenges in compliance for effective long-term hormonal contraception. Weekly TE injections required for maximal suppression of spermatogenesis (256) are far from ideal (284) and cause supraphysiological blood testosterone levels risking both excessive androgenic side effects and preventing maximal depletion of intratesticular testosterone for optimal efficacy (285, 286). Other currently available oil-based testosterone esters (cypionate, cyclohexane-carboxylate, propionate) do not differ from the enanthate ester (287), but longer-acting depot preparations such as injectable testosterone undecanoate are a substantial improvement. Subdermal testosterone pellets sustain physiological testosterone levels for 4-6 months (288) and the newer injectable preparations testosterone undecanoate (17), testosterone-loaded biodegradable microspheres (289) and testosterone buciclate (290) provide 2-3 months duration of action. Depot androgens suppress spermatogenesis faster, at lower doses and with fewer metabolic side effects than TE injections but azoospermia is still not achieved uniformly (291) although when combined with a depot progestin, this goal is achievable (18).

Oral synthetic 17-a alkylated androgens such as methyltestosterone (292), fluoxymesterone (293), methandienone (294)and danazol (295, 296) suppress spermatogenesis but azoospermia is rarely achieved and the inherent hepatotoxicity of the 17-a alkyl substitutent (297, 298) renders them unsuitable for long-term use. Athletes self-administering supratherapeutic doses of androgens also exhibit suppression of spermatogenesis (294, 299). Synthetic androgens lacking the 17-a alkyl substituent have been little studied although injectable nandrolone esters produce azoospermia in 88% of European men (300, 301) whereas oral mesterolone is ineffective (302). On the other hand, nandrolone hexyloxyphenylpropionate alone was unable to maintain spermatogenic suppression induced by a GnRH antagonist (303) in a prototype hybrid regime (where induction and maintenance treatment differ) whereas testosterone appears more promising (304). A 7-methyl derivative of nandrolone (MeNT), which is partly aromatisable but resistant to 5α-reductive amplification of androgenic potency, has been studied as a non-oral androgen for hormonal male contraceptive regimens (305). While its non-amplification by 5α reduction may be theoretically prostate-sparing (306), dose titration to achieve essential androgen replacement at each relevant tissue might be more difficult to achieve than for testosterone (307) or impossible. Non-aromatisable androgens which lack estrogen- receptor-mediated effects such as on bone density maintenance (308) and sexual function (309, 310) may not be ideal for long-term hormonal contraception. More potent, synthetic androgens lacking 17-a alkyl groups (311, 312) remain to be evaluated.

Adverse effects due to testosterone administration in prototype hormonal contraceptive regimens include (asymptomatic) polycythemia, weight (muscle) gain, and acne as well as changes in mood or sexual behavior. These are usually minor in severity, reversible upon cessation of treatment and of minimal clinical significance (11).

The safety of androgen administration concerns mainly potential effects on cardiovascular and prostatic disease As the explanation for the higher male susceptibility to cardiovascular disease is not well understood, the risks of exogenous androgens are not clear (313, 314). In clinical trials, lipid changes are minimal with depot (non-oral) hormonal regimens (18, 291, 315, 316). Changes in blood cholesterol fractions observed during high hepatic exposure to testosterone and/or progestins, due to either oral first pass effects or high parenteral doses, have unknown clinical significance but, in any case, maintenance of physiological blood testosterone concentrations is the prudent and preferred objective. The real cardiovascular risks or benefits of hormonal male contraception will require long-term surveillance of cardiovascular outcomes (317).

The long-term effects of exogenous androgens on the prostate also require monitoring since prostatic diseases are both age and androgen dependent. Exposure to adult testosterone levels is required for prostate development and disease (318-320). The precise relationship of androgens to prostatic disease and in particular any influence of exogenous androgens remains poorly understood. Ambient blood testosterone or DHT levels do not predict development of prostatic cancer over future decades in prospective studies of adults (321). A genetic polymorphism, the CAG (polyglutamine) triplet repeat in exon 1 of the androgen receptor, is an important determinant of prostate sensitivity to circulating testosterone with short repeat lengths leading to increased androgen sensitivity (322), however the relationship of the CAG triplet repeat length polymorphism to late-life prostate diseases remains unclear (323). Among androgen deficient men, prostate size and PSA concentrations are reduced and returned towards normal by testosterone replacement without exceeding age-matched eugonadal controls (322, 324-326). In healthy middle-aged men without known prostate disease, very high doses of the potent natural androgen DHT for 2 years did not increase prostate size or age-related growth rate compared with placebo indicating that effects of even high dose exogenous androgen treatment has much less effect than age on the human prostate (308). Similarly, self-administration of massive androgen over-dosage does not increase total prostate volume or PSA in anabolic steroid abusers although central prostate zone volumes increases (327). In-situ prostate cancer is common in all populations of older men whereas rates of invasive prostate cancer differ many-fold between populations despite similar blood testosterone concentrations. This suggests that early and prolonged exposure to androgens may initiate in-situ prostate cancer, but later androgen-independent environmental factors promote the outbreak of invasive prostate cancer. Therefore, it is prudent to maintain physiological androgen levels with exogenous testosterone, which then might be no more hazardous than exposure to endogenous testosterone. Prolonged surveillance comparable with that for cardiovascular and breast disease in users of female hormonal contraception would be equally essential to monitor both cardiovascular and prostatic disease risk in men receiving exogenous androgens for hormonal contraception.

Extensive experience with testosterone in doses equivalent to replacement therapy in normal men indicates minimal effects on mood or behavior (14, 15, 256, 328-330). A careful placebo-controlled, cross-over study showed that a 1000 mg TU injection in healthy young men produces minor mood changes without any detectable increase in self or partner-reported aggressive, non-aggressive or sexual behaviors (331). By contrast, extreme androgen doses used experimentally in healthy men can produce idiosyncratic hypomanic reactions in a minority (332). Aberrant behavior in observational studies of androgen-abusing athletes or prisoners are difficult to interpret particularly to distinguished genuine androgen effects from the influence of self-selection for underlying psychological morbidity (333).

ANTI-ANDROGENS

Antiandrogens have been used to selectively inhibit epididymal and testicular effects of testosterone without impeding systemic androgenic effects (334). Cyproterone acetate, a steroidal antiandrogen with progestational activity, suppresses gonadotropin secretion without achieving azoospermia but leads to androgen deficiency when used alone (335). In contrast, pure non-steroidal antiandrogens lacking androgenic or gestagenic effects such as flutamide, nilutamide and casodex fail to suppress spermatogenesis when used alone (336, 337). Two studies evaluating the hypothesis that incomplete suppression of spermatogenesis is due to persistence of testicular DHT have reported no additional suppression from administration of finasteride, a type II 5a reductase inhibitor (338, 339); however as testes express predominantly the type I isoforms (340), further studies are required to conclusively test this hypothesis using an inhibitor of type I 5-a reductase (341).

ANDROGEN COMBINATION REGIMENS

Combination steroid regimens use non-androgenic steroids (estrogens, progestins) to suppress gonadotropins, in conjunction with testosterone for androgen replacement, have shown the most promising efficacy with enhanced rate and extent of spermatogenic suppression compared with androgen alone regimens (315, 342, 343). Synergistic combinations reduce the effective dose of each steroid and minimizing testosterone dosage could enhance spermatogenic suppression if high blood testosterone levels counteract the necessary maximal depletion of intratesticular testosterone (344, 345) as well as reducing androgenic side-effects (Figure 4).

Figure 4. Plot of time course of sperm output (expressed as sperm density in millions of sperm per mL) before and after implantation of two 200-mg testosterone pellets (400 mg total; closed circles), four 200-mg testosterone pellets (800 mg total; closed squares), four testosterone pellets plus depot progestin (800 mg total testosterone plus 300 mg DMPA; closed diamonds) and six 200 mg testosterone pellets mg (1200 mg total; open hexagons) in healthy fertile men (262). Results expressed as the mean and SEM. Note the cube root transformed scale on the y-axis.

Progesterone is a key precursor and steroidogenic intermediate for all bioactive natural steroids and the progesterone receptors A and B are structurally and evolutionarily the closest members of the nuclear receptor superfamily to the androgen receptor. Yet, although progesterone has crucial gestational and lactational roles in female reproductive physiology, it has no well-established role in male reproductive physiology apart from a possible role in sperm function (200, 346), possibly via non-genomic rather than a classically genomic mechanism (347). Nevertheless functional nuclear progesterone receptors are expressed in male brain, smooth muscle and reproductive, but not most non-reproductive, tissues (348). Synthetic progestins, steroidal structural agonistic analogs of progesterone, are potent inhibitors of pituitary gonadotropin secretion used widely for female contraception and hormonal treatment of disorders such as endometriosis, uterine myoma and mastalgia. Used alone, progestins suppress spermatogenesis but cause androgen deficiency including impotence (349, 350) so androgen replacement is necessary. Non-human primate studies indicate that this is mediated via a central hypothalamic-pituitary site of action rather than direct effects on the testis (351). Extensive feasibility studies concluded that progestin-androgen combination regimens had promise as hormonal male contraceptives if more potent and durable agents were developed (256, 352). The largest and most detailed multi-center study of the contraceptive efficacy of a androgen-progestin combination involving 320 healthy men aged 18-45 years together with their 18-38 year old female partners, both without known fertility problems, studied for up to 56 weeks while having intramuscular injections of 200 mg norethisterone acetate and testosterone undecanoate every 8 weeks that produced near-complete suppression (96% declined to <1 million/ml by 24 weeks) and reversible recovery (95% recovered by 52 weeks) of sperm output (353). Four pregnancies occurred in partners of 266 men with a pregnancy failure rate of 1.6% (95% confidence interval 0.6-4.1%). Prior information on androgen/progestin regimens derives from studies with medroxyprogesterone acetate (MPA) combined with testosterone. Monthly injections of both agents or daily oral progestin with dermal androgen gels produce azoospermia in ~60% of fertile men of European background with the remainder having severe oligozoospermia and impaired sperm function (256, 352, 354). Nearly uniform azoospermia is produced in men treated with depot MPA and either of two injectable androgens in Indonesian men (230, 231) or testosterone depot implants in Caucasian men (315). Smaller studies with other oral progestins such as levonorgestrel (342, 355, 356) and norethisterone (357, 358) combined with testosterone demonstrate similar efficacy to oral MPA whereas cyproterone acetate with its additional anti-androgenic activity has higher efficacy in conjunction with TE (343, 359) but not oral testosterone undecanoate (360). Highly effective suppression of spermatogenesis is reported with depot progestins in the form of non-biodegradable implants of norgestrel (361-363) or etonorgestrel (364, 365) or depot injectable medroxyprogesterone acetate (18, 315, 366, 367) or norethisterone enanthate (368, 369)coupled with testosterone (370). A comparative study showed that all four progestins (cyproterone acetate, levonorgestrel, norethisterone acetate and nesterone) displayed significant short-term gonadotrophin suppression when combined with testosterone (371). The pharmacokinetics of the testosterone preparation is critical to efficacy of spermatogenic suppression with long-acting depots being most effective while transdermal delivery is less effective than injectable testosterone (361). Progestin side-effects are few if sexual function and well-being are maintained by adequate doses of testosterone replacement. The metabolic effects depend on specific regimen with oral administration and higher testosterone doses exhibiting more prominent hepatic effects such as lowering SHBG and HDL cholesterol. After treatment ceases with depletion or withdrawal of hormonal depots, spermatogenesis recovers completely but gradually consistent with the time-course of the spermatogenic cycle (234, 282). Steroids with dual androgen and progestin properties may be potentially well suited to male contraception if the balance between these two bioactivities is properly balanced (372). A study comparing a gel combining nesterone (8.3 mg) with testosterone (62.5 mg) with the same dose of testosterone alone for daily transdermal application for 28 days demonstrated suppression of sperm output and serum gonadotropin to levels consistent with contraceptive efficacy and superior to testosterone alone indicating the suitability of the combination gel for further product development (373). Two orally active nandrolone derivatives, dimethandrolone (374-376) and 11-methyl nandrolone dodecylcarbonate (377), are undergoing promising, early stage clinical development showing high acceptability in a short-term, placebo-controlled clinical trials (378, 379). Whether such daily use oral or transdermal gel (239, 380)-based male contraceptives will have sufficient user compliance to attain high contraceptive efficacy remains to be evaluated.

Estradiol augments testosterone-induced suppression of primate spermatogenesis (381) and fertility (382) but estrogenic side-effects (gynecomastia) and modest efficacy at tolerable doses make estradiol-based combinations impractical for male contraception (383). The efficacy and tolerability of newer estrogen analogs in combination with testosterone remain to be evaluated.

GONADOTROPIN-RELEASING HORMONE BLOCKADE

The pivotal role of GnRH in the hormonal control of testicular function makes it an attractive target for biochemical regulation of male fertility. Blockade of GnRH action by GnRH receptor blockade with synthetic analogs or GnRH immunoneutralization would eliminate LH and testosterone secretion requiring testosterone replacement. Many superactive GnRH agonists are used to induce reversible medical castration for androgen-dependent prostate cancer by causing a sustained, paradoxical inhibition of gonadotropin and testosterone secretion and spermatogenesis due to pituitary GnRH receptor downregulation. When combined with testosterone, GnRH agonists suppress spermatogenesis but rarely achieve azoospermia (344, 345, 384) being less effective than androgen/progestin regimens. By contrast, pure GnRH antagonists create and sustain immediate competitive blockade of GnRH receptors (385, 386) and, in combination with testosterone, are highly effective at suppressing spermatogenesis. Early hydrophobic GnRH antagonists were difficult to formulate and irritating, causing injection site mast cell histamine release. Newer more potent but less irritating GnRH antagonists produce rapid, reversible and complete inhibition of spermatogenesis in monkeys (387-389) and men (390, 391) when combined with testosterone. The striking superiority of GnRH antagonists over agonists may be due to more effective and immediate inhibition of gonadotropin secretion and thereby more effective depletion of intratesticular testosterone. Due to their highly specific site of action, GnRH analogs have few unexpected side-effects. Depot GnRH antagonist plus testosterone formulations suitable for administration at up to 3-month intervals could be promising as a hormonal male contraceptive regimen. Whether GnRH antagonists are more cost-effective than progestins as the second, non-androgenic component of combination male hormonal contraceptive regimens remains to be established (278, 303, 367, 392). The drawback of higher cost might be overcome by hybrid regimens using GnRH antagonists to initiate suppression followed by a switch to more economical steroids for maintenance of spermatogenic suppression in humans (304). A GnRH vaccine could intercept GnRH in the pituitary-portal bloodstream preventing its reaching pituitary GnRH receptors. A limitation of a GnRH vaccine is its uncertain and/or unpredictable reversibility. If it is not irreversible, the offset of contraceptive efficacy may vary between individuals and create practical difficulties in ensuring reliable contraception. Gonadotropin-selective immunocastration would require androgen replacement in men (393) and pilot feasibility studies in advanced prostate cancer are underway (394)but the prospects for acceptably safe application for male contraception remain doubtful (395). By contrast there are growing applications for anti-hormonal contraceptive vaccines in control of companion (pet), agricultural, zoo, feral and wild animal populations (156, 396).

FOLLICLE STIMULATING HORMONE BLOCKADE

Selective FSH blockade theoretically offers the opportunity to reduce spermatogenesis without inhibiting endogenous testosterone secretion. FSH action could be abolished by selective inhibition of pituitary FSH secretion with inhibin (397)or novel steroids (398), by FSH vaccine (399, 400) or by FSH receptor blockade with peptide antagonists (401). Although FSH was considered essential to human spermatogenesis, spermatogenesis and fertility persist in rodents (402-404) and humans (405) lacking FSH bioactivity. Even complete FSH blockade alone might produce insufficient reduction in sperm output and function required for adequate contraceptive efficacy (406). In addition to the usual safety concerns of contraceptive vaccines including autoimmune hypophysitis, orchitis or immune-complex disease, an FSH vaccine might be overcome by reflex increases in pituitary FSH secretion. Hence, FSH suppression is a necessary but not sufficient for a hormonal male contraceptive regimen.

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Non-Exercise Activity Thermogenesis in Human Energy Homeostasis

ABSTRACT

Low levels of physical activity combined with food intake in excess of daily energy expenditure over extended time periods precede weight gain and promote increases in body fat. Obesity and related insulin resistance are common sequelae of a chronically positive energy balance, potentially resulting in type 2 diabetes (T2D) and nonalcoholic/metabolic dysfunction associated fatty liver disease (NAFLD/MAFLD). The percentage of individuals considered as obese and morbidly obese is continuously rising and developing countries are catching up quickly as compared to industrialized nations. If the observed trend continues, global obesity prevalence will prospectively reach more than 21% in women and 18% in men by 2025. In addition to poor dietary habits, physical activity levels have decreased in recent decades in parallel with an increase in sedentary behavior. Given the technological advances in domestic, community, and working spaces in the last century it is not uncommon for people in industrialized countries to spend one half of their day sitting. As well, for a majority of people, voluntary physical exercise remains of minor importance. Non-Exercise Activity Thermogenesis (NEAT) refers to that portion of daily energy expenditure resulting from spontaneous physical activity that is not specially the result of voluntary exercise. Levels of NEAT ranges widely, with variance of up to 2000 kilocalories per day between two individuals of similar size. These differences are related to complex interactions of environmental and biological factors, including people’s differing occupations, leisure-time activities, individual molecular and genetic factors, and evidence that food intake has independent effects on spontaneous physical activity. Available data support the hypothesis that targeting NEAT could be an essential tool for body weight control. This comprehensive review systematically describes the definition, evaluation methods, and environmental and biological factors involved in the regulation of NEAT. It further emphasizes the association with obesity and related disorders and suggests practical relevant implications, but also potential limitations for the integration of NEAT in daily life.

INTRODUCTION

The prevalence of obesity and resultant adverse health implications continues to rise in both sexes in the United States and worldwide (1). In 2011 Finucane et al. estimated international trends of mean, age-standardized, body mass index (BMI) and suggested that an estimated 1.46 billion adults worldwide had BMI of 25 kg/m² or greater, of these 205 million men and 297 million women were considered obese (2). Indeed, between 1980 and 2008 the prevalence of adult obesity (BMI ≥ 30 kg/m²) has risen in every sub-region of the world except for Central Africa and South Asia (2). The mean BMI increase was reported to be 0.4 kg/m² per decade for men and 0.5 kg/m² for women (2). Comparable data have been presented by the NCD Risk Factor Collaboration Trial (3). With a focus on industrialized countries, the most prominent increases of mean BMI have been observed in the United States, followed by the United Kingdom and Australia. The United States had the highest BMI of all studied high-income countries, recent data from the U.S. National Health and Nutrition Examination Survey indicate that obesity affects approximately 35% of the male and 40% of the female population (1, 4, 5). Similarly, populations of European and Eastern Mediterranean countries show an overweight prevalence of up to 50% (6). Further data indicate that compared to 1975, childhood obesity is much more common in 2016, with one obesogenic key driver in this group representing reduced physical activity (3, 7). This continuous rise in the incidence and prevalence of overweight and obesity in children, adolescents, and adults in the past century has created major burdens for national health care systems worldwide.

Obesity-related diseases are typically of chronic nature and include type 2 diabetes mellitus (T2D), nonalcoholic/metabolic dysfunction associated fatty liver disease (NAFLD/MAFLD), peripheral artery and cardiovascular disease, sleep apnea, hypertension, as well as various cancers. In the United States, seven out of the leading ten causes of premature death and disability are represented by chronic diseases related to obesity (8). In fact, obesity is one of the most important avoidable risk factors for significant morbidity and premature mortality, leads to impaired quality of life, can elevate disability rates, and statistically reduces expected life span by seven years (9, 8, 10, 11).

The etiology of obesity is multifactorial, resulting from genetic and epigenetic, physiological, behavioral, sociocultural, and environmental factors leading to an imbalance between energy intake and expenditure (12). To this end excess body fat consistently results from a period of sustained net-positive energy balance, followed by homeostatically regulated weight maintenance. The roles of diet and caloric intake in these processes have been the principal focus of most mechanistic studies. In countries with high obesity prevalence rates, nutritional quality is mostly poor, but there is a controversy as to whether increased energy intake alone can explain the obesity epidemic. Trends for improved dietary quality and reduced energy intake have been observed in recent decades in the United Kingdom and among parts of the U.S.-population (13–15). For instance, since the 1980s obesity prevalence has substantially risen in Great Britain, while caloric intake appeared to decline on a population level (12). In order to explain such findings it was hypothesized that if physical activity decreases, body weight may increase if energy intake is not altered (16). Therefore, under conditions of unchanged caloric intake, the amount of daily physical activity emerged as major predictor (1, 17). Accordingly, the National Weight Control Registry of the United States has identified strategies for maintaining weight loss, which include engagement in higher levels of physical activity (1). Therefore, greater attention has been paid to addressing the energy expenditure side of the caloric balance equation, in particular with regards to prevention of body weight regain after successful weight loss.

NON-EXERCISE ACTIVITY THERMOGENESIS: A SIGNIFICANT COMPONENT OF DAILY ENERGY EXPENDITURE

Daily total energy expenditure (TEE) is the net amount of energy utilized by animals and humans to maintain core physiological functions and locomotion. Three main components of energy balance determine TEE: Basal metabolic rate (BMR), the thermic effect of food (TEF), also called diet-induced thermogenesis, and the energy expended for physical activity (18). Additional components may exist (e.g. energy costs of emotion), but apparently play a minor role with respect to energy balance (19–22). Multiple factors significantly affect our daily energy needs, including age, body composition, thyroid hormone status, catecholamine levels/sympathoadrenergic activity, ambient and body temperature, disease states, and certain medications (19, 18).

Basal Metabolic Rate

Basal metabolic rate (BMR) represents the minimal amount of energy expended to maintain all the body’s vital processes at rest (the basal state). BMR is essentially a function of lean body mass (LBM), which is estimated to account for 80% of BMR within and across species (23–29) . BMR is sometimes misinterpreted to imply the lowest level of energy expenditure during the day. This is, however, not true, since during sleep or undernutrition metabolic functions may be lower than that observed under basal conditions (27). In free-living individuals, BMR accounts for the main percentage of TEE (Figure 1), in healthy subjects with mainly sedentary occupations it predicts around 60% of the variance (22). Otherwise it is proportionally larger in individuals with minimal physical activity (e.g., in sedated and ventilated patients on an intensive care unit) (30).

Of note, it is not uncommon that the term resting energy expenditure (REE) is synonymously interchanged with BMR. However, BMR is measured in completely rested subjects, both before and during the measurements. Therefore, measurements are classically taken in the morning after 8 hours of sleep. Subjects should be lying supine, fully awake, and be fasted for at least 10-12 hours. The environment in which the measurements are taken should be thermo-neutral (22–26 °C) so that no thermoregulatory effect on heat production biases the results (19); and subjects should be free from emotional stress and familiar with the measurement apparatus (27). By contrast, REE is equivalent to postabsorptive energy expenditure at complete rest at any time of the day and can vary as much as 10% from BMR (21).

Some studies suggest BMR is reduced in those prone to become obese (31–33). It is, however, a matter of controversy as to whether a reduction of energy expenditure due to a decrease in BMR sufficiently explains the positive net energy balance that contributes to excess weight gain in the majority of subjects who become obese (34–36). On the other hand, LBM can be enlarged or even preserved by means of regular physical activity, which in turn can favorably modulate BMR (1, 37–39).

Thermic Effect of Food

Thermic effect of food (TEF) is the increment of energy expenditure above REE following meal ingestion that reflects the energy cost (burned) during food digestion, absorption, and storage. It is a relatively stable component of total energy expenditure (Figure 1). Thermic effect of food typically ranges between 8-15% of TEE and the variance of TEF has been associated with nutrient composition and energy content of consumed foods (40).

Physical Activity: EAT and NEAT

Physical activity is the second most significant contributor to TEE in most people (Figure 1). It is defined as the additional energy expenditure above REE and TEF that is required for performing daily activities and therefore also called physical activity-related or simply activity-related energy expenditure (PEE/AEE). PEE/AEE can be categorized into exercise-related activity thermogenesis (EAT) and non-exercise activity thermogenesis (NEAT). Both vary widely within and between individuals.

For the majority of subjects in industrialized countries, exercise is believed to be negligible (20). According to NHANES data, 36.1% of the studied US population was categorized as sedentary, while a further 47.6% were physically active at low levels (40, 41). Remarkably, only around 16% of subjects in NHANES met recommended guidelines for physical activity or were highly active. Even so, the latter subjects did not necessarily exercise (42, 43, 41). Thus, it is reasonable to conclude that on a population level the percentage of subjects engaging in regular, intense physical exercise is low. In those who habitually participate in purposeful physical training, EAT is believed to maximally account for 15-30% of TEE (18, 44). Other authors suggest that the majority of subjects undergoing regular physical training, defined as “bodily exertion for the sake of developing and maintaining physical fitness,” do not exercise more than two hours a week, accounting for an average energy expenditure of 100 kilocalories (kcal) per day(45). Such expenditure would contribute to only 1-2% to the variance of TEE. Taken together, for most human subjects EAT seems to not be a major contributor to TEE variance.

Figure 1. Model of human energy expenditure components (adapted from (46)). Exercise-related physical activity is comparable to exercise-related activity thermogenesis (EAT), while spontaneous physical activity is comparable to non-exercise activity thermogenesis (NEAT); for further explanations see text. Note that parts of spontaneous physical activity are beyond voluntary control, also called “fidgeting.” Arrows symbolize the multiple and varying impacts of individual factors, genetic background, and environment.

In contrast, NEAT comprises the largest share of daily activity-related thermogenesis, including for most subjects engaging in regular physical training. It is important to note that both NEAT and spontaneous physical activity are not interchangeable but represent complementary concepts (47): NEAT refers to energy expenditure, while spontaneous physical activity describes the types of bodily activity that are not defined as purposeful movements but still contribute to NEAT. Some authors categorize NEAT into three main subcomponents comprising body posture, ambulation, and all other spontaneous movements including “fidgeting” (48). Accordingly, a certain percentage of spontaneous physical activity (and therefore NEAT) is beyond voluntary control (e.g., “fidgeting”).

NEAT corresponds to all the energy expended with occupation, leisure time activity, sitting, standing, stair climbing, ambulation, toe-tapping, shoveling snow, playing the guitar, dancing, singing, cleaning, and more (20, 22). These activities do not characteristically involve moderate- to vigorous-intensity activities in comparable manner as voluntary exercise, but occur at a low energy workload for minutes and up to hours (12).

The importance of NEAT becomes apparent when considering the following points: The variability in BMR between individuals of similar age, BMI and of equal gender ranges around 7-9% (49), while the contribution of TEF is maximally 15%. Thus, BMR and TEF are relatively fixed in amount and account for approximately three quarters of daily TEE variance. As EAT is believed to be negligible on a population level, NEAT consequently represents the most variable component of TEE within and across subjects. It is responsible for 6-10% of TEE in individuals with a mainly sedentary lifestyle and for 50% or more in highly active subjects (47, 22, 29).

Taken together, with respect to body mass regulation and modification of energy balance, NEAT must be considered as a factor with potentially major impact. This is of particular interest in terms not only of developing therapeutic strategies for the patient with obesity, but also for designing obesity-prevention strategies for populations. The following sections will focus on these issues, including the environmental and biological modification of NEAT.

Measurement of NEAT

For a better understanding of the potential role of NEAT in the context of obesity it is essential to recognize the strengths and limitations of available techniques used for the quantification of NEAT.

NEAT can be principally measured by two approaches: 1) by assessing total NEAT, and 2) by using the factorial approach (for extensive reviews of available techniques see (50, 51)).

TOTAL NEAT

Assuming that EAT is negligible, total NEAT can be calculated by subtracting BMR and TEF from TEE (16):

Equation 1: NEAT = TEE – (BMR + TEF)

To complete equation 1, TEE, BMR and TEF need to be measured. Under free-living conditions TEE can be reliably measured by using the doubly-labeled water method (16). This approach requires the application of stable isotopes (deuterium/²H₂ and O¹⁸) containing water (²H₂O¹⁸). The difference in the clearance rate of the two isotopes equals carbon dioxide (CO₂) production generated during energy production and thus represents quantification of TEE (52)(Figure 2).

One necessary precondition of the doubly-labeled water method is that the O₂ of expired CO₂ is in equilibrium with the O₂ in body water. Once ingested, O¹⁸ will be readily distributed in the systemic H₂O, H₂CO₃ and CO₂pools. ²H₂will be dispensed in body water and the H₂CO₃ pool (equation 2).

Equation 2: CO₂ + H₂O ↔ H₂CO₃

The concentration of labeled O₂ in body water will then decrease over time due to a loss of CO₂ with expiration, perspiration, and in excreted body water (urine). ²H₂is mainly lost through excreted body fluids, yet a small percentage can be incorporated into body fat or protein. Since body water becomes tagged with known amounts of tracers at the same time at the beginning of the measurement period, the difference of elimination rates of both tracers equals CO₂ excretion.

Doubly labeled water is usually administered at baseline after samples of blood, urine and saliva have been collected (21). Subsequent sample collections occur within 7-21 days after administration, when the isotopes are completely dispersed within body compartments. Isotope-ratio mass spectroscopy is used to measure ²H₂ and O¹⁸enrichments in collected samples, which are needed to finally calculate CO₂ production. Thereby, TEE can be ascertained under free-living conditions with an error of about 6-8%. This error can be minimized by repeatedly gathering samples over the measurement period rather than relying on only two time points.

Figure 2. Schematic picture of the doubly labeled water method. CO2, carbon dioxide; 2H2, deuterium; O18, labeled oxygen.

It should be mentioned that the study of TEE classically relied on direct calorimetry (49, 18). Direct calorimetry, or direct measurement of heat loss from a subject, is achieved by using well-controlled environmental chambers. However, this requires participants to be confined to small rooms, which fails to capture the enormous variety of NEAT components in free-living individuals (21, 22).

By contrast, indirect calorimetry is an easily accessible and portable method commonly used to assess BMR, RMR and TEF. Indirect calorimetry is based on the principle that by measuring oxygen consumption (VO₂), CO₂excretion (VCO₂), or both over a defined time span, the metabolic conversion of fats, carbohydrates, and proteins into energy can be calculated using established formulae (18). The net energy released is typically expressed as kcal or Kilojoule (kJ). The small percentage of physiological protein oxidation can be estimated by nitrogen excretion in urine, or can be neglected without adding a substantial error in subjects who are in stable nitrogen balance (18).

Indirect calorimetry is accordingly one of the most commonly used approaches in clinical investigations, either in controlled metabolic-ward studies or in field settings. For instance, Levine et al. in their study on human NEAT applied indirect calorimetry to measure BMR and TEF (53). To measure TEF they provided participants with a meal containing a third of the subject’s daily weight maintenance energy needs. To estimate TEF over 24 hours, the measured energy expenditure area-under-the-curve obtained when they ate the standardize meal was multiplied by three (16). Alternatively, study protocols may ignore TEF as a non-substantial variable, or simply multiply TEE by 0.1 to yield a crude estimate.

When sensitive techniques are not available, BMR can be alternatively estimated by using validated age-, gender- and population-specific equations. For instance, the Harris-Benedict equation is widely used in clinical settings (18). However, this equation was derived from about 300 healthy, normal-weight Caucasian adults between 1907 and 1917 and its application, therefore, has limitations (18). Substantial error could be introduced by applying it to an inappropriate collective, including subjects who are with obesity, which is also true for other validated equations estimating BMR (18).

The physical activity level (PAL) is frequently used in studies to provide an index of activity when measurement of total NEAT is not possible. It is calculated by expressing TEE relative to BMR (15). Sedentary subjects in industrialized countries typically have a PAL of approximately 1.5 (see Table 1). The PAL rises to values of 2.0-2.4 with strenuous work and under certain conditions it can increase to values up to 3.5-4.5 (22). Using PAL, however, can also introduce significant bias in the assessment of NEAT, as under free-living conditions the cumulative error of PAL measurements can be as high as 7% (21).

Table 1. Physical Activity Levels (PAL) Predicted From Lifestyle (from 16).
Chair or bed bound	1.2
Seated work with no option of moving around and little or no strenuous leisure activity	1.4 – 1.5
Seated work with discretion and requirement to move around but little or no strenuous leisure activity	1.6 – 1.7
Standing work (e.g., homemaker, shop assistant)	1.8 – 1.9
Strenuous work or highly active leisure	2.0 – 2.4

THE FACTORIAL APPROACH OF NEAT MEASUREMENT

Measuring total NEAT or using PAL provides no information regarding the individual components contributing to PEE/AEE. Therefore, the factorial approach is widely used to estimate NEAT constituents. Accordingly, all physical activities of a subject of interest are recorded over a defined time span, typically seven days. The energy equivalent of each activity is determined, and these equivalents are then apportioned according to the time spent by the individual with the respective activities. Finally, the data is totaled to get an estimate of the energy expenditure attributable to NEAT (21, 22). The first step when using the factorial approach is the quantification of a subject’s physical activities. Several methods are available to obtain such information, including questionnaires, interviews, and activity diaries. These approaches might be useful for estimates of particular activities, such as those related to occupational duties. Otherwise, they have substantial limitations, including inadequate or incomplete data recording, alteration of habits during assessment periods, and others (21, 22).

Measuring NEAT is a complex task when intensities of transition movements and fidgeting-like activities are the matter of research interest. By applying a combination of direct calorimetry and motion detectors, Ravussins’s group have shown that there is a considerable inter-person variability of daily energy expenditure, even after adjustment for differences in LBM (29). A large percentage of this variability among individuals was due to variability in the degree of e.g., "fidgeting." Thus, estimation of fidgeting-like activities represents a substantial subcomponent under conditions of NEAT measurement. However, subcomponents such as “fidgeting” might not be captured by many methods. Levine et al. proposed an accelerometer method that could predict 86% of activity related energy expenditure for the posture and locomotion component of NEAT compared with a room calorimeter, but predicted only 50% of the variance in fidgeting and did not discriminate various types of fidgeting (50). Therefore, more technically advanced approaches have been developed. Among these are motion detectors, floor-pressure-pad displacement, cine photography, pedometers, accelerometers, and global positioning systems (GPS) (21, 22). Recently, portable intelligent devices for energy expenditure and activity assessment were used to study subtypes of NEAT by the method of mechanical modeling (54).

However, depending on the degree of sophistication, all these approaches have limitations. While data obtained from triaxial accelerometers typically found in wearable activity monitors is often used as an estimate of activity-related energy expenditure, they only measure actual movements (not energy expended), and a combination of methods probably yields the most appropriate estimates (21, 22). For example, energy costs of single NEAT components are typically measured by means of indirect calorimetry, for which highly sophisticated portable systems are now available. Alternatively, tables with listed energy costs of NEAT activities may be used. This latter method is convenient and inexpensive, but for similar reasons as pointed out for PAL, substantial systematic errors can be introduced (21). Similar problems will arise when NEAT is assessed by multiple linear regression approaches at a population level (55).

Furthermore, principal problems ascend with the NEAT measurement per se, as little validated information is available concerning the time necessary to representatively assess spontaneous physical activity. Many studies report extrapolation of measured results of an individual’s energy expenditure obtained during a limited timeframe to longer intervals. In this regard, experienced investigators believe that measurements of approximately seven days will likely provide a representative assessment regarding a 2-4 month time span (21).

Taken together, the difficulty of getting true estimates under free-living conditions is a major reason why available information on human NEAT physiology is limited. Representative studies are needed that are conducted over appropriate time spans (months) and utilize a combination of the factorial approach and total NEAT measurement to overcome major limitations in current estimates.

INDIVIDUAL AND ENVIRONMENTAL DETERMINANTS OF NEAT

There is a close interplay between environmental factors and individual characteristics with respect to NEAT as discussed below.

Effects of Occupation, Gender, and Age

Highly physically-active individuals expend up to three times more energy in 24-hours than subjects with negligible bodily activity (56). As previously discussed, NEAT accounts for a significant proportion of the observed differences at the population level TEE. Thereby, occupation clearly represents the key determinant of NEAT (45). Indeed, NEAT can vary by as much as 2000 Kcal per day when comparing two adults of similar body size, lean body mass, age and gender (Figure 3).

For an average worker spending most of her/his time in a seated position, occupational NEAT is relatively low and associated energy costs range at a maximum of 700 kcal per day (Figure 3). A comparable person working mainly in a standing position can increase their occupational NEAT to up to 1400 kcal per day, while an agricultural occupation would theoretically result in NEAT categories ranging around 2000 kcal per day or more (Figure 3) (45). Thus, occupations relying on intense physical activity can expend 1500 kcal per day more than a sedentary job.

Figure 3. The effect of occupational intensity on NEAT (adapted from (45)). The data assume a BMR of 1600 kcal per day.

This variation in daily NEAT between individuals and populations becomes even more accentuated when considering research from non-industrialized countries (57, 58). For instance, a study by Levine and colleagues included more than 5000 inhabitants of agricultural regions of the Ivory Coast of Africa (Figure 4).

Figure 4. Gender- and age-dependent physical activity levels (time engaged in work activities) of people living in agricultural communities of the Ivory Coast in Africa (adapted from reference (58)).

Each subject was studied over seven days and all of their daily tasks were recorded by a trained evaluator (58). Several lines of available evidence regarding NEAT were established from this study. First, a substantial effect of gender was observed as women apparently worked more than men in these societal constructs. Women were responsible for more than 95% of domestic and for an additional 30% of agricultural tasks. Otherwise, men exclusively worked in agricultural occupations and had more leisure time, resulting in substantially lower NEAT than women (45, 58). In contrast, men in Canada, Australia or the United Kingdom are reported to be up to three times more physically active than females, while data from the United States indicate comparable activity levels of genders (59, 60). Even less data is available on NEAT in children, but in general boys seem to be reproducibly more active than girls (61, 62).

A second important observation from the study of Levine et al. is the fact that decreased occupational NEAT was observed with aging (Figure 4) (58). An age-related decline of NEAT had been previously reported across species (63). The underpinnings for this age-effect on NEAT in free-living humans has been reported by Harris et al. (64), who showed that the substantial decrease in elderly as compared to younger subjects was mainly attributable to the ambulation subcomponent of NEAT. Elderly subjects walked less distance despite having a comparable number of daily walking periods compared to their younger counterparts. Indeed, elderly subjects performed 29% less non-exercise activity, corresponding to three miles less ambulation per day (64).

A further factor that should be considered to potentially impact NEAT could be sleep restriction. It was shown that subjects with 5.5 compared to 8.5 hours nighttime sleep opportunity had 31% fewer daily activity counts, spent 24% less time engaged in moderate plus vigorous physical activity and became more sedentary (65). However, the significance of these findings is currently limited to time spans over few weeks and it remains therefore unclear as to whether the results are also valid in terms of common problems, such as chronic sleep insufficiency or shift working. Although, some short-term experiments support the latter findings, conflicting data have also been reported on that matter (66–69). Future research will need to validate these associations and, if true, better understand mechanisms underlying restricted sleep time and alterations in PEE/AEE. Sleep restriction should be nevertheless regarded as potential biasing factor in future research projects on NEAT.

Industrialization and Societal Status

Industrialization and societal status are distinctive factors affecting NEAT. Urban environment and mechanization are associated with a decrease in daily physical activity. For example, it was shown that sales of labor-saving domestic machines (e.g. washing machines) and obesity rates are closely correlated in the U.S. population, while this association was not present with respect to energy intake (20, 22). Furthermore, it is known that poverty and societal aspects comprising neighborhood and educational status are associated with increased consumption of energy-dense foods, which is thought to contribute to higher obesity rates reported in lower socioeconomic groups (70, 71). Highly educated individuals report more leisure-time physical activity and are three times more likely to be physically active as compared to less educated individuals (20, 22).

Seasonal Effects

Seasonal variations also play a role for NEAT. Time spent in activity is twice as likely during the summer as compared to winter months and contributions to activity from agricultural or construction work plays a greater role in the summer season (20).

Together, available data indicate close relationships between environmental conditions and biological aspects of NEAT. Evidence suggests that the contribution of occupational activity to NEAT can vary by 2000 kcal per day. Since the accumulation of excess body fat is a result of positive energy balance, subtle perturbations of energy balance over sustained time periods are theoretically capable of contributing to unwanted weight gain. But once weight stability is again established at a higher weight, energy expenditure and energy intake need to be precisely matched to achieve a long-term persistency of body mass, as an error of 1% would lead to a gain or loss of 1 kg per year or some 40 kg between the ages of 20-60 years. Therefore, small alterations of energy balance by means of NEAT could theoretically result in significant effects with respect to severity of obesity in an individual and the prevalence of obesity in a population. Consequently, when considering how to influence NEAT as a strategy for the treatment or prevention of obesity, a central question arises: In what fashion does NEAT interact with another environmentally-influenced factor involved in energy balance, that of energy intake?

Alteration of NEAT with Varying Energy Availability from Foods

Alteration of energy balance is followed by multiple adaptations, but relatively few studies have addressed modulation of spontaneous physical activity and NEAT by variations in nutritional energy intake. The latter is part of the concept of adaptive thermogenesis (72).

Adaptive thermogenesis refers to changes in REE, EAT and NEAT, which are independent of changes in LBM and are, instead, modulated by other factors, including increased or decreased caloric intake (72). With caloric restriction, both REE and non-resting energy expenditure substantially contribute to changes in adaptive thermogenesis (72). Interestingly, in most studies, alterations in adaptive thermogenesis in response to overfeeding are in large part explained by raised non-resting energy expenditure (e.g., NEAT). However, the individual ability to adapt NEAT to manipulations of energy balance is highly variable between subjects and situations, particularly with respect to overfeeding (39). But if an individual is able to generate a significant upregulation of NEAT in the face of positive energy balance brought about by an increase in food intake, they may be more able to prevent obesity (34).

NEAT UNDER CONDITIONS OF OVERFEEDING

As discussed above, by using a combination of direct calorimetry and motion detectors, Ravussin et al. have shown that the inter-person variability of TEE is considerable and remains significant even after adjustment for LBM (29). A substantial percentage of this variability is explained by differences in spontaneous physical activity including “fidgeting” (29). Fidgeting-like activities accounted for a range in energy expenditure of 100-800 kcal per day in the subjects. Interestingly, the researchers observed elevated spontaneous physical activity levels in subjects with obesity as compared to those who were lean, indicating a positive relationship with elevated body mass. However, the latter study was performed under isocaloric conditions and over a short observation period. Therefore, the importance of this report was to establish the significance of spontaneous physical activity for energy balance, but whether acute variability in energy intake affects NEAT was not addressed. In their “weight clamping” study, Leibel et al. (73) overfed adults who were lean and overweight/obese by 10%, or approximately with 5000-8000 kcal per day over 4-10 weeks, followed by a weight maintenance period. Body composition, fecal caloric loss, TEE, REE, TEF and spontaneous physical activity were measured using multiple technical approaches including indirect calorimetry, the doubly labeled water method, and activity trackers in a subgroup (73). They showed that maintaining a 10% elevated body mass induced a significant increase of TEE by 9±7 kcal per kg LBM per day in subjects who had not been previously obese and by 8±4 kcal per kg LBM per day in those who had formerly been obese (73). The majority of this rise in TEE was attributable to non-resting energy expenditure. The magnitude of this effect was comparable in those who had previously not been obese and those who had been overweight or obese, indicating similar adaptations to overfeeding conditions (73). However, a related review pointed out the large inter-individual variability regarding the capability to adjust energy expenditure with overfeeding in the “weight clamping” experiments (46) and emphasized that in other human over-feeding studies there is a wide range of individual weight gain per unit of excessively consumed energy. Therefore, it can be proposed that some individuals show a remarkable capacity to increase energy expenditure in response to overfeeding, while others do not (46). It was further suggested that spontaneous physical activity, and accordingly NEAT, likely play an important role regarding the impact on body weight under such conditions.

Levine’s group was the first to systematically investigate the effect of overfeeding on the individual ability to adapt NEAT in free-living subjects (53). Using sophisticated methods and measuring NEAT over a representative time span, the authors overfed 16 volunteers (12 males, 4 females; age ranging from 25-36 years) by 1000 kcal per day over their weight maintenance requirements (20% of the calories came from protein, 40% from fat, 40% from carbohydrates). The energy surplus was paralleled by a mean TEE increment of 554 kcal per day. In this study 14% of this TEE increase was attributable to a rise in REE, approximating by 79 kcal per day. A further 25% of the TEE increment (136 kcal per day) corresponded to an increase in TEF, probably due to the relatively high percentage of protein intake. The most prominent effect was, however, attributable to enhanced physical activity thermogenesis, corresponding to around 336 kcal per day. As volitional exercise of the study subjects remained at a constant low level and since the authors did not detect changes in exercise efficiency, they concluded that about 60% of the increase in TEE due to overfeeding was attributable to NEAT (53). Again, however, the change in NEAT varied remarkably between subjects, ranging from -98 to +692 kcal per day. The maximal individual increase of NEAT constituted 69% of the excessively consumed 1000 kcal per day in this study (74). Moreover, the change in NEAT was directly predictive of the individual vulnerability or resistance to body fat accumulation (53).

A recent randomized study enrolled young aged female and male participants with variable BMI (19-30 kg/m²) and used gold standard methods to investigate not just the effect of caloric overfeeding, but also of protein intake on PEE/AEE (75). 140% of caloric needs including 5, 15, or 25% of energy from protein was fed for 56 days. Caloric overfeeding resulted in increased physical activity and correspondingly PEE/AEE including NEAT, even after adjustment for changes in body composition. By contrast, changes in physical activity were not related to protein intake. The authors concluded that increased PEE/AEE in response to weight gain might be one mechanism of modulating adaptive thermogenesis (75). But the results indicate also that the observed increase in activity was not likely effective at attenuating weight gain.

Other recent reports have failed to detect compensatory increases in spontaneous physical activity during short-term overfeeding in humans (76–80). While in several cases, methodological differences make them less directly comparable to Levine’s study, (76, 56, 77, 79), a study by Siervo et al. used state-of-the-art methods and applied a highly standardized 17-week protocol with progressive overfeeding from 20-60% energy in excess of maintenance needs (81) in lean, healthy men. Three sequential intervals of stepwise overfeeding were each separated by one week of ad libitum energy intake. In agreement with previous findings, between-subject variability concerning weight change during ad libitum phases was high (63). However, in contrast to Levine’s study (53), Siervo et al. failed to detect a significant systematic change in spontaneous physical activity (81). The authors therefore concluded that systematic elevations in energy intake induce very limited counter-regulatory responses in energy expenditure (63).

In summary, these data underscore the highly individual compensatory response regarding adaptive thermogenesis to overfeeding. From Levine and Apolzan et al., supportive evidence is provided for the susceptibility to gain body mass under standardized overfeeding conditions and identifies an individual’s ability to adapt NEAT as a potential modulator (75, 53, 20, 34). In accordance with findings of other overfeeding studies (reviewed in (74)), adaptations in thermogenesis by changes in NEAT observed between individuals may explain why some individuals are particularly susceptible or resistant to weight gain. The potential impact of age on these adaptational mechanisms remains unclear and should be addressed by future research.

THE IMPACT OF CALORIC DEPRIVATION ON NEAT

While evidence with respect to systematic NEAT upregulation under overfeeding conditions remains somewhat controversial, TEE and spontaneous physical activity are consistently influenced with energy deprivation in the majority of available studies (72, 80). This is of importance as most subjects who are overweight or have obesity try to lose body mass through various calorie-restricted dietary strategies.

The first study to quantitatively investigate adaptive thermogenesis under conditions of food restriction was the Minnesota Starvation Experiment (reviewed in (46, 49, 72)). This evaluation showed a marked reduction in TEE that was later reproduced by numerous studies, independent of pre-starvation BMI or method used to induce weight loss (reviewed in (72)). For instance, Leibel et al. in a second arm of their “weight clamping” experiment evaluated the effect of a 10% body mass reduction, followed by weight maintenance at this level (73). While overfeeding was accompanied by a 16% increase of adjusted TEE, weight reduction from underfeeding induced a 15% decrement (57). As compared to overfeeding, it is well accepted that both REE and non-resting energy expenditure are affected by energy deprivation in humans and animal models (82, 72, 80).

These findings are supported by results from the so-called Biosphere 2 experiment, where the authors demonstrated a major reduction of TEE and spontaneous physical activity with body mass reduction over a two-year time period, which persisted after six months of body weight regain (46, 83). Demonstrated TEE reductions by adaptive thermogenesis in weight-regainers has been reproduced in other human studies (72). Of note, when subjects recover from starvation they spontaneously overeat and, moreover, if weight loss-induced adaptive thermogenesis persists (e.g. reduced NEAT), there is a significant risk of regaining weight and “overshooting” pre-starvation body mass (72). Thus, changes in non-resting energy expenditure with underfeeding (or overfeeding) appear to occur in a direction tending to return subjects to their initial body mass and to conserve body energy as recently described in humans as so-called “thrifty phenotype” compared to a “spendthrift phenotype” (84).

Regarding this, findings from a recent study in sedentary overweight females are of potential practical relevance (85). In this investigation, 140 pre-menopausal females reduced their body mass by an average of ~11 kg while undergoing a total of 800 kcal per day diet. Subjects were randomly assigned to one of three groups: no exercise, a structured aerobic training schedule, or an exercise program incorporating resistance training. Body composition, strength, TEE, REE and NEAT were measured using various approaches. One main finding was that TEE, REE and NEAT all declined with weight loss in the no-exercise group. Non-exercise activity thermogenesis was reduced by 150 kcal per day, which was equivalent to 27% compared to baseline levels. However, in subjects undergoing the exercise regimens only REE was reduced, while NEAT remained unaffected. Thus, since caloric deprivation appears to consistently decrease spontaneous physical activity, untrained subjects with obesity who plan to lose body mass by means of dietary intervention alone would be advised to engage in a concomitant exercise program so as to avoid negative effects on NEAT and mitigate weight cycling after reaching a lower body weight. Of note, the authors of this study emphasized that, consistent with the concept that “more is not always better,” exercising two days a week was capable of increasing NEAT but, paradoxically, an intense three days weekly schedule was followed by a substantial decrement in NEAT (85).

At this end, one further study of Levine’s group should be considered (78). NEAT as measured by gold standard methods and posture allocation was evaluated in mildly obese subjects and normal weight controls over a period of ten days. This baseline evaluation showed a 164 minute per day longer seated time in obese compared to lean subjects. A subset of eight obese subjects then underwent an intentional weight loss program over a time span of eight weeks and thereby lost significant body weight. Posture allocation was measured for another ten days after intervention. Interestingly, the weight losing obese subjects did not further reduce their NEAT in this experiment (78). The latter study has several limitations, e.g., the small number of enrolled subjects and the relatively short intervention period when compared to some of the above-mentioned trials. Levine and colleagues themselves emphasized that it was a pilot experiment (78). The data otherwise suggest that interindividual differences in posture allocation could at least in part be biologically determined and are therefore not necessarily influenced in every individual by e.g., reduced energy intake over short time periods. Over- and underfeeding studies in identical twins indeed suggest an important role of genetics regarding the variability underlying body mass regulation (86, 87). Specifically regarding NEAT, it is likely that due to the polygenetic nature of obesity, numerous pathways are involved in the regulation of spontaneous physical activity, as the latter has multiple environmental cues and affects a multitude of behaviors (8, 88). Zurlo et al. have shown that spontaneous physical activity levels cluster in families and could prospectively help to explain the propensity for weight gain (89). Moreover, according to a recent review up to 57% of the variability of spontaneous activity has been attributed to inheritance (90).

Otherwise, biological and genetic determinants can only partially explain NEAT variance. Models of human energy budgets related to exercise can provide a conceptual framework to better understand NEAT regulation in humans (reviewed in (39)). For instance, the so-called independent model predicts that changes in basal TEE have no impact on the energy budgeted for behavior, e.g., spontaneous physical activity. This model is analogous to the factorial model of exercise in humans that assumes that exercise has an additive effect on TEE (39). In contrast, the allocation model suggests that the total energy budget is constrained, and therefore, an increase in the energy cost related to maintaining basal functions will reduce the amount of available energy to support other functions, finally altering an individual’s behavior (39). The independent model would for example predict that exercise additively increases TEE, while the allocation model predicts that exercise leads to a reduction in some components, e.g., NEAT. Indeed there is evidence that each of these models is evident in different human populations, although studies suggest that e.g. allocation may be affected by confounders such as age (older more likely to reallocate), sex (males more likely to reallocate), and exercise volume (allocation more likely with higher volumes) (39). Such aspects have to be considered and could help to integratively explain the contradictory findings of Levine et al. and from other researchers.

In summary, an integrative view of existing human overfeeding studies proposes that the magnitude of adaptations regarding energy expenditure varies largely between individuals. Subjects capable of responding with increased spontaneous physical activity to overfeeding can be categorized as “compensators” and are therefore less susceptible to obesity (“spendthrift phenotype”). Subjects unable to respond to a continued energy surplus with increased NEAT (e.g., “non-compensators” or “thrifty phenotype”) seemingly represent the majority of subjects on a population level, of which a major percentage will attempt dietary weight-loss strategies at some time in their lives. However, REE and non-resting energy expenditure including NEAT are reduced with underfeeding in the majority of subjects. They therefore represent a risk factor for weight regain after dietary intervention. Mild voluntary exercise during and maybe shortly after periods of dietary weight loss could be a promising strategy to avoid decreases in NEAT, although this alone will probably not fully compensate for the obligate decline in REE. Whether the majority of dieting subjects with obesity will continuously adhere to a structured exercise program during extended periods of weight maintenance as suggested by current recommendations is also uncertain (and unlikely) (42, 8, 43, 41).

Therefore, to advance anti-obesity strategies that systematically increase or even conserve NEAT it is essential to understand the mechanisms contributing to regulation of spontaneous physical activity.

PHYSIOLOGICAL AND MOLECULAR DETERMINANTS OF NEAT

Principles of NEAT Regulation

The ability to adapt thermogenesis, and specifically NEAT, presupposes existing mechanisms in the body that sense, accumulate, and integrate internal and external directionality signals with respect to energy balance (20). Figure 5shows a proposed model of the principal regulation of spontaneous physical activity and correspondingly NEAT. In this model, physiological data regarding caloric intake, energy depots and energy expenditure are compiled centrally by the brain. These signals converge at the data acquisition center, are rectified to a common signal, and directed to a NEAT accumulator, which is constantly summing the net amount of NEAT per unit of time. The latter continuously refers to the energy balance integrator, which, in turn, modulates spontaneous physical activity in response to changes in energy intake. Thereby, energy balance resulting from these three components is in constant flux.

Figure 5. The NEAT hypothesis of energy balance (adapted from (20)).

Continuous arrows represent energy flow through the system, broken arrows stand for putative signaling pathways. BMR, basal metabolic rate; NEAT, non-exercise activity thermogenesis; TEF, thermic effect of food.

For example, under circumstances of positive energy balance during increased food intake, the energy balance integrator may mediate deposition of the caloric surplus in energy stores (adipose tissue) or alternatively dissipate the excess energy in the form of NEAT. More likely, since BMR is essentially fixed, some energy will be spent on food digestion and absorption (increased TEF), some will be deposited as body fat or in other depots, and the rest will be dissipated as NEAT (20). This model also supports the observation that although BMI and energy balance are continuously oscillating around a weight set point (or “set range”) under daily life conditions, body mass is remarkably stable in the long term. This is exemplified by data from the Framingham Study showing a mean body mass increase of only 10% over a 20 year time span in average adults (cited in (91, 92)).

Thus, it appears that the regulation of energy balance and body mass is realized by a complex network of precisely working auto-regulatory structures controlling effector systems, one of which being spontaneous physical activity. However, while ample available data detail the regulation of food intake, there is lack of information concerning the biological mechanisms driving NEAT (34). Most animal evidence supports the concept that NEAT can help to protect against obesity (93). Therefore, spontaneous physical activity has certainly potential for impacting body mass and energy balance. It is very likely that many of the biological systems involved in the regulation of energy intake are also involved in gathering and integrating information regarding NEAT and determining NEAT adaptation in humans (34).

Tissues, Organs, and Central Mechanisms Involved in NEAT Regulation

Several well-described central neuroendocrine systems are thought to regulate NEAT through interactions with peripheral tissues/organs known to affect energy balance (Figure 6).

Figure 6. Model for the neuroendocrine regulation of NEAT in the service of energy balance (adapted from (34)). Multiple external and internal signals are sensed and integrated, whereby defined brain structures (e.g., arcuate nucleus of the hypothalamus, area postrema and nucleus of the solitary tract of the hindbrain, dopamine pathway of the mesolimbic system) interpret a multitude of sensory cues of energy availability. The involved brain systems have multiple ascending and descending projections affecting the amount of physical activity through arousal and limbic pathways, and descending neural projections and endocrine signals to modulate the energy efficiency of physical activity. Thereby, the central nervous system could adapt NEAT to adjust energy balance under conditions of caloric excess or starvation. Ach, acetylcholine; AgRP, agouti-related peptide; CART, cocaine- and amphetamine-regulated transcript; CCK, cholecystokinin; LC, locus ceruleus; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; (P)SNS, (para-)sympathetic nervous system; SNS, sympathetic nervous system; TMN, tuberomammilary nucleus; VP, ventral pallidum

These associated neural mechanisms of brain regions involve a multitude of neurotransmitters and neuropeptides (for extensive review see (34) and (94)). While all are important, this section will focus on the biological role of central orexin peptides as promising and potentially targeted regulators of spontaneous physical activity (for extensive review see (47)).

Experimental data indicate that injections of orexin A into defined brain regions reproducibly induce a dose dependent increase in spontaneous physical activity along with significant increments in NEAT. Moreover, current knowledge suggests a role for the orexin system in the control of arousal and sleep, and for reward and stress reactions. This has led to conduction of extensive research showing that the orexins A and B are produced by cleavage from a single pro-peptide (44). The majority of orexin A is synthesized in the lateral hypothalamus and perifornical area. Orexin A binds to two G protein-coupled receptors, namely orexin receptor type 1 (OXR1) and 2 (OXR2). OXR1 has high affinity for orexin A, while OXR2 has equal affinity for the A and B peptide. Receptor binding of orexin A is associated with increased intracellular calcium levels and followed by enhanced neuronal signaling (44).

Physiologically, orexin A is capable of modulating spontaneous physical activity, food intake, and sleep as evidenced by an obesity-resistant rat model with high orexin A activity (34). These animals show more ambulatory and vertical movement, independent of age or food availability (34). Intriguingly, this rat model exhibits lower body weight gain when fed a high-fat diet, despite consuming significantly more kcal per gram body mass. From this study, it appears that orexin A simultaneously enhances feeding behavior and induces physical activity, but the consumed calories are outweighed by those expended with spontaneous physical activity (47, 34). These data were confirmed by a mouse model with postnatal loss of orexin neurons (47). These mice exhibit hypophagia, decreased spontaneous physical activity, and develop spontaneous-onset obesity while consuming a regular chow-diet. Furthermore, in recent years so called DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) have been developed and were applied to manipulate orexin neuron activity in specific animal models. Compared with previous work of targeted infusions of orexin and/or orexin agonists into the brain, the use of DREADDs allows for the sustained activation of the neurons of interest without needing to inject directly into the brain (95).

A high-fat diet model was used to evaluate the effect of DREADDs on NEAT, since spontaneous physical activity can be decreased under such circumstances. It was shown that NEAT was decreased in animals on the high-fat diet, and was increased to the NEAT level of control animals following activation of orexin neurons with DREADDs (95). Thus, these preliminary results clearly support the hypothesis that the orexin signaling system could be a promising target of modulation by drugs.

However, from the perspective of humans, data on orexin are currently scarce. Otherwise it was recently shown in a pilot study that plasma levels of orexin A correlated with physical activity levels including many NEAT features (96). Therefore, plasma orexin A could be considered as a biomarker of NEAT in future studies. Furthermore, the orexin system has been shown to be defective in the sleep disorder narcolepsy. Since obesity is a recognized comorbidity of narcolepsy in both animal models and humans (47), regulation of this signaling system has the potential to impact clinical endpoints, probably including the development of overweight and obesity.

The Role of Peripheral Tissues for NEAT Regulation: Adipose Tissue and Adipokines

Peripheral tissues are also believed to contribute to NEAT regulation (Figure 6). That NEAT can be influenced by over- and underfeeding has been extensively discussed in earlier sections; but it is essential to also reconsider that longitudinal studies of fasting humans show significant decreases in adaptive thermogenesis with starvation, while refeeding may induce a variable elevation (72). These adaptations are likely the result of feedback mechanisms between thermogenesis regulation and energy depots (Figures 5 and 6). It can be further hypothesized that adaptations of thermogenesis in response to fasting or overfeeding result, at least in part, from an adipose tissue-specific impact on thermogenesis (reviewed in (46)). Indeed, studies of white adipose tissue (WAT) have led to the recognition that this energy store represents an important endocrine organ communicating with the brain through secretion of so-called “adipokines.” Accordingly, adipokines could play a role for NEAT regulation (Figure 7) (97).

Figure 7. Essential elements in the regulation of thermogenesis including effects of WAT (adapted from (34, 98)). BAT, brown adipose tissue; cAMP, cyclic 5” adenosine monophosphate; COX, cyclooxygenase; IL, interleukin; LPS, lipopolysaccharide; PAMPs, pathogen associated molecular pattern; PGE, prostaglandin E; PKA, protein kinase A; SNS, sympathetic nervous system; UCP, uncoupling protein; WAT, white adipose tissue.

Leptin is such an adipokine signal that is the product of the rodent obese (ob) gene and human homolog (LEP) first characterized in 1995 (99). Due to its role regarding the sensing of energy stores and regulation of food intake, it was also suggested as an important signal with respect to adaptive thermogenesis (reviewed in (34, 98)). For instance, Dauncey and Brown studied ob⁄ob mice lacking leptin production and their lean littermates at comparable body weights (100). Leptin-deficient mice expended less energy and showed less motor activity as compared to control animals. The energy expended relative to metabolic size was greater in lean littermates, who were also more active than the ob⁄ob mice. It was calculated that activity-related energy expenditure accounted for at least part of the body mass difference observed between ob⁄ob and wild-type mice (100, 101). A central mediator of leptin action on NEAT partly includes the orexin signaling system, since the activity of orexin neurons has been shown to be modulated by leptin among other metabolic indicators (reviewed in (93)).

With respect to humans, Franks et al. have shown that fasting leptin levels were significantly associated with physical activity-related energy expenditure (101). Moreover, it was pointed out in a recent review that replacement of leptin in weight-reduced subjects was capable of reversing neuroendocrine adaptations that accompany caloric reduction as evidenced by an increase in SNS activity, suggesting that this component of the non-resting energy expenditure and adaptive thermogenesis is at least in part under endocrine control (72). In response to caloric restriction, the authors therefore suggest an adaptation model where in an initial fast “first set,” reduced insulin secretion and glycogen stores are accompanied by a reduction of both REE and non-resting energy expenditure within days, while in the long term, with maintenance of reduced body weight, a “second set” mediated by a drop in leptin levels becomes active that keeps energy expenditure low so as to preserve fat depots and maintain reproductive function (72). This endocrine action resulting from the drop in leptin levels with related activation of multiple pathways then enhances the risk of weight regain (72).

Overall, available data suggest that leptin’s central actions include not only reducing food intake but potentially also increasing energy expenditure mediated, in part, through increasing physical activity levels (reviewed in (34)). The potential role of leptin on NEAT in human obesity is of particular interest given the phenomenon of central leptin insensitivity under conditions of overfeeding and related obesity (102, 103). However, it must be also kept in mind that the leptin system represents only one component of peripheral tissue signaling that impacts NEAT. Other endocrine systems, such as thyroid hormone status, also have major influences on energy expenditure. For example, hyperthyroid rats have been shown to have significantly increased NEAT (104).

Energy availability from food and internal energy stores, ambient and body temperature, and systemic inflammation are further determinants of thermogenesis regulation. In WAT, two relevant factors converge: WAT represents the principal energy storage depot and, under conditions of significant obesity, WAT is a site of low-grade inflammatory activity that contributes to systemic inflammation. Recent epidemiological data from humans support this hypothesis (105). It was shown that levels of systemic low-grade inflammation (e.g., circulating interleukin-6 and interleukin-1β) were related to non-exercise physical activity. Although no causal relationship can be drawn from these findings, inflammation is theoretically one signaling system involved in NEAT regulation.

Signals from WAT can induce enhanced sympathetic nervous system (SNS) activity and affect downstream regulators of adaptive thermogenesis (Figure 7). Indeed, experimental data indicate that NEAT is one component affected by WAT signaling (93). Related to the hypothesis of “adaptive thermogenesis”, Dulloo et al. in their review regarding human starvation and overfeeding suggest SNS as key regulatory effector mechanism (46). Of note, activation of brown adipose tissue, which has recently gained a lot of interest with respect to human obesity (106–108), is suggested to be an additional site of adaptive thermogenesis.

Skeletal Muscle as Peripheral NEAT Modulator

Skeletal muscle is a major contributor not only to REE, but also to spontaneous physical activity and has been newly recognized as important NEAT effector. Human subjects maintaining a 10% weight loss in response to caloric restriction have increased skeletal muscle work efficiency (92, 109). This mechanism is believed to be responsible for the phenomenon that, even after adjustment for the reduced body mass, weight-loss maintaining subjects need proportionally fewer calories for performing comparable physical-activity tasks compared to before weight loss (92, 109). Therefore, adaptations of skeletal muscle work efficiency can contribute to reduced NEAT after low-calorie induced weight loss. Several recent animal models are in line with this hypothesis (82, 110). For example, a recent study in rats investigated the contribution of REE and non-resting energy expenditure to reduced TEE observed after three weeks of 50% energy deprivation (82). They reported a 42% reduction in TEE, even after correction for the loss in body weight and LBM (82). Moreover, 48% of the detected TEE reduction was explained by a significant decline in non-resting energy expenditure, e.g. spontaneous physical activity (82). Reduced baseline and activity-related muscle thermogenesis was also found in this study. Reduced skeletal muscle norepinephrine turnover as a surrogate of SNS drive and increased expression of the more energy “economic” myosin heavy chain (MHC) 1 isoform were identified potential mechanisms for the observed changes (82, 92, 109). Thus, according to these findings, restriction of caloric intake apparently not only suppresses spontaneous physical activity, but in addition reduces related caloric demand, which is mediated by reduced SNS activity to skeletal muscle and altered expression of MHC isoforms. These findings are supported by data from another recent animal study investigating rodents with “high intrinsic physical activity” and counterparts with “low intrinsic running capacity” under eucaloric conditions (87). It was found that animals with high intrinsic physical activity were lean, had lower skeletal muscle economy along with increased skeletal muscle heat dissipation during activity. This resulted in higher TEE and NEAT and was related to increased activation of skeletal muscle by SNS (110).

Taken together, skeletal muscle is an intrinsic site for NEAT and not only of great importance quantitatively, but also represents an important effector system for NEAT mediated through modifications on the level of SNS activity and gene expression, particularly under conditions of altered caloric intake. Therefore, evidence indicates that NEAT is modified by a complex network regulation of central and peripheral systems. The latter involve a variety of redundantly organized immediate and delayed mechanisms, including central neuropeptides and signaling systems to and from peripheral tissues. Involvement of such biochemical signaling systems identifies spontaneous physical activity and NEAT as a potential site for pharmaceutical targeting. Pharmaceutical manipulation of such a system could be envisioned to either increase the amounts of activities people undertake, or elevate activity-related energy expenditure.

As stated in the introduction, obesity is associated with a many adverse health outcomes (1, 111). Energy expenditure in occupational activities has declined by a mean of 140 kcal/d since 1960 in the United States and this reduction is thought to account for a significant portion of the observed increase in mean BMI (reviewed in (1)).The latter hypothesis is supported by observational data showing that reduced energy expenditure of 100 kcal/d below expected values corresponds to a 0.2 kg/year weight gain, of which 0.1 kg/year are fat mass (112). In addition there is ample evidence from epidemiological, cross-sectional, and longitudinal studies showing that individuals with higher physical activity levels have lower mean body weight, gain less weight and fat mass over time, and show reduced weight regain after intended loss in body mass (113–117). Moreover, regular physical activity can moderate or even eliminate weight gain among those carrying a risk at the FTO (fat mass and obesity associated) gene variants ((118), reviewed in (93)). It has further been evidenced that levels of habitual or spontaneous physical activity are positively related to reduced risk of major cardiovascular disease and premature mortality, while an increase of sitting time correlates with a raise in complications (119–123).

Associations of NEAT with Type 2 Diabetes Mellitus and NAFLD/MAFLD as Common Sequelae of Obesity

In industrialized countries the daily time spent sitting is estimated to be 6-7 hours per day, which is closely correlated not only with obesity rates but also with the incidence of T2D (120, 124). A plethora of data suggest, on the other hand, beneficial effects of lifestyle interventions on insulin resistance, metabolic control, pain and clinical endpoints in these patients (e.g. (125, 126), reviewed in (127)). In subjects with impaired glucose tolerance or prediabetes, lifestyle interventions have proven to decrease the incidence of T2D, of which prominent examples comprise the DaQuing Study, the Finish Diabetes Prevention Study, the Diabetes Prevention Program and others (128–131). This was newly confirmed by the lifestyle intervention and impaired glucose tolerance Maastricht (SLIM) trial, which has shown to impact clinical endpoints even four years after stopping the intervention (132, 133).

The prevalence of NAFLD/MAFLD parallels the pandemic rise in T2D. A recent meta-analysis indicates that on a global perspective more than 55% of T2D patients suffer from bland liver steatosis, while a further 37% show signs of non-alcoholic steatohepatitis (NASH) (reviewed in (127)). “NAFLD” was defined in the 1980s to describe excess hepatocellular lipid accumulation in absence of significant alcohol intake, autoimmune, or viral liver disease, and the course of this pathology was long believed to follow the so-called “two-hit hypothesis” (134). Manifestation of bland steatosis was defined as first hit, while histological signs of liver inflammation, fibrosis and hepatocyte injury were proposed as succeeding second hit (NASH). Insulin resistance has now been recognized as the most valid predictive parameter of NAFLD/MAFLD progression to NASH. Moreover, presence of insulin resistance puts these patients to an elevated risk of morbidity and mortality, while sedentary behavior constitutes a complementary risk factor and correlates with clinical outcomes (reviewed in (135, 127)).

Since physical exercise is capable of improving and maybe reversing insulin resistance, while short term decreases in physical activity reduce multiorgan insulin-sensitivity and in parallel increase liver fat (reviewed in (127)), it is reasonable to assume that physical activity represents a potent treatment modality for NAFLD/MAFLD. There is indeed some evidence from prospective controlled randomized intervention studies that liver fat can be reduced independently from changes in body weight by structured exercise regimens in a limited, yet significant manner, while effects on liver inflammation and fibrosis remain to be investigated (extensively reviewed in (127)). Although no direct research of NEAT effects on NAFLD/MAFLD is currently available, one well-designed study using brisk walking as an intervention has shown reduced liver fat after 6 and 12 months (136). This suggests that low to moderate intensity activities potentially impact hepatic steatosis, which should be further evaluated.

For weight maintenance purposes, current recommendations encourage subjects with obesity to engage in 200-300 minutes per week in activities as e.g. walking (8). Yet, patients with T2D show markedly reduced TEE (< 300 kcal per day), number of steps taken (<1500 per day), physical activity duration (<130 min per day), and activity related energy expenditure (<300 kcal per day) as compared to subjects without diabetes (137). Accordingly, only 28% of patients with T2D in the U.S. achieve the recommended physical activity levels and less than 40% engage in regular voluntary exercise (120, 138, 139). In line with this, recent observational studies have shown that reduced NEAT is related to measures of insulin resistance, glycated hemoglobin A1c, and other features of the metabolic syndrome, consistent with the hypothesis that increasing spontaneous physical activity could be beneficial for those with T2D and NAFLD/MAFLD (140–142). This is supported by data coming from the Nurses’ Health Study showing that regular walking at normal pace (e.g. 3.2-4.8 km h^-1) was associated with a 20-30% relative risk reduction of T2D, while in another study, frequent walking was correlated with an equally remarkable risk reduction of mortality in T2D (120, 143, 144).

On the other hand, it must be acknowledged that data on NEAT, T2D and NAFLD/MAFLD are limited. First, evidence suggests interindividual differences in the response to a standardized exercise schedule at a given dose and there is, moreover, strong indication of genetic components to the variation in human trainability (reviwed in (145)). Second, available evidence almost exclusively comes from observational and cross-sectional studies. Furthermore, the data on lifestyle interventions on sequalae of insulin resistance and manifest T2D also have limitations (146). For instance the 10-year prospective randomized LookAHEAD study in T2D patients was stopped early based on a futility analysis, since the trial was unable to detect substantial effects on cardiovascular events, although the intervention group experienced significant weight loss (147). Otherwise, secondary analyses of this hallmark study were able to show that the magnitude of weight loss may be predictive of outcome measures (148). In more detail, obese and overweight subjects suffering from T2D were examined regarding the association of the magnitude of fitness change (n=4406 and weight loss (n=4834) over a median of 10 years of follow-up for the primary outcome. The primary outcome was a composite of death, cardiovascular disease, myocardial infarction, hospitalization for angina, and stroke. Subjects losing more than 10% of body mass in the first year of the intervention had a 21% lower risk of the primary outcome relative to participants with stable body mass or weight gain. Interestingly, achieving a > 2 metabolic equivalents (MET) fitness change was not significantly associated with the primary, but with the secondary endpoint (composite of coronary–artery bypass grafting, carotid endartectomy, percutaneous coronary intervention, hospitalization for congestive heart failure, peripheral vascular disease, or total mortality (148). Therefore, it appears that life style modifications are principally capable of beneficially modulating clinical endpoints in, while the optimal exercise (and lifestyle intervention) therapy for individuals with T2D, particularly with co-existing complications, remains unknown (120). These findings suggest that priority should be made for subjects who are obese to engage in any type of regular physical activity before T2D and NAFLD/MAFLD become manifest ((144), reviewed in (120, 8)). Accordingly, the “Step It Up! (The Surgeon General’s Call to Action to Promote Walking and Walkable Communities)” program was recently released, focusing on promoting optimal health before disease occurs (149).

In summary, since EAT is negligible in the vast majority of subjects and NEAT represents the main contributor to daily PEE/AEE and therefore TEE in Western populations NEAT can be recognized as a major potential target for lifestyle modification in subjects suffering from T2D and probably also under conditions of NAFLD/MAFLD.

Personalized Approaches versus Environmental Re-engineering for Promoting NEAT

In free-living individuals, counterbalancing effects of an obesity-promoting environment involves addressing several central questions:

How can the amount of NEAT be increased or preserved under conditions of caloric restriction?

What are realistic goals for daily spontaneous physical activity and what are appropriate activities for increasing NEAT?

When aiming to prevent or treat obesity, it can be argued that in an obesogenic environment each person needs to increase physical activity levels (45). Alternatively, it could be argued that the epidemic of obesity needs to be addressed at the population level, since obesity has emerged as a result of environmental pressures that have led to decreased population-wide activity levels (e.g., more sedentary jobs, more time at work and less in leisure) (45). In reality, these contributors are not mutually exclusive. However, in order to substantially increase NEAT it is useful to categorize these perspectives as the “individualized approach” versus the “environmental re-engineering approach”(45). Accordingly, Levine and Kotz have developed the “egocentric” and the “geocentric” models, which provide a theoretical framework to understand important environmental determinants of NEAT from these two perspectives (20).The egocentric model focuses on a single person. Accordingly, environmental factors that impact a particular person’s spontaneous physical activity levels are considered, such as “my occupation”, “my transportation to work”, or “my leisure time activities” (Figure 8) (20). By contrast, the geocentric model is focused on how the environment impacts NEAT of multiple subjects, such as city planning to ensure walk-friendly or bike-accessible environments (Figure 8) (20). These models may help to elucidate how NEAT can be effectively modulated.

Figure 8. Environmental determinants of NEAT – the egocentric versus the geocentric model (adapted from (20)).

In the geocentric model a variety of environmental factors impact spontaneous physical activity. The most prominent negative influences on NEAT are represented by urbanization and mechanization, which are largely a phenomenon of high- and middle-income countries. Examples include televisions, drive-through restaurants, clothes washing machines, motorized walkways, and others (20).

Accordingly, when comparing daily energetic costs of mechanized tasks with the same tasks performed manually a century ago, the difference in daily energy costs approximates 111 kcal, or more than 40,000 kcal per year (150). Evidence such as this has been used in support of the proposal that urbanization and mechanization have likely had a dramatic impact on energy balance (20). Hence, proposals to increase NEAT on a population level have included environmental “re-engineering,” including provisions for adequate walkways, to build schools within walking distance, and to adapt employment laws for promoting office fitness. The problem with this argument is that it includes imposing high economic costs on business and localities without agreed upon standards for success (45).

It is self-evident that to intentionally increase NEAT over a protracted time period presupposes a significant amount of self-discipline. To facilitate behavior modification Levine has published approaches for promoting NEAT that focus on behavioral economic theory (for extensive review see (45)). Behavioral economic theory is a framework for conceptualizing how people make behavioral choices based upon their perceived relative value. When applied to NEAT, behavioral economic theory is concerned with how people choose between various activity/inactivity options. Consequently, Levine proposes four key elements (45): 1) It is critical to provide individuals with free-choice. By contrast, forcing a subject to choose a specific NEAT-promoting activity is likely to have the opposite to the intended effect. Otherwise, if an activity is self-selected, it is likely to be more reinforcing and consequently self-selected more often; 2) The delay between performing a NEAT-promoting behavior and the outcome needs to be minimized. While sedentary behaviors that people enjoy have immediately reinforcing consequences, health benefits of standing or ambulation may take longer to manifest. Therefore, it is important to choose NEAT-promoting physical activities; that are pleasing. For example, walking while listening to music or walk-and-talk with a friend. 3) Behavioral “costs” determine which sorts of activity/inactivity become selected. If a person has to work strenuously to participate in a given activity, they will be less likely to do it. For example, it is unlikely that many people will drive 40 minutes to the gym long-term just to engage in fitness training. People are more likely to choose physical activities that are easily accessible, such as home- or even office-based activities that do not require changing location or clothes; and 4) For an individual to choose a NEAT-promoting activity, it has to be more attractive than available alternatives. For example, behavior will change when providing a competing behavior that is more valued: a given person may prefer to surf the internet while seated rather than visiting the gym for a fitness workout. If, however, walk-and-talk with a friend was an option, the individual could choose that instead of internet-surfing. These behavioral components have been synthesized into a simplified approach termed, STRIPE (45): STRIPE is an acronym that represents S = Select a NEAT-activity that is enjoyed and start it; T = targeted, specific individual goals must be defined; R = rewards need to be identified for reaching the defined goals; I = identify barriers and remove them; P = plan NEAT-activity sessions; E = evaluate adherence and efficacy.

Overall, to increase NEAT in the prevention and treatment of obesity, egocentric and geocentric approaches should be considered. Occupation and leisure time are the two principal time frames that have to be targeted for promoting individual NEAT. To increase NEAT by means of changing behavior, the STRIPE approach is considered as safe and well-grounded in conceptual evidence. Unfortunately, there is no evidence as to whether an individualized or the population-based approach is more effective in terms of increasing physical activity levels and/or to affect body mass. Despite this, however, it is our opinion that both approaches should be still considered when counseling patients regarding ways they can improve their daily life conditions (45).

Increasing NEAT: Realistic Goals, Pitfalls and Appropriate Activities

Obesity is not a problem in only one component of energy balance and food restriction alone is surely not the complete answer on a long-term perspective. Hill et al. hypothesized that a person who is physically highly active could maintain energy balance and a healthy body weight by eating and expending at, say, an estimated 3000 kcal per day. This person, if adopting a sedentary lifestyle, could alternatively maintain energy balance and consequently body weight by eating and expending 2000 kcal per day. If, however, this sedentary person fails to strictly keep energy intake at 2000 kcal per day to match reduced energy expenditure over time, experiencing weight gain would be the unavoidable consequence (16). In other words, for a majority of subjects in a given population, maintaining energy balance at substantial higher levels by increasing NEAT could be in the long term much more realistic under free-living conditions (e.g. in an environment with high access to energy dense foods), than maintaining caloric intake at lower levels (16). Therefore, when attempting to increase NEAT, it is important to establish what goals have to be addressed for prevention or treatment of obesity. According to a review, the amount of physical activity necessary for weight loss approximates 2000-2500 kcal per week or about 2 ½ hours of additional daily ambulation, which is considered realistic for a majority of subjects with obesity (45). This is in line with current recommendations, suggesting ≥ 150 minutes per week of aerobic exercise in combination with a hypocaloric diet (8).

However, evidence is lacking from randomized, controlled studies as to whether strategies to promote NEAT in absence of dietary manipulation are effective for obesity prevention or treatment. Moreover, some lines of evidence suggest a compensatory upregulation of energy intake with rising physical activity energy expenditure, especially at higher workloads and energy expenditure levels (reviewed in (90, 112, 151, 80). Therefore, it remains unclear whether substantially increasing NEAT for body mass control under free-living conditions will be capable of inducing weight loss without caloric restriction. Probably this shows large inter-individual variability, since it was recently shown that subjects losing weight over a two year time span increased their daily activity by about 35 minutes, while subjects gaining weight showed a reciprocal decrease (113). Energy intake was comparable in both of these groups. Contrasting with that, subjects either losing body weight or weight maintainers did not show alterations in daily activity as compared to weight gainers in another study (152). Thus, even under real life conditions there might be “responders” and “non-responders” in terms of daily activity/NEAT and body weight control. These groups remain, however, to be better defined.

Recent work could help to better categorize “responders” and “non-responders” by applying the above mentioned constrained energy expenditure model (153). The authors hypothesized that total energy expenditure would increase with physical activity at low levels, while plateauing at higher activity levels. It was found that after adjusting for body size and composition, TEE is indeed positively associated with PEE/AEE, but the relationship was markedly stronger over the lower range of physical activity. For subjects in the upper range TEE plateaued (Figure 9). According to the author’s hypothesis, body fat and activity intensity appeared to modulate the metabolic response to physical activity (153). In other words, subjects who maintain high levels of physical activity in their daily lives could show a smaller response to attempts of further increasing NEAT as it contributes to TEE increment, while subjects with habitually low activity levels could increase NEAT contribution and can therefore possibly be categorized as “responders”.

Figure 9. Effects of physical activity on PEE/AEE and its components (adapted from (153)).

Since hypocaloric diets can significantly reduce NEAT (see previous sections), a major goal has to include prevention of this reduction in NEAT. There is strong evidence that physical activity is essential for the prevention of weight regain after weight loss, and for weight maintenance (16, 80). For instance, it was shown that low physical activity levels are related to significant weight regain at follow up after hypocaloric diets (154). Furthermore, when subjects gaining weight under free-living conditions are compared to weight stable counterparts, the “weight gainers” demonstrated markedly lower physical activity related energy expenditure and less muscle strength (155). Of note, after a one year period, lower physical activity energy expenditure explained approximately 77% of body mass increase in the “weight gainers” as compared to “weight maintainers” (155). A recent meta-analysis on weight loss maintenance shows that physical activity has a significant treatment effect of approximately 1.6 kg in the short term (156). However, long term this effect was lost, probably due to reduced compliance since effects of voluntary physical exercise were examined (156).

As discussed above, for prevention of a decrease in NEAT resulting from hypocaloric dieting, an effective strategy could therefore be to recommend moderate voluntary exercising during weight reduction, and then to engage in NEAT-enhancing activities at the initiation of weight maintenance. This strategy could mitigate poor long-term compliance and substantially contribute to lower body mass preservation. However, when purposeful training is initiated in combination with a hypocaloric diet, the exercise schedule must be moderate as too intense of an exercise regimen during caloric restriction can disproportionally reduce NEAT rather than stabilize it (157, 85). Otherwise, even purposeful exercise training cannot completely overcome hypocaloric diet-induced reductions of TEE, as the REE-component remains largely unaffected by this kind of intervention even when LBM is preserved (85, 72, 36, 80).

From a practical point of view this is a central issue, as after weight loss reduced TEE will persist over years. This was recently shown by a follow up study on participants of the “The Biggest Loser” televised weight loss competition in the U.S.(158). Fourteen subjects who were severely obese lost approximately 59 kg during a 30 week period of strenous physical exercise in combination with dieting. Fat mass was primarily lost, while LBM was largely preserved (158). Compared to baseline, by the end of the 30-week competition TEE decreased significantly by 800 kcal per day, which was mainly explained by the decline in RMR, approximating 600 kcal per day (158). Unfortunately, NEAT was not directly measured. However, keeping in mind that subjects underwent an intense physical exercise schedule it can be hypothesized that the observed difference of TEE and RMR (e.g. 200 kcal per day) was contributed to by a decline in spontaneous physical activity, which is comparable to earlier studies (157, 39, 72). After six years, the participants had regained 41 kg, TEE was reduced by roughly 400 kcal day-, and in spite of the significant weight regain, RMR remained reduced by 600 kcal per day (158). Therefore, after six years, “The Biggest Loser” televised weight loss competition resulted in a total weight loss of approximately 12 kg in subjects who were previously severely obese. The data indicate that these individuals will have to live with remarkably reduced dietary energy needs, even after adjustment for body weight and age (158) that contributes to their ongoing weight regain. Remarkably, the fact that after six years the reductions in RMR were lower than TEE indicates that some form of compensation is occuring, but it is unclear which component of daily energy needs is accounting for it (Figure 1). From the previously reported findings it can surmised that decreased NEAT was one of the factors contributing to the large weight regain (158, 39). Indeed, it was shown that participants of the “The Biggest Loser” television show with high levels of physical activity over years had significantly lower body mass regain compared to subjects with low physical activity levels. Weight loss maintainers had physical activity thermogenesis of roughly 12 kcal/kg/d as compared to weight regainers with approximately 8 kcal/kg/d, which was reflectetd by a significant negative correlation of body mass regain and physical activity levels (114).

Overall, moderate voluntary exercise results in significantly preserved weight loss during hypocaloric dieting (159, 160), which can be a helpful tool for preventing significant decreases in NEAT that will later help to stabilize body weight loss. With initiation of weight maintenance, subjects should be encouraged to engange in actvities increasing NEAT, to compensate at least in part for the obligatory weight loss-induced reduction in RMR. As stated previously, additional voluntary exercise is a useful tool for weight maintenance, but engagement in NEAT-related activities can be more easiliy integrated in daily life and will therefore likely result in higher adherence and compliance rates.

In terms of appropriate activities, it can be relatively easy to increase NEAT (Figure 10). Standing instead of sitting burns three times more kcal per hour, gum chewing increases energy expenditure four times, and stair climbing more than 40 times above resting levels (45). Ambulation (e.g., walking) in particular can raise NEAT and is easily performed at almost any place and at any time. But how can a goal of 2.5 hours of additional daily ambulation/standing time be realistically be integrated into daily routine? A key problem is that people’s occupations and personal lives regularly contribute to prohibit this degree of adaptation (45). As one’s occupation is the principal determinant of NEAT in adulthood and can represent an efficient means of promoting physical activity (45), how NEAT can be primarily integrated at the site of occupation needs examining. One important issue in this context addresses transportation to work. Similar to the NHANES data for the United States (42, 43, 41), recent results from the English and Welsh 2011 Census show that among the 23.7 million adult commuters, approximately 67% used private motorized transport as their usual main commute mode, while about 18% used public transport (161). In contrast, only 10.9% walked and 3.1% cycled (161). Thus, the majority of subjects rely on motorized transport. Promoting cycling or walking as a daily routine, not only with regard to transportation to work but also with respect to other daily needs (such as going to the grocery store), could represent a promising and realistic way for a majority of subjects to increase individual NEAT levels.

Figure 10. Effects of physical activity on PEE/AEE and its components (adapted from (153)).

Together, 2.5 hours of additional ambulation and standing time per day are proposed as goals for those considering weight loss. However, potential beneficial effects on weight loss could be at least partially compensated for by increasing energy intake from food. Increasing NEAT as an adjunct for weight maintenance could overcome potential compensatory effects to the low-calorie state and hypothetically represent an alternative strategy for body mass control.

Limitations to Increasing NEAT

A number of factors potentially affect time spent in NEAT, of which some (sociological and environmental factors) can be influenced, while others cannot (genetic and endocrine factors) (Figure 11). Two points are important to make. First, as was shown previously in this chapter, skeletal muscle can increase in fuel economy and work efficiency not only in response to the loss of body mass, but also so that benefits of NEAT may be diminished. Moreover, it has also been reported that under weight-loss conditions, subjects with obesity oxidize proportionally more carbohydrates and less fat as compared to lean counterparts, and that differences in skeletal muscle metabolism and SNS activity underlie some of the observed differences between subjects who are and are not obese (reviewed in (21)). And second, even though regular physical activity can increase the capacity of skeletal muscle to oxidize lipids and to store glycogen (85), regular physical activity can also increase work efficiency and thereby possibly reduce the energetic costs of NEAT (85). Even so, given the known contribution of physical activity to body weight maintenance, the recommendation to patients who are overweight and with obesity to increase NEAT-related activities such as ambulation or bicycling is logical, even before definitive evidence is reported.

Figure 11. Factors interfering with spontaneous physical activity and NEAT (adapted from (120)).

CONCLUSION

The epidemic of worldwide obesity in past decades is contributing to serious health concerns. Apart from poor diet, reduced physical activity and increased sedentary behavior contribute to the pathogenesis of obesity. Non-exercise activity thermogenesis is a highly variable component of daily total energy expenditure and essentially a function of environmental and individual factors. Whether caloric overfeeding systematically affects non-exercise activity thermogenesis is a matter of debate, while a negative energy balance due to voluntary caloric restriction and/or exercise can decrease it. As physical activity contributes to weight maintenance and prevention of body mass regain after hypocaloric dietary interventions, overcoming current obesogenic environmental pressures and increasing non-exercise activity thermogenesis could holds tremendous promise as a tool for body weight control.

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The Control of Food Intake in Humans

ABSTRACT

Knowledge of the factors influencing food intake is crucial to form an understanding of energy balance and obesity. Classical physiological feedback models propose that eating behavior is stimulated and inhibited by internal signaling systems (for the drive and suppression of eating, respectively) to maintain stability of the internal environment (usually energy or nutrient stores). However day-to-day food intake involves complex interactions of both internal and external inputs coordinated through homeostatic, hedonic, and cognitive processes in the brain. Twenty-five years ago, the term ‘obesogenic environment’ entered into scientific discourse and implies that prompts, cues, and triggers from the external environment are largely responsible for the increases in food intake that underlie the epidemic of obesity. This approach revitalized interest in the sensory and external stimulation of food intake and has drawn attention to the hedonic dimension of appetite. There is now a very strong current of thought that energy balance regulation is asymmetric and excess food intake is due to poor homoeostatic defense against positive energy balances in an environment rich in available, accessible, and readily assimilated food energy. This does not mean that regulatory signals concerned with energy balance regulation are unimportant. Indeed, they appear to be of incremental importance in prolonged negative energy balances and help explain control and loss of control of food intake as a dynamic continuum.

INTRODUCTION

Traditionally food intake has been researched within the homeostatic approach to physiological systems pioneered by Claude Bernard (1), Walter Cannon (2) and others, and because eating is a form of behavior, it forms part of what Curt Richter referred to as the behavioral regulation of body weight (or behavioral homeostasis) (3). This approach views food intake as the vehicle for energy supply whose expression is modulated by the metabolic requirement to replenish energy stores. The idea was that eating behavior is stimulated and inhibited by internal signaling systems (for the drive and suppression of eating, respectively) in order to maintain stability in the internal environment (energy stores, tissue needs). Since this time, it has become clear that energy intake is not as tightly regulated under modern environmental conditions as symmetrical negative feedback models initially suggested. Compensatory changes in physiology and behavior are more pronounced in response to negative than positive energy balances. Furthermore, energy intake is determined by eating behavior, which itself, may be determined by a number of complex physiological, environmental, social, and cultural factors. One should not perhaps expect to see strict regulation of food or energy intake on a day-to-day basis in modern environments.

IS EATING BEHAVIOUR REGULATED?

The control of appetite is often viewed to operate within an energy balance model of body weight regulation, but this should not lead to the view that appetite is controlled simply as an outcome of energy balance. Eating (food, energy, and nutrient intake) is a form of behavior, and like many aspects of volitional behavior, the factors leading to changes in that behavior are complex and are the outcome of factors which can be described by behavior change models. Using a COM-B model of behavior (Capability, Opportunity, Motivation, Behavior) (4), these factors include capability (psychological or physical ability to enact the behavior), motivation (reflective and automatic mechanisms activate or inhibit behavior), opportunity (physical and social environments that enables the behavior). Given the interacting and complex nature of these influences it is to be expected that unless homeostatic or other physiological feedback signals are particularly powerful, their effects on eating behavior would be difficult to detect. It is often implied (but not explicitly stated) that eating behavior is part of a feedback loop that is subsumed to the regulation of energy balance, but under the conditions of modern environments, there is little rationale for why eating behavior per se should be a regulated phenomenon as described by classic homeostatic models. Eating behavior may show repeated patterns which are stable over time, but these patterns may be quite different from the concept of regulation described in typical homeostatic models of blood glucose regulation or thermoregulation for example.

While eating behavior is influenced by a number of factors, it is reasonable to argue that some of those factors may become particularly salient under specific physiological or environmental circumstances. This is important as energy balance regulation appears asymmetric, with compensatory changes in physiology and behavior more pronounced in response to negative than positive energy balances. The exact mechanisms that oppose energy deficits are complex, inter-related, and individually subtle (5, 6). While energy expenditure and its components change in response to energy deficits in a quantitatively important manner, it is likely that changes in energy intake (EI) have a greater capacity to produce relatively large alterations in energy balance and body composition (7). The physiological and psychological impacts of weight loss likely occur on a continuum, with the point on this continuum influenced by (i) the degree of energy deficit, (ii) its duration, (iii) body composition at the onset of the energy deficit, and (iv) the psychosocial environment in which it occurs (8). In contrast, as weight is progressively gained there is very little evidence of physiological or behavioral systems exerting negative feedback to actively limit further weight gain. However, there is considerable heterogeneity in the rate of weight gain during overfeeding (9), which may in part, reflect inter-individual variations in physical activity and/or partitioning the excess energy between fat mass (FM) and fat-free mass (FFM) (10, 11).

The implication of asymmetric energy balance regulation would be that appetite is under stronger physiological control in relation to negative energy balances, whereas in a state of energy balance or positive energy balances weak linkages (negative feedback) exist between physiological functioning, food intake, and the motivation to eat. If energy balance regulation is asymmetric and modern environments are spatially and temporally rich in energy dense foods, it is logical to assume that many of the factors that shape eating behavior and energy intake are due to environmental influences such as sensory and environmental cues for food intake (which are presumably mediated through hedonic and other affective mechanisms). There is now a strong current of thought that a major cause of an increase in food intake associated with the rise of obesity resides in the hedonic rather than the homeostatic system. Some authors argue that in the resource limiting environments in which we evolved, hedonic and homeostatic systems functioned in a synchronized manner to facilitate over consumption during relatively brief periods of food abundance. In an environment where food resources are unpredictable and finite, overconsumption would have been an adaptive behavior limited (capability, opportunity) by environmental uncertainty. Natural selection would favor such behaviors. There would have been little need to evolve systems that protect against weight gain as it would be an improbable outcome in resource limited environments. This does not mean that the so-called ‘energy homeostasis system’ is no longer important in modern environments. Modern day environments have changed very rapidly and radically relative to the environment shaping energy balance regulation. Therefore, to understand how homeostasis and hedonics may influence food intake in modern environments requires an appreciation of the asymmetry of energy balance regulation, the time course over which such regulation may operate, and the very rapid time-course over which the modern food environment has changed.

FOOD INTAKE AND APPETITE CONTROL

The Motivation to Eat

Within the COM-B model of behavior, motivation is an important factor influencing behavior if capability and opportunity do not constrain behavior (as is the case in today’s so called ‘obesogenic’ environment). In the field of ingestive behavior, motivation to eat usually refers to reflective processes that are subjectively experienced or expressed. They are often believed to relate to underlying physiological or external environmental influences, but have the status of a self-reported, subjectively expressed psychological construct. Appetite has been defined as the subjective expression of willingness or motivation associated with qualitative selection and quantitative consumption of specific foods during an ingestive event (12). Appetite is not necessarily solely related to situations of nutritional depletion and can be influenced by a number of physiological and non-physiological factors. Appetites are specific to certain foods, often learned and frequently sensory specific (13). Unfortunately, the term appetite control is often used without specifying what is being controlled. Does eating behavior change to maintain some constancy and motivation to eat? Does appetite change in order to control food intake around some central tendency? Does appetite change with the aim of changing food intake to regulate energy balance? Often it is the latter view that is implied but not clearly articulated.

Table 1. Key Psychobiological Components of Appetite (14, 15)
COMPONENT OF APPETITE	DEFINITION
Hunger	The subjective sensation described as the primary motivation to eat. An increase in subjective hunger usually predicts meal initiation under ad libitum feeding situation. It does not necessarily predict type or amount of food eaten.
Satiation	The process during a meal that generates the negative feedback leading to its termination (within-meal inhibition).
Satiety	The degree of satisfaction and/or fullness following food consumption. This bears some reciprocal relationship to hunger and inhibits further motivation to eating.
Liking	The sensory pleasure elicited by contact with food contributing to the hedonic motivation to consume (wanting).
Wanting	The motivation to consume a specific food, manifesting explicitly (desire to eat) or implicitly.

Because a great deal of human behavior is both reactive and learned, it is possible that the environment can produce prompts, cues, and stimuli that influence learned patterns of motivation to eat. Because eating behavior is a significant determinant of energy balance it is often argued that manipulation or control of motivation to eat (commonly termed appetite control) can be used as a means to prevent excess energy intake and obesity, usually via putative mechanisms of satiety. This is a logical proposition particularly if appetite is not actually very tightly controlled with reference to overconsumption and the development of obesity. Some characteristics of the expression of appetite do appear to render individuals vulnerable to over-consumption of food - these characteristics can be regarded as risk factors that vary between individuals (16). Other significant and salient environmental, acquired, and inherited influences on eating behavior aside, there has been considerable work dedicated to trying to conceptualize and understand putative mechanisms that may link motivation to eat to food and energy intake.

A CONCEPTUAL THEORETICAL MODEL LINKING PHYSIOLOGY, MOTIVATION, AND BEHAVIOR TO FOOD INTAKE

One of the most commonly accepted theoretical models for the control of appetite is the satiety cascade, a putative network of interactions between physiological, psychological, and behavioral factors which form a psychobiological system. The term psychobiological assumes that physiological functions provide internal cues that may impact motivation to eat, and can be conceptualized on three levels (Figure 1). These are the levels of psychological events (hunger perception, cravings, and hedonic sensations) and behavioral operations (meals, snacks, energy, and macronutrient intakes); the level of peripheral physiology and metabolic events; and the level of neurotransmitter and metabolic interactions in the brain (17) (see for Andermann & Lowell (18) for a review of the central control of food intake). Appetite reflects the synchronous operation of events and processes in the three levels. Implicit in this theoretical model is the notion that the physiological signaling systems influence motivation to eat and that motivation to eat shapes eating behavior. The model suggests that neural events trigger and guide behavior, but each act of behavior involves a response in the peripheral physiological system. In turn, these physiological events are translated into brain neurochemical activity that is related to the strength of motivation to eat and the willingness to refrain from eating. In this model it is assumed that motivations to eat change with the aim of changing food intake to regulate energy balance. The lower part of the psychobiological system (Figure 1) illustrates the satiety cascade links motivation and behavior to peripheral and central signals related to eating. It also includes those behavioral actions which actually form the structure of eating, and those processes which follow the termination of eating and which are referred to as post-ingestive or post-prandial events.

Figure 1. The satiety cascade, as originally presented (17), showing the expression of appetite as the relationship between three levels of operations: the behavioral pattern, peripheral physiology and metabolism, and brain activity. See for Andermann & Lowell (18) for a more recent review of the central control of food intake. PVN, paraventricular nucleus; NST, nucleus of the tractus solitarius; CCK, cholecystokinin; FFA, free fatty acids; T: LNAA, tryptophan: large neutral amino acids.

EPISODIC AND TONIC SIGNALS OF APPETITE CONTROL

Traditionally a distinction has been drawn between episodic and tonic signals in the control of appetite (19). Episodic signals are mainly inhibitory (but can be excitatory) and are usually generated by episodes of eating. These signals oscillate in accordance with the pattern of eating, and most are closely associated with the signaling of satiety. Tonic signals arise from tissue energy stores such as adipose tissue and metabolically active tissues to exert some degree of feedback on the expression of appetite to match day-to-day food intake with longer-term energy needs. These two sets of signals, one set responding sharply to nutrient flux and the other providing a slow modulation of appetite and food intake, are integrated within complex brain networks that control the overall expression of appetite. Examination of these putative mechanisms tend to be more common in acute or short-term studies of ingestive behavior in which the primary change is motivation to eat or eating behavior rather than longer term studies. Short-term experiments are valuable for mechanistic understanding, but such studies often make the assumption that changes in the motivation to eat or eating behavior will translate in the long term into aspect of energy balance regulation. However, concentrating only on short-term effects without considering the longer-term time scale may fail to reveal the way that food intake is affected or energy balance is regulated as the experimental time window is much narrower than is relevant for such regulation to occur (e.g., weeks and months rather than minutes, hours or days). Put simply, many investigators may be looking for evidence of regulation over a period where no such regulation is likely to occur. It is therefore important to distinguish between longer term (tonic) mechanisms of putative energy balance regulation and shorter term (acute, episodic) mechanisms that may affect motivation to eat or EI.

Episodic Appetite Signals

Episodic signals are those physiological events that are triggered as responses to the ingestion of food. These form the inhibitory processes which first of all stop eating and then prevent its re-occurrence and are therefore termed satiety signals. The types of signals involved in terminating a meal (satiation) and preventing further consumption (post meal satiety) can be represented by the satiety cascade. Initially the brain is informed about the amount of food ingested and its nutrient content via sensory input. The gastrointestinal tract is equipped with specialized chemo- and mechano-receptors that monitor physiological activity and pass information to the brain mainly via the vagus nerve (20). This afferent information constitutes one class of ‘satiety signals and forms part of the pre-absorptive control of appetite. It is usual to identify a postabsorptive phase that arises when nutrients have undergone digestion and have crossed the intestinal wall to enter the circulation. These products, constitute the flux of energy and nutrients into the circulation, may be metabolized in the peripheral tissues or organs, or may enter the brain directly via the circulation. In either case, these products constitute a further class of metabolic satiety signals. Additionally, products of digestion and agents responsible for their metabolism may reach the brain and bind to specific chemoreceptors, influence neurotransmitter synthesis or alter some aspect of neuronal metabolism. In each case the brain is informed about some aspects of the metabolic state resulting from food consumption. It seems likely that chemicals released by gastric stimuli or by food processing in the gastro-intestinal tract are involved in the control of appetite (21). Many of these chemicals are peptide neurotransmitters, and many peripherally administered peptides cause changes in food consumption (22). (Please refer to ENDOTEXT chapter ‘Endocrinology of The Gut and the Regulation of Body Weight and Metabolism’ by Andrea Pucci and Rachel L. Batterham for additional information on gut hormones physiology).

Cholecystokinin

Cholecystokinin (CCK) is a hormone released in the proximal small intestine mediating meal termination (satiation) and possibly early phase satiety. CCK reduces meal size and also suppresses hunger before the meal; these effects do not depend on the nausea that sometimes accompanies an IV infusion (23). Food consumption (mainly protein and fat) stimulates the release of CCK (from duodenal mucosal cells), which in turn activates CCK-A type receptors in the pyloric region of the stomach. Fat in the form of free fatty acids (FFA) of carbon chain lengths C12 and above produce pronounced CCK releases (24, 25). This signal is transmitted via afferent fibers of the vagus nerve to the nucleus tractus solitarius (NTS) in the brain stem. From here the signal is relayed to the hypothalamic region where integration with other signals occurs.

Animal data suggest that endogenous CCK release mediates the pre-absorptive satiating effect of intestinal fat infusions, and may in turn be critical in regulating the intake of fat (26). As in rats, intestinal infusions of fat produce a reduction in food intake and promote satiety in humans (27). In humans the satiety effect of fat infused directly into the duodenum can be blocked by the CCKA receptor antagonist loxiglumide (28). High-fat breakfasts have been shown to produce both greater feelings of satiety (signified by reduced levels of hunger, desire to eat and prospective consumption) and elevated endogenous plasma CCK levels. Collectively, these studies support the theory that CCK plasma levels are a potent fat (or fatty acid) -stimulated endogenous satiety factor, whose effects on food intake and eating behavior are mediated by CCKA receptors.

It has also been shown that synthetic CCK-A type agonists suppress food intake in humans. A drug, known by the number ARL1718, caused a significant reduction in meal size and had a longer duration of action than observed after infusions of CCK itself. A number of other CCK analogues / CCK 1 receptor agonist treatments have been developed including most recently GW181771 (GlaxoSmithKline) and SR146131 (Sanofi-Aventis). Studies with such drugs, together with those on the peptide hormone itself, do suggest that CCK has the properties of a true satiation signal which contributes, under normal circumstances, to the termination of a meal. However, CCK is not uniquely involved in the expression of satiety and is also involved in a spectrum of physiological responses generated following nutrient consumption. The action of CCK certainly acts in concert with other meal-related events, such as gastric distention for example.

Glucagon-Like-Peptide-1

Glucagon-like peptide (GLP)-1 is an incretin hormone, released from the gut into the blood stream in response to intestinal nutrients. Endogenous GLP-1 levels increase following food intake, particular of carbohydrate (29, 30). These studies suggest a role for GLP-1 in mediating the effects of carbohydrate (specifically glucose) on appetite. In healthy men of normal weight, infusions of synthetic human GLP-1 (7-36) during the consumption of a fixed breakfast test meal, enhanced ratings of fullness and satiety when compared to the placebo infusion (31). During a later ad libitum lunch, food intake is also significantly reduced by the earlier GLP-1 infusion. Intravenous GLP-1 also dose-dependently reduces spontaneous food intake and adjusts appetite in healthy weight male volunteers. This marked reduction in food intake and enhancement in satiety is also observed in male patients living with overweight or obesity and type 2 diabetes. In men living with obesity, intravenous GLP-1 potently reduces food intake either during or post-infusion (32) and, at lower sub-anorectic doses, slows gastric emptying. Reductions in intake and slowed gastric emptying are accompanied by decreased feelings of hunger, desire to eat and prospective consumption, and a prolonged period of post-meal satiety. These data demonstrate that exogenous GLP-1 reduces food intake and enhances in satiety in humans, both those healthy weight and living with obesity. However, it should be kept in mind that the doses of GLP-1 often administered are usually higher than the normal values seen in blood after a meal. Consequently, although GLP-1 receptors could be a possible target for anti-obesity drugs, the physiological role of GLP-1 itself in the normal mediation of satiety is still not confirmed. Nonetheless, GLP-1 through its action as an incretin which prompts the release of insulin, will certainly have some indirect role on the pattern of eating behavior. Interestingly, two of the most promising drug options for people living with obesity are liraglutide and semaglutide; both GLP-1 agonists. These drugs have been shown to decrease hunger (33) and/or increase satiety (34). The action of the pharmacological agents may mean that people living with obesity gain some control over their eating behaviors, however longer-term studies are required to investigate their true potential. In addition, it should be noted that the GLP-1 receptors responsible for the anti-obesity action of semaglutide and liraglutide are located in the brain rather than the periphery.

Peptide YY _3-36

Peptide YY _3-36 (PYY _3-36) is one of the two main endogenous forms of PYY. It is produced from the cleavage of PYY _1-36 (the other major form of PYY) by dipeptidyl peptidase IV (DPP IV). PYY is a 36 amino acid ‘hind gut’ peptide released from endocrine cells in the distal small intestine and large intestine. This hormone is similar in structure to the orexigenic neuropeptide NPY (70% amino acid sequence identity), and in the past, PYY has been regarded, like NPY, as a potent stimulator of food intake. However, in a series of studies in rats, mice and in one human study (all included in one paper), Batterham et al. (35) have demonstrated that peripheral PYY _3-36 administration reduces food intake and inhibits weight gain in rodents. These effects on intake and body weight are not observed in transgenic animals lacking NPY Y2 receptors (the NPY Y2 receptor knock-out), thereby implicating these receptors in mediating the anorectic effects of PYY. PYY release in the distal intestine is triggered by a variety of nutrients, including fats (particularly FFA), some forms of fiber and bile acid (24, 25). In humans, endogenous PYY is released predominantly after, rather than during a meal (35, 36) and causes a decrease in gastric emptying (the so-called ‘ileal brake’). Thus, it is more associated with post-meal satiety. PYY (including PYY _3-36) can cross the blood brain barrier via a non-saturable mechanism. Moreover, some of the effects of peripheral PYY _3-36 on food intake are either independent of or dependent on vagal afferents running from the periphery to the brain (37, 38).

With regard to the effect of PYY on human appetite, Batterham et al. (35) demonstrated that in healthy humans a 90-minute PYY _3-36 infusion reduced hunger and subsequent food intake two hours later. In a further report, PYY infusions in people who were either a healthy weight or living with obesity caused a 30% reduction in lunch intake post infusion and decreased the 24 h energy intake by 23% in those with a healthy weight and by 16% in those living with obesity (36). The natural plasma levels of PYY were lower in those with living with obesity than in the healthy weight participants, and were inversely correlated with the body mass index. The lower levels of PYY in those with living with obesity could mean a weaker satiety signaling through this hormone and therefore a greater possibility of over-consumption. However, as the authors noted these effects required doses greater than the normal physiological range of endogenous PYY and marked nausea was observed in one experiment (39-41).

Amylin

Research has also focused on amylin, a pancreatic rather than a gastrointestinal hormone, which also has a potent effect on both food intake and body weight (42). Peripheral administration of amylin reduces food intake in mice and rats, and meal size in rats. Chronic or peripheral administration of amylin over a period of 5 to 10 days produces significant reductions in cumulative food intake and body mass of rats (43). Thus, amylin appears to be a component part of the appetite regulation system. The effects of amylin on human food intake, food choice or appetite expression has yet to be fully assessed. However, pramlintide (a human amylin analogue), given to replace deficits in endogenous amylin in people with diabetes, has been shown to alter body weight in people living with obesity and diabetes treated with insulin (44-46) and people with obesity without diabetes (47). In healthy weight participants pramlintide induces reductions in meal intake and duration, and reduces pre meal appetite (48). Similar effects of pramlintide on intake and eating behavior are reported in people living with obesity (with and without type 2 diabetes) (49, 50).

Ghrelin

In contrast to the peptides mentioned above, ghrelin is the only known excitatory peptide released in the gastrointestinal system. Ghrelin is a 28-amino acid peptide that stimulates the release of growth hormone from the pituitary (51). Secreted primarily from the stomach, it is also found in a number of other tissues (52, 53). Ghrelin was the first excitatory peptide discovered and acts upon the hypothalamic arcuate nucleus (51, 54, 55). The composition and action of ghrelin is uniquely modified by the addition of an octanoyl group to the serine residue at position three. Some studies suggest this acylation is crucial for ghrelin to bind to the growth hormone-secretagogue receptor (GHS-R) and cross the blood-brain barrier (56). Ghrelin’s effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein in the central nervous system (57).

The majority (80-90%) of circulating ghrelin is in the deacylated form (51). Two theories have been proposed, firstly that deacylated ghrelin could result from incomplete acylation of the peptide, with both forms utilising differently regulated pathways, or secondly, that DG could result from the deacylation of ghrelin (58). More recently, deacylated ghrelin has been termed the inactive form. Ghrelin is thought to be involved in meal initiation as it is high during periods of fasting and decreases in response to food intake, thus suggesting a physiological role for ghrelin in meal initiation (59). Intravenous infusion or subcutaneous injection of ghrelin in humans increases both feelings of hunger and food intake (56, 60) and to promote increased food intake, weight gain and adiposity in rodents (60).

Satiety Cascade Peptides

In the overall control of the eating pattern, the sequential release and then de-activation of the peptides described above, can account for the evolving biological profile of influence over the sense of hunger and the feeling of fullness (61). The actions of these hormones therefore contribute to the termination of an eating episode (thereby controlling meal size) and subsequently influence the strength and duration of the suppression of eating after a meal. Evidence of this is shown by Gibbons et al. (62) whereby the post meal period was separated into early and late phases of satiety. It should be noted that whilst the profiles of peptide response shown in papers often show the expected increase and decrease (in the case of satiety peptides) and decrease and increase (in the case of ghrelin), there is a wide degree of individual variability in peptide responses. This is not often commented on, or shown. Furthermore, the majority of papers do not measure a range of peptides but rather focus on one or two. Individual variability in the release and maintenance of the levels of hormones (or the sensitivity of receptors) may determine whether some individuals are prone to snacking between meals or to other forms of opportunistic eating. The overall strength or weakness of the action of these peptides will help to determine whether individuals are resistant or susceptible to weight gain. Individual variability in the response of gut peptides to different food types has been shown more recently (63) and can be seen in Figure 2. At present, it does not appear that a poor response in one peptide means a poor response in all peptides, and it is likely that the cumulative response of the peptides (of which there are many) is key for the modulation of appetite and EI. Since different foods may produce the same effect on hunger and fullness but display quite distinctive profiles of post-prandial peptides, this suggests that the satiety signaling system is complex and there is no single unique pattern of peptides that defines satiety. This questions to what extent short term satiety can be accounted for by individual changes in putative satiety peptides, and what other factors may contribute to subjective satiety or cessation of eating behavior in humans.

Figure 2. Panel A shows the average ghrelin suppression after high fat and low-fat meals and Panel B and C shows the individual profiles of ghrelin for each participant after both high and low-fat meals. Adapted from (63).

TONIC SIGNALS OF APPETITE CONTROL

Ghrelin and the Hunger Drive

As noted above, ghrelin is an episodic peptide increasing during periods of fasting and decreasing in response to food intake. However, ghrelin is also unique since it has been proposed that in addition to being linked to the initiation of eating, ghrelin also acts as a compensatory hormone. Circulating ghrelin decreases in response to overfeeding and increases in response to chronic negative energy balance such as occurs with exercise or anorexia nervosa (64). This means that in people living with obesity and in animals experimentally made fat, circulating ghrelin levels would be reduced in an apparent attempt to restore a normal body weight status. Therefore, ghrelin illustrates the characteristics of both an episodic and tonic signal in appetite control. From meal to meal the oscillations in the ghrelin profile act to initiate and to suppress hunger; over longer periods of time, some factor associated with fat mass applies a general modulation over the profile of ghrelin and therefore, in principle, over the experienced intensity of hunger. At some point, it seems likely that people living with obesity are insensitive to lower ghrelin levels and/or other factors outweigh the relative importance of circulating ghrelin. When weight is lost, for example following a period of food restriction and weight loss, ghrelin levels would rise (or normalize), and therefore promote the feeling of hunger. This is likely to be one of the signals that makes the loss of body weight difficult to maintain. Ghrelin blockade therefore may prove a useful anti-obesity treatment. Whilst people living with obesity have lower fasting ghrelin levels, they have been shown to show a similar response to infused ghrelin as normal-weight participants, that is, increased food intake (65). Ghrelin levels in people living with obesity do fall after food, but not to the same degree as healthy weight participants in whom different calorie loads were shown to decrease ghrelin levels in a dose-response manner, but in people living with obesity this clarity was not shown as clearly (66). This points towards a potential mechanism for weight gain to be a consequence of a down regulation of gut peptide signaling and that the sensitivity to ghrelin is being overridden by other factors, for example, hedonic control of appetite.

The Role of Leptin

One of the classical theories of appetite control has involved the notion of a long-term signal, leptin, which informs the brain about the state of energy stored in adipose tissue (67). In 1994 a mouse gene that controls the expression of a protein by adipose tissue which could be measured in the peripheral circulation (leptin) was discovery. It is now well accepted that there is a good correlation between the plasma levels of leptin and adipose tissue (68), and leptin interacts with NPY, one of the brain’s most potent neurochemicals involved in appetite, and with the melanocortin system, in the central control of appetite. Alongside other neuromodulators involved in a peripheral-central circuit, leptin links adipose tissue with central appetite mechanisms and metabolic activity. A number of mutations in the genes controlling molecules in the leptin-insulin pathways are associated with the loss of appetite control and obesity. For example, the MC4-R mutation (melanocortin concentrating hormone receptor 4) leads to an excessive appetite and massive obesity in children, similar to leptin deficiency. Studies have shown for individuals with a genetic form of leptin deficiency that leptin treatment produces dramatic weight loss, an effect associated with marked decreases in hunger (69-71). Further, the administration of exogenous leptin to humans, with either an insufficiency in or a specific deficit of endogenous leptin appears to strengthen within meal satiation and post meal satiety (71, 72). However, leptin-based obesity treatments for most individuals with obesity seem inappropriate given they appear leptin insensitive rather than leptin deficient. Indeed, despite extensive literature on leptin and other putative feedback signals arising from adipose tissue (73, 74), there appears to be limited evidence in humans of the extent to which changes in adipose tissue exert strong negative feedback on motivation to eat or EI in individuals in approximate daily energy balance or modest positive imbalances (as characterize the majority of individuals in the modern environment). As a number of questions still exist regarding the applicability of a 'lipostatic' control system to the regulation of appetite in humans free from congenital leptin deficiency (75). Consequently, recent models of human appetite have attempted to integrate the role of both FM and FFM into the control of appetite and energy balance to better account for the peripheral signals of appetite.

Fat-Free Mass and Resting Metabolic Rate and Associations with Appetite

A conceptual model of human appetite that incorporates the energetic demands of metabolically active tissues has been proposed, with a tonic drive to eat arising from components of energy expenditure (e.g., resting metabolic rate; RMR) and its main determinants (e.g., FFM). This model is based on a series of studies demonstating that FFM, but not FM, is associated with hunger and EI under conditons of energy balance (76-81) (see Figure 3). For example, Blundell et al. (78) reported that FFM was associated with self-selected (ad libitum) meal size and total daily EI in 93 individuals living with overweight or obesity. In contrast, no associations were found between FM and EI. Resting metabolic rate, of which FFM is the main determinant (82), has also been found to be associated within-day hunger sensations and EI (80, 81, 83). These findings have been replicated in studies employing a wide range of participants and under a variety of experimental conditions, with the associations between FFM and EI observed under laboratory (79-81, 84) and free-living settings (85, 86), in new born babies (87), adolescents (88, 89), normal weight women (90),people living with moderate (91) or extreme obesity (92), and people of varying ethnic origin (93, 94). These studies provide evidence that the relationship between FFM and EI exists across the entire age spectrum from birth (87), through childhood and adolescence (88) and into adulthood (78, 81, 84, 93, 95) and older age (96). There is also limited evidence that losses of both FFM and FM may be associated with changes in appetite (97) and weight regain (98) following weight loss, suggesting that integrated models of weight loss that account for FFM and FM losses may better explain changes in appetite during prolonged energy deficit (see below sections). However, there are few prospective longitudinal studies relating changes in functional body composition to appetite or EI, and where there is evidence, it often from studies with extreme weight loss induced via semi-starvation or military training.

Figure 3. Association between fat-free mass, resting metabolic rate and daily energy intake (top panels), and a path diagram illustrating a mediation model for the direct effects of FM and FFM on RMR and RMR on EI, the indirect effect of FM and FFM on EI mediated by RMR, and the squared multiple correlations (R2) for RMR and EI. Data originally reported in Hopkins et al. (80, 85).

While often not explicitly discussed in relation to models of human appetite, the concept that energy needs exert influence on food intake is not new. In 1962, Kenneth Blaxter (99) noted that the basal metabolism of mature animals of different species increases to a fractional power of weight, with small animals have a higher basal metabolism per kilogram of weight than the large ones. This implies that to maintain body weight, small animals must obtain each day from food a larger number of calories per unit of the weight the large ones. If the total habitual energy expenditures of different species are all about the same multiples of their basal energy expenditure, then both the calorie intake and the basal metabolism are likely to be proportional to the same fractional power of bodyweight. Furthermore, the energetic demands of individual tissue-organs such as the liver (100) or the growth and maintenance of lean tissues (101, 102) have previously been suggested as sources of appetitive feedback. For example, Millward’s protein-stat theory suggests that lean mass, and in particular skeletal muscle mass, is tightly regulated such that food intake (specifically, dietary protein) is directed to meet the needs of lean tissue growth and maintenance (101). This theory is based on the existence of an ‘aminostatic’ feedback mechanism in which food intake is adjusted in response to amino acid availability to meet the protein demands of lean tissue growth and maintenance.

Studies Examining the Associations between Fat-free Mass, Resting Metabolic Rate and Energy Intake

Using statistical mediation models, a number of cross-sectional studies conducted under conditions of energy balance have demonstrated that the effect of FFM on EI is mediated by RMR (80, 103) and total daily energy expenditure (TDEE) (104), suggesting that energy expenditure per se may exert influence over EI. For example, Hopkins et al. (80) reported that the effect of FFM on EI was fully mediated by RMR i.e. FFM had no ‘direct’ effect on EI but rather ‘indirectly‘ influenced EI via its effect on RMR. In agreement with these findings, Piaggi et al. (104) reported that TDEE accounting for 80% of the observed effect that FFM exerted on EI in 107 healthy individuals. Such findings suggest that the associations between FFM and RMR with EI may reflect a ‘mass-dependent’ effect arising from the energetic demands of FFM and its constituent tissue-organs rather than a specific endocrine signal secreted by these tissue-organs. However, it should be acknowledged that such findings represent statistical rather than biological pathways. Furthermore, skeletal muscle, a major component of FFM by weight, secretes a large number of myokines (105)which provide a molecular signal for bi-directional communication with other organs (106). While myokines such as interleukin 6 (107) and irisin (108) have been linked to food intake and energy expenditure, the specific role that these (and other myokines) play in the control of appetite is unclear.

An important consideration in the proposed relationship between FFM and EI is that FFM is a heterogeneous tissue compartment that is comprised of numerous individual tissue-organs with wide ranging metabolic functions and mass-specific metabolic rates (109-111). Tissue-organ structure and function are tightly coupled and determine their tissue-specific metabolic rate (112). In turn, the tissue-specific metabolic rates of individual organs summate to determine whole-body metabolic rate (e.g., RMR). The maintenance of tissue-organ structural integrity and function is therefore a metabolic priority (112), but to date there has been little attempt to integrate individual tissue-organs and their mass-specific energy expenditures into homeostatic models of human appetite. Recently however, Casanova et al., (113)used whole-body magnetic resonance imaging to examine whether the masses of high-metabolic rate organs (brain, liver, heart and kidneys) were associated with fasting hunger in 21 healthy males (age= 25 ± 3 years; BMI = 23.4 ± 2.1 kg/m²) (114). As expected, fasting hunger was associated with FFM (r = 0.39; p = 0.09) but not FM (r = -0.01; p = 0.99). Interestingley, the association between the combined masses of the high-metabolic rate organs and fasting hunger (r = 0.58; p = 0.01) was stronger than with FFM as a single uniform body compartment. In particular, liver (r = 0.51; p = 0.02) and skeletal muscle mass (r_s= 0.57; p = 0.04) were strongly associated with fasting hunger. As the masses of the liver and skeletal muscle explained ~17% and ~21% of the variance in RMR, respectively, these findings again suggest that energy expenditure per se may exert influence over food intake.

Another important consideration is how behavioral components of total daily energy expenditure (e.g., physical activity or activity energy expenditure) influence energy intake. The effects of physical activity and/or exercise on appetite is discussed below. As noted, physical activity may influence the control of appetite via a number of physiological and psychological pathways (e.g., alterations in gastric emptying (115), appetite-related hormones (116), food reward (117), and eating behavior traits (118)). In addition, physical activity or exercise may also exert influence, albeit modestly, on appetite and EI via its contribution to TDEE. Hopkins et al. reported in 242 individuals in which physical activity and EI were measured under free-living conditions that activity energy expenditure was independently associated with daily EI alongside FFM and RMR (86). As activity energy expenditure only explained 3% of the variance in total daily EI, its effect on daily EI was much more modest than that seen for FFM or RMR. This is perhaps not surprising given the smaller and more variable contribution of physical activity energy expenditure to TDEE as compared to FFM and RMR (119). It could be argued that while FFM and RMR are well placed to exert stable influence over day-to-day food intake, the contribution of physical activity energy expenditure to daily EI is likely to be weaker and more variable (and therefore, also harder to quantify).

Factors Affecting the Strength of Association Between Fat-Free Mass and Energy Expenditure with Energy Intake

It has been suggested that excess FM may disrupt the coupling between FFM and EI, with associations between FFM and EI weaker in those living with obesity than in healthy weight individuals (90, 95, 104, 120). Early work by Cugini et al. reported that a positive association existed between FFM and hunger, while FM and hunger were negatively associated, in healthy weight individuals (121). However, no such associations were seen between FFM or FM and hunger in those living with obesity (120). Based on these data, the authors suggested that FM accumulation may disrupt the feedback mechanisms linking these tissues to hunger. More recently, Grannell et al. reported a positive association between FFM and EI during an ad libitum test meal in 43 individuals living with severe obesity, but the strength of this association was weaker in individuals with a higher BMI (92). To further explore the moderating effect of FM, Casanova et al., (90) examined the linear and non-linear associations between body composition (FFM and FM), energy expenditure (RMR and TDEE) and EI (ad libitum test meal intake and free-living 24-hour EI) in 45 healthy weight and 48 individuals living with obesity. Percentage body fat moderated the associations between RMR (β=-1.88; p=0.02) and TDEE (β=-1.91; p=0.03) with free-living 24-hour EI. Furthermore, FM was negatively associated with test meal EI only in the leaner group (r=-0.43; p=0.004), with a weak non-linear association observed between FM and EI in the whole sample (r²=0.092; p=0.04).

Such findings point to a non-linear relationships between FM and EI, and this may help account for why negative associations between FM and EI have been observed in healthy weight individuals (121, 122), but studies in those living with overweight or obesity often report no association between FM and EI (79, 88, 91, 123). A weaker negative association between FM and EI at higher body fatness is in line with the notion of leptin and insulin resistance (124, 125), which may alter central and peripheral sensitivity to appetite-related feedback signals (126-128). Furthermore, while the contribution of FM to RMR is smaller than FFM (129), its contribution to RMR becomes proportionally larger as FM increases with excessive weight gain. Therefore, differences in the strength and direction of association between FM and EI at higher body fatness may reflect the increased contribution of FM to body weight and RMR alongside a blunting of its inhibitory influence on EI. It should also be acknowledged that the associations between FM and EI likely reflects both biological and psychological factors. Indeed, Hopkins et al., (85) have demonstrated that psychological factors such as cognitive restraint are robust predictors of EI when considered alongside physiological determinants of EI (e.g. FFM and RMR), and have the potential to play a mediating role in the overall expression of EI (85). A recent paper also suggests that the associations between FFM and TDEE with EI may become weaker with age (96). Based on a secondary analysis of the Interactive Diet and Activity Tracking in AARP Study, a biomarker validation study of self-reported diet and PA measures in older adults, Hopkins et al., reported that FFM and TDEE (derived from doubly labelled water) predicted self-reported EI in 590 older adults (mean age 63.1 ± 5.9 years). Interestingly, while the associations between FFM or TDEE and EI existed across age quintiles, age moderated the associations between FFM and TDEE with EI such that these associations weakened with increasing age (96). Please refer to ENDOTEXT chapter ‘Control of Energy Expenditure in Humans’ by Klaas R Westerterp for additional information).

Associations Between Body Composition and Energy Intake During Prolonged Negative or Positive Energy Balances

Another important point to note is that the aforementioned associations between body composition, energy expenditure, and EI are from cross-sectional analysis performed in weight stable individuals at or close to energy balance. However, the effect of FFM on appetite appears to be dependent on energy balance status, with evidence suggesting that losses of FFM may also act as an orexigenic signal during energy deficit. During the Minnesota semi-starvation study (130), 32 healthy men undertook 24 weeks of semi-starvation (25% of weight loss), 12 weeks of controlled refeeding and 8 weeks of ad libitum refeeding. During the last phase (n = 12), hyperphagia remained until baseline levels of FFM were restored. This led to FM accumulation that surpassed baseline levels (i.e., “fat-overshoot”), a phenomenon that has been reported elsewhere following underfeeding or military training (131, 132). As hyperphagia persisted until FFM had been restored to pre-weight loss levels, it was suggested that independent appetitive feedback signals from both adipose tissue and FFM (e.g., a ‘proteinostatic’ mechanism) contributed to the changes in hunger and food intake seen and restoration of body weight (133, 134).

While the demands imposed by semi-starvation or military training on energy balance clearly exceed those experienced during common diet and/or weight loss interventions, evidence also exists to suggest that FFM loss during clinically relevant weight loss may also act as an orexigenic signal. Following weight loss, it is commonly suggested that subjective hunger and orexigenic hormone concentrations increase (135, 136), but studies have also reported no change or reduced hunger following weight loss (137-139). The composition of the weight lost, which is a function of initial body fat, the rate and extent of weight loss, diet composition and exercise (140, 141), may also influence any accompanying changes in appetite. It has been suggested that greater FFM loss during weight loss is associated with increased hunger (97) and weight regain (98) following weight loss. To assess whether changes in body composition occurring during weight loss were associated with subsequent energy balance behaviors under conditions of therapeutic weight loss, Turicchi et al. conducted a systematic review and meta-regression examining weight loss studies in weight clinical weight loss was achieved (mean = 10.9%) and weight regain occurred in the follow-up period (mean = 5.4%) (98). They found that while both greater rate and amount of WL predicted weight regain, the composition of weight loss i.e. the (amount of FM and FFM) explained greater variance in weight loss alone (40% vs 29%) (98). Furthermore, Turicchi et al., reported that greater FFM loss following a 12% reduction in body weight via a low-calorie diet was associated with greater increases in hunger in men (r = 0.69, p = 0.002) but not women (r = 0.25, p = 0.24) (97). In line with these findings, after 5 weeks of very-low calorie diet (500kcal/d) or 12 weeks of low-calorie diet (1250kcal/d) Vink (142) reported that greater FFM loss during energy restriction was associated with greater weight regain during a subsequent 9-month follow-up period. Data examining FFM loss during extensive periods of energy deficit are therefore suggestive that FFM loss may be part of an integrated response driving post-weight loss increases in EI and weight regain, potentially as a means to restore the structural integrity of FFM compartments (although the influence of FFM loss appears more modest than FM loss). These data also emphasize the importance of developing integrative models of energy balance that consider the dynamic relationships between body structure, physiological function, and the way these mechanistic interactions influence key psychological and behavioral determinants of energy balance such as appetite. However, there are few prospective longitudinal studies relating changes in functional body composition to appetite or EI, and further research using advanced imaging methods for tissue-organ composition and multi-compartmental body composition models across a range of initial body compositions and weight losses would provide additional mechanistic insight (97).

Taken together, cross-sectional research in weight stable individuals indicates that greater FFM is associated with increased EI, but research also indicate that FFM loss is associated with increased appetite and EI. If greater FFM is associated with increased appetite, how is it that FFM loss during weight loss is also associated with increased appetite? One explanation is that FFM exerts ‘passive’ and ‘active’ effects on appetite under situations of differing energy balance (6, 143). At or near to energy balance, Dulloo et al. (143) has suggested that the energy demand of FFM and its constituent components create a ‘passive’ background pull on EI that ensures the energetic demands of metabolically active tissues are met through day-to-day food intake. In contrast, during weight loss, FFM loss may act as an ‘active’ orexigenic signal that stimulates increased hunger and EI in an attempt to ensure the preservation of FFM and the functional integrity of its constituent tissue-organs (143). However, it should be noted that long-term studies with longitudinal tracking of appetite, body composition and energy expenditure are rare, particularly under-conditions in which body composition is systematically manipulated.

Body weight gain leads to an expansion of FFM which increases RMR, but how such changes causally influence appetite or food intake has not been examined. As weight is gained, both FM and FFM expand but at different rates. While such changes may not drive weight gain per se, a higher FFM and associated RMR may increase the background tonic drive to eat, favoring maintenance of a higher body weight. Expansion of FM over the long term induces insulin and leptin resistance, expansion of FFM and, in extremis, some slight elevation of RMR, could account for the apparent diminishing negative feedback from FM as adipose tissue expands (90, 95, 104, 120). Therefore, it may be argued that any putative effect of FFM or RMR on the drive to eat may decrease with increasing BMI, since FFM and RMR increase at a decelerating rate with increase in weight, while the energy content of the body expands disproportionately as FM expands. While factors associated with energostatic models of appetite may be unlikely to drive body weight up in the first place, it is not inconsistent with maintenance of a higher body weight once this is achieved by other means. Thus, it may be that RMR is associated with EI at or close to energy balance, but that RMR (and its primary determinant FFM) become dissociated from the process of overconsumption during significant weight gain as the signal(s) becomes ‘overwhelmed’ by other stimuli important in driving weight gain e.g., food availability, sensory variety, dietary energy density and food reward.

HOMEOSTATIC AND HEDONIC PROCESSES OF APPETITE CONTROL

Food intake is clearly influenced by homeostatic processes of hunger and satiety, modulated according to short term and long-term signals of nutritional and energy status and moderated by lifestyle factors such as physical activity. However, human eating behavior is a complex phenomenon and people eat for many other reasons too: for friendship and celebration, in response to sadness or stress. All these cognitive and emotional motives converge on the fact that eating is a potent source of reward. It provides the eater with an instant but temporary hit of gratification. The greater the reward, the harder it is to resist the behaviors that produce it even when the consequences are risky or harmful and especially when the risk is removed in time. Therefore, a key issue in the study of appetite control is the relationship between hedonic and homeostatic drives (144). Historically, hedonic processes have been viewed as a function of nutritional need-state. In a state of depletion, the hedonic response (experienced palatability or pleasure) to energy providing foods is enhanced and when replete, the hedonic effect of these foods is reduced (145). This view is compatible with the association between energy density and palatability (146) and also that the consumption of fats and sugars “energy-dense nutrients“ may be under neuro-regulatory control (147). However, the idea of reward as merely serving the fulfilment of nutritional need is not sufficient to explain non-homeostatic food intake and it is perhaps more useful to try and distinguish the substrates of homeostatic and hedonic systems and to assign them separate identities (148).

Homeostasis and Hedonics: Cross-Talk and Interaction

Advances in our understanding of the molecular and neural mechanisms behind food intake regulation and appetite control are revealing how the reward system can interact with homeostatic mechanisms. For example, cannabinoid receptors and their endogenous ligands (e.g., anandamide) are implicated in the reward system. Peripheral and central administration of anandamide increased appetite in rodents, and this seemed to be related to alterations in incentive value (wanting) for palatable foods (149). However, the cannabinoid system has been shown to interact with homeostatic processes in a number of ways: Leptin signaling becomes defective when hypothalamic endocannabinoid levels are high (150); activation of CB1 receptors prevent the melanocortin system from altering food intake (151); furthermore, CB1 receptors can be found on adipocytes where they may directly increase lipogenesis (152). Opioid neurotransmission also forms part of the biological substrate mediating reward processes of consumption. For example, endogenous opioids are associated with the reinforcing effect of food (especially when palatable) (153, 154). However, there is evidence to show that in a fasted state, the reinforcing effect of food can be reinstated in enkephalin and Î²-endorphin knock-out mice (155). Therefore, homeostatic processes may interact with hedonic signaling to override selective reward deficit.

Hence although homeostatic and hedonic systems can be given separate identities (148), they are also - to an extent - inseparable, with neural cross-talk permitting functional interactions which may influence the organization of eating behavior. From this standpoint, the interaction of homeostatic and non-homeostatic pathways in the neuro-regulatory control of eating may be more important than the two systems studied in isolation. From behavioral and anatomical observations (156), it has been suggested that projections from the hypothalamus to the nucleus accumbens may modulate the motivation to eat via metabolic signals. Furthermore, direct and indirect projections from the accumbens to the hypothalamus may explain the ability for mesolimbic processes “activated by relevant environmental cues and incentives” to essentially hijack the homeostatic regulatory circuits and drive-up energy intake. Further research is necessary to identify the pathways that mediate such interactions; however progress has been made (157).

Liking vs. Wanting Food

The hedonic perspective on appetite control accounts for eating behavior motivated by the expectation or experience of pleasure from consuming specific foods and involves dissociable processes of “wanting” and “liking”. The “liking” component refers to the subjective experience of pleasure elicited by the sensory perception of food and is associated with the release of endogenous opioids acting on localized clusters of neurons termed “hedonic hotspots” (158) The “wanting” component of reward refers to the process by which food is assigned motivational significance or “incentive salience attribution” and is associated with the release of the neurotransmitter dopamine in the mesocorticolimbic pathway. This latter component can be activated by thoughts or cues signaling food and often precedes the actual receipt of food (159).

In human neuroimaging studies, regional differences in the neural activation to food stimuli during either anticipatory or consummatory phases of reward processing are broadly supportive of the distinction between liking versus wanting. Response to passive viewing of high- versus low-calorie foods or cues signaling the imminent receipt of a tasty food are more reliably observed in the amygdala and ventral striatum, whereas the response to the actual taste and consumption of a palatable food is associated with activation in the primary taste cortex in the insular and opercular cortices (160). Some researchers have proposed that differences between individuals who are healthy weight and those with obesity in neural activation to palatable food can be understood as a dissociation in both the direction and region of responding during the anticipatory and consummatory phases of food intake- with greater striatal activation in individuals living with obesity compared to healthy weight controls when a food is wanted but lower activation in liking-related regions when a food is actually tasted (161). However, several inconsistencies in the brain imaging literature have been noted (162), and further research is needed to substantiate this hypothesis.

Liking and wanting for food are often viewed in relation to subjective states or explicit feelings that refer to the everyday understanding of these terms in the context of food choice and food intake (163). Wanting might describe subjective states of desire, craving or perceived deprivation of pleasure, whereas liking is characteristically understood as the perceived hedonic effect of a food, the appreciation of its sensory properties or some evaluative judgment of its potential to give pleasure. As the subjective sensations of liking and wanting often overlap and are subject to interference or misinterpretation, their relationship with behavior is often difficult to discern (163). However, liking and wanting responses to food are not necessarily consciously monitored or even always accessible to the individual. Although people tend to be very good at estimating and reporting their liking for food, they are often unable to accurately gauge their implicit wanting for food (i.e., why they are unconsciously drawn to one food over another).

The hedonic aspect of eating is important in a well-functioning homeostatic system for the directing and motivating an adequate supply of nutrients and energy. Increasingly, evidence for the interplay between liking and wanting with hunger and satiety is helping to clarify the role of hedonics in the control and loss of control over food intake (164). This extension to the conventional homeostatic model recognizes that hedonic processes are affected by acute nutritional need states and might modulate food intake through their interaction with other physiological processes involved in satiation and satiety. Likewise, cognitive and sensory inputs implicated in food liking and wanting can modulate the metabolic processes associated with homeostatic control over food intake (165). In addition to the effect of liking and wanting on episodic appetite responses, more recent evidence is emerging to suggest that tonic signals of nutritional status might affect liking and wanting for food to influence food preference and the composition of the diet (164).

MODULATION OF APPETITE THROUGH PHYSICAL ACTIVITY

Physical Activity and Control of Food Intake

Some individuals believe that the energy expended during exercise will automatically drive-up hunger and food intake to compensate for the energy deficit incurred. However, evidence shows that interventions of acute exercise generate little or no immediate effect on levels of hunger or daily EI (see (166-170) for reviews). One reason that studies do not demonstrate an increase in EI following acute exercise could be that they fail to track EI for sufficiently long periods after the bout of exercise, or that the exercised-induced energy expenditure is not large enough to stimulate appetite. However, even with a high dose of exercise (gross exercise-induced increase in energy expenditure = 4.6 MJ) in a single day and following tracking of EI for the following two days, there is no automatic compensatory rise in hunger and EI (171).

Exercise training interventions have shown similar findings with regards to EI (169, 172). In a recent systematic review and meta-analysis of exercise training interventions in people living with overweight or obesity, Beaulieu et al. (172)found that among 25 exercise groups, exercise training (ranging 2-72 weeks) did not lead to any significant post-intervention differences in EI compared to non-exercise control groups. Meta-regression showed this was not affected by intervention duration. However, due to the high number of poor-quality studies (i.e., using self-reported measured of dietary intakes), further analyses were conducted in only fair/good quality studies (reduced to 5 exercise groups), and found a 102-kcal post-exercise difference between exercisers and controls. It is important to note that this is an effect observed on the average and does not illustrate inter-individual variability (discussed below). Therefore, over time, on average there may be small compensatory increases in EI in response to the increased energy demands from greater physical activity levels. Indeed, the review also found a small increase in fasting hunger after exercise training in 19 exercise groups (172). Nevertheless, depending on the daily energy expenditure accrued with exercise training, this should still lead to some degree of weight loss and favorable changes in body composition, as reviewed by Bellicha et al.(173). Small positive changes were also observed in relation to eating behavior traits, with a reduction in disinhibition (13 exercise groups) and an increase in restraint (12 exercise groups) assessed by the Three-Factor Eating Questionnaire (172). A small number of studies in the review also suggested that exercise training may reduce reward or preference for high-fat foods (172). Overall, most of the evidence indicates that exercise training in people living with overweight or obesity appears to have a relatively small but positive influence on eating behavior. This effect may be heightened with longer-term physical activity by altering the sensitivity of appetite regulation. It should be kept in mind that most exercise training studies are of modest duration (e.g. up to 72 weeks the meta-analysis of Beaulieu et al. (172)), and the long-term changes in appetite and eating behavior, and the time-course over which these occur, are yet to be fully understood. Fewer studies have examined the relationship between changes in energy expenditure and eating behavior in non-obese participants who do not have a preconceived goal associated with weight reduction.

Does Habitual Physical Activity Level Affect Appetite Sensitivity?

Appetite sensitivity refers to the capacity to detect over- or under-consumption with the potential to subsequently adjust intake accordingly. This can be achieved by compensating for a particular EI by adjusting the size of the next meal. There is some evidence to suggest that regular exercisers, or habitually physically active individuals, have a better capacity to control food intake and energy balance due to increased appetite sensitivity. Long et al. (174)demonstrated that habitual exercisers have an increased accuracy of short-term regulation of EI in comparison to non-exercisers. In this study, participants were given either a low or high energy preload for lunch, and were then asked to eat ad libitum from a test meal buffet. Energy intake did not significantly differ following the two preloads in the non-exercise group, indicating a weak compensation. However, the habitual exercisers demonstrated nearly full compensation (~90%) by reducing their EI following the high energy preload compared to the low energy preload. This study has been replicated in groups varying in objectively-assessed habitual physical activity levels (175). Similarly, Martins et al. (176) reported that the sensitivity of short-term appetite control increased in previously sedentary individuals following 6 weeks of aerobic exercise training, with participants again better able to adjust subsequent EI following high and low energy pre-loads following the exercise intervention. Furthermore, King et al. (177) examined the effects of 12 weeks of supervised aerobic exercise on hunger and satiety in 58 individuals living with overweight or obesity. Two separate processes were revealed that acted concurrently to influence the impact of exercise on appetite regulation. Post-intervention, a significant increase in fasting hunger was seen, but this increased orexigenic drive was offset by a parallel increase in post-prandial satiety (as measured in response to a fixed energy meal). Therefore, this ‘dual process’ may reflect the balance between the strength of tonic and episodic signaling following chronic exercise, and may be an important factor which determines whether individuals successfully lose weight or not. Interestingly, these improvements in appetite control appear to be related to satiety and perhaps not to satiation (178), but more research on the interplay between diet composition, satiation/satiety and physical activity level is required, as well as underlying mechanism.

Findings of improved appetite sensitivity with increased physical activity are in line with Jean Mayer’s work over 60 years ago. Mayer et al. (179) demonstrated a non-linear relationship between energy expenditure and EI in Bengali jute mill workers. Daily occupational physical activity and EI were closely matched in those performing physically demanding jobs. However, in those performing light or sedentary occupational roles, this coupling was lost such that daily EI exceeded expenditure. Such work has led Blundell et al. (180) to suggest a J relationship between physical activity and appetite regulation, with ‘regulated’ and ‘non-regulated’ zones of appetite regulation seen across the physical activity spectrum. Sedentary or low levels of physical activity coincide with an ‘unregulated zone’ of appetite in which EI and energy expenditure are disassociated (thereby promoting overconsumption of food at low levels of physical activity). At higher levels of physical activity however, stronger regulation of appetite and food intake exists such that EI better matches energy expenditure. This would promote the better maintenance of energy balance, albeit at higher levels of absolute intake and expenditure, i.e. higher ‘energy turnover/flux’ (180). This non-linear relationship has since been replicated in a systematic review (181) and in a study of over 400 participants (182). The model proposed by Blundell was recently updated by Beaulieu et al. (183), as shown in Figure 4. Furthermore, emerging evidence from a highly-controlled metabolic chamber study suggests that appetite control is indeed improved at higher levels of energy turnover (184). Longer-term studies are required to understand the impact of high energy turnover on appetite control, but it is believed that high levels of EI relative to expenditure may improve weight management via a favorable impact on physiological adaptations (185).

Figure 4. Regulated and non-regulated zones of appetite with varying levels physical activity from Beaulieu et al. (183). Model based on Jean Mayer’s study in Bengali jute mill workers (179) and previously published in Blundell (180).

While the mechanisms behind an improvement in appetite control with regular physical activity remains unclear, insulin sensitivity has been proposed as one mechanism by which activity-induced improvements in appetite regulation may occur. Exercise is known to increase insulin sensitivity (186-188), and insulin sensitivity is known to be involved in satiety induced by particular foods (189) and in the compensatory response to high energy loads (190). A further mechanism by which exercise could affect appetite is through altering gut peptide action. For example, CCK is implicated in the short-term regulation of appetite, and levels of CCK have been shown to rise after exercise (191). Interestingly, Martins et al. (192) measured fasting and post-prandial levels of orexigenic (total and acylated ghrelin) and anorexigenic (PYY, GLP-1) peptides in 15 individuals living with overweight or obesity during 12 weeks of supervised aerobic exercise. A significant increase in fasting hunger was again seen following the intervention, but this was offset by greater satiety in response to a fixed energy meal following the intervention. Interestingly, there was also a significant increase in the suppression of acylated ghrelin following the fixed energy meal, and a tendency toward an increase in the post-prandial release of GLP-1 following the exercise intervention. These hormonal responses would have acted to augment satiety during the post-prandial period. Further work by Gibbons et al. (193)revealed that exercise training had no overall impact on pre and postprandial appetite peptide release; however, differences emerged when participants were classified as ‘responders’ and ‘non-responders’. Compared to non-responders, responders had overall greater suppression of acylated ghrelin and greater increase in GLP-1 and total PYY. This effect was independent of the exercise intervention as differences were observed from baseline. Therefore, the specific role that appetite-related peptides play in activity-induced improvements in appetite regulation remains to be fully understood.

In addition to gastrointestinal mechanisms, it is important to understand how physical activity timing and patterns impact energy balance and appetite control. Physical activity can be prescribed by the FITT principle: frequency, intensity, time (duration) and type, and more recently, timing as another parameter has been proposed, as emerging evidence suggests that when exercise is performed relative to a meal or the time-of-day may influence obesity and cardiometabolic health in both adults and children (194). Indeed, physical activity is also another cue that influences circadian rhythms (195). In a systematic review and meta-analysis of exercise training interventions in individuals living with overweight or obesity (117), only 3 studies (1 RCT) on diurnal exercise timing were identified (196-198).These studies, in addition to observational studies (reviewed in (199)), suggest that early relative to late day exercise timing may lead to greater weight/fat loss. However, a recent RCT suggests no effect of exercise timing on weight loss, but this may have been due to the relatively low dose of aerobic exercise (200). The underlying mechanisms the greater weight loss in response to early exercise timing likely involve an impact on both behavioral and physiological processes regulating energy balance and appetite control (201). Performing morning exercise may lead to greater weight loss by enhancing the satiety response to food throughout the day, reducing the desire for and intake of high-energy-dense foods, and shifting food intake timing to earlier in the day, thus reducing daily energy intake (199).Evidence for this hypothesis is scarce, with an acute study showing that morning exercise led to greater post-exercise satiety; however, this was not examined in response to actual food intake (196). More rigorous research in this area is needed to clarify these proposed effects.

CONCLUSION

The control of food intake in humans is a biopsychological phenomenon. Motivation to eat is an important factor influencing food intake if the environment does not constrain behavior. Food intake is not necessarily solely related to situations of nutritional depletion and can be influenced by a number of homeostatic and non-homeostatic factors. Appetites are often learned and frequently sensory specific. The implication of asymmetric energy balance regulation is that food intake is under stronger physiological control in relation to negative energy balances, whereas in a state of energy balance or positive energy balances the linkages between physiological signaling and subjective motivation to eat are weaker. In obesogenic environments where palatable, energy dense food is ubiquitous, intake can easily exceed energy requirements due to motivation underpinned by food reward. Another consideration is how physical activity or exercise influence energy intake. Physical activity may influence the control of appetite via a number of pathways (e.g., alterations in gastric emptying, appetite-related hormones, food reward, circadian synchrony).

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Diagnostic Testing for Diabetes Insipidus

ABSTRACT

Diabetes insipidus (DI) is a disorder characterized by excretion of large volumes of hypotonic urine. The underlying cause is either a deficiency of the hormone arginine vasopressin (AVP) in the pituitary gland/hypothalamus (central DI), or resistance to the actions of AVP in the kidneys (nephrogenic DI). In most circumstances, DI is also characterized by excessive consumption of water (polydipsia). A third condition called primary polydipsia can clinically show overlapping features with DI. Both DI and primary polydipsia are collectively referred to as ‘polyuria-polydipsia syndromes. Like other endocrine disorders, an accurate diagnosis of DI can be challenging. This is mainly because the results obtained from diagnostic testing can show significant overlap among the different forms of DI and primary polydipsia. When a case of DI is suspected, the initial step involves the confirmation of the presence of hypotonic polyuria, which is the hallmark of DI. Once hypotonic polyuria is established, the next step is to identify the type of polyuria-polydipsia disorder (central DI vs. nephrogenic DI vs. primary polydipsia). This can be determined either through the water deprivation test or through the hypertonic saline infusion test along with plasma AVP or plasma copeptin measurements. Lastly, a detailed history and physical examination must be performed and appropriate laboratory and imaging studies must be undertaken to identify the underlying etiology of DI. This chapter describes the diagnostic steps to be pursued to identify the presence of DI, distinguish the various forms of polyuria-polydipsia disorders, identify the underlying disorders responsible for the DI, the challenges faced with diagnostic testing for DI in clinical practice, and future prospects in the field of DI diagnosis.

INTRODUCTION

Diabetes insipidus (DI) is a disorder of water homeostasis that is characterized by excretion of large volumes of hypotonic urine either due to the deficiency of the hormone arginine vasopressin [AVP, also known as antidiuretic hormone (ADH)], or due to resistance to the action of AVP on its receptors in the kidneys (1, 2). Large volumes of urine excretion, also known as polyuria (typically over 4 L per day), is the hallmark of DI. The urine of DI has been classically described as insipid (tasteless), hypotonic and dilute (3). Polyuria is defined as excretion of a urinary volume >150 ml/Kg/24 hours at birth, >100-110 ml/Kg/24 hours up to the age of 2 years, and >50 ml/Kg/24 hours in older children and adults (1). DI is typically classified into 3 forms: 1. Central DI, 2. Nephrogenic DI and 3. Gestational DI. Primary polydipsia is a disorder that is characterized by excessive intake of water which results in hypotonic polyuria. Clinically, this condition can manifest with symptoms of DI and adds to the diagnostic challenge of identifying DI. Primary polydipsia and DI together are referred to as ‘polyuria-polydipsia syndromes’, characterized by hypotonic polyuria (4, 5).

Central DI is caused by a variety of disorders that arise from either the pituitary or the hypothalamus. These conditions are characterized by defective production, transport or secretion of AVP (3, 6). This results in inappropriately low AVP levels in the setting of increased plasma osmolality. Nephrogenic DI is a form of DI that results from resistance by the kidney towards the action of AVP, due to AVP receptor defects or as an adverse effect of certain medications (3, 6). A third, rare form of DI occurs during pregnancy. Also known as gestational DI, this type of DI results from the enzymatic breakdown of the endogenous AVP by a placental cysteine aminopeptidase (3, 7). Therefore, this enzymatic degradation of plasma AVP during pregnancy can unmask sub-clinical DI in those women with borderline-low plasma AVP levels (8). Although primary polydipsia is not a true DI state, long-standing primary polydipsia can give rise to a DI-like picture on laboratory evaluation (described later) (4). There are several etiologies that give rise to each of these forms of polyuria-polydipsia syndromes (6). They have been listed in Table 1.

Table 1. Etiologies of the Various Polyuria-Polydipsia Syndromes

Central Diabetes Insipidus (involvement of pituitary and/or hypothalamus)

· Neoplastic:

o Craniopharyngioma

o Germinoma

o Meningioma

o Pituitary macroadenoma (invasive)

o Metastasis to the pituitary and/or the hypothalamus

· Vascular:

o Hypothalamic infarction/hemorrhage

o Cerebral infarction/hemorrhage

o Anterior communicating artery ligation

o Anterior communicating artery aneurysm

o Sheehan’s syndrome

o Sickle cell disease

· Trauma:

o Deceleration injury

o Intracranial surgery

o Transsphenoidal pituitary surgery

· Autoimmune/inflammatory:

o Lymphocytic hypophysitis

o IgG4 disease

o Xanthogranulomatous hypophysitis

o Anti-vasopressin neuron antibodies

o Guillain-Barré syndrome

· Infectious:

o Meningitis

o Encephalitis

o Tuberculosis

o Pituitary or hypothalamic abscess

· Granulomatous:

o Sarcoidosis

o Granulomatous hypophysitis

o Langerhans’ cell histiocytosis

o Erdheim-Chester disease

· Drug/toxin-induced:

o Phenytoin

o Ethyl alcohol

o Snake venom

· Congenital/genetic:

o Autosomal dominant AVP-neurophysin II gene alterations

o Wolfram (DIDMOAD) syndrome

o Septo-optic dysplasia

o Schinzel-Giedion syndrome

o Culler-Jones syndrome

o Alstrom syndrome

o Hartsfield syndrome

o Webb-Dattani syndrome

o X-linked recessive defects with subnormal AVP levels

Nephrogenic Diabetes Insipidus

· Metabolic:

o Hypokalemia

o Hypercalcemia

· Drug-induced:

o Lithium

o Demeclocycline

o Methoxyflurane

o Cisplatin

o Pemetrexed

o Aminoglycosides

o Amphotericin B

· Renal disease:

o chronic kidney disease

o Polycystic kidney disease

o Obstructive uropathy

· Systemic disease:

o Amyloidosis

o Sarcoidosis

o Sjogren’s syndrome

o Multiple myeloma

· Vascular:

o Renal infarction

o Sickle cell disease

· Congenital/genetic:

o Autosomal recessive aquaporin-2 channel gene alterations

o X-linked recessive V-2 receptor gene alterations

o Polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE) syndrome

o Type 4b Bartter syndrome

Pregnancy-Induced/Gestational Diabetes Insipidus

· Increased vasopressin metabolism induced by placental cysteine aminopeptidase

Primary Polydipsia

· Psychogenic polydipsia

· Drug-induced:

o Anticholinergics

o Phenothiazines

· Intracranial etiology:

o Hypothalamic tumors

o Tuberculous meningitis

o Intracranial surgery/trauma

o Sarcoidosis

· Lowering of hypothalamic threshold for thirst (‘Dipsogenic DI’)

The typical clinical presentation of DI is in the form of polyuria and polydipsia. On several occasions, co-existing conditions or other aspects of the patient’s history can provide clues towards the possible etiology causing DI. Patients with hypophysitis can present with concomitant symptoms of anterior pituitary hormonal dysfunction including fatigue, erectile dysfunction, weight changes, loss of libido, galactorrhea, and amenorrhea (9). Headaches, visual field defects, or cranial nerve palsies can be observed along with DI in case of central nervous system (CNS) tumors, hypophysitis, or a pituitary adenoma (1, 9). In most circumstances, despite losing large volumes of water in the urine, individuals with DI do not manifest dehydration. This is due to the activation of the thirst center in the hypothalamus that induces a strong sense of thirst with rising plasma osmolality which results in the intake of large volumes of water (6). Therefore, although an intact thirst mechanism helps to compensate for the urinary water loss, the polyuria and polydipsia persist, and this can cause considerable distress for the patient unless the underlying DI is treated. A plasma osmolality of about 285mOsm/Kg usually acts as a trigger for thirst, with further increments in plasma osmolality resulting in a linear increase in thirst (6, 10). DI becomes more challenging to manage if this thirst response is diminished or if the patient is unable to access water for drinking. Patients with an attenuated thirst response have reduced urge to drink water despite rising plasma osmolality and loss of large volumes of water in the urine, resulting in ‘adipsic DI’ (11). Although being classically associated with craniopharyngioma, adipsic DI can also manifest with CNS trauma, CNS tumors, or after CNS neurosurgical or neurovascular procedures (12). Moreover, the thirst mechanism may be lost in hypothalamic lesions, whereby DI may present acutely with signs and symptoms of dehydration. Patients who are unable to voluntarily access water include infants and young children, and individuals with altered consciousness. In young children, lack of adequate water intake can lead to dehydration, sleeplessness, irritability, enuresis, failure to thrive and impaired growth (1). Similarly, among patients with altered/reduced consciousness, rapid output of large volumes of urine can result in dehydration and acute, sometimes severe, hypernatremia can ensue (2). The shrinkage of the brain induced by excessive water loss and severe hypernatremia could potentially lead to intracranial bleeding, obtundation, convulsions or coma (13).

Diagnosing the type of DI/polyuria-polydipsia syndrome is essential for making the optimal treatment decision. A potential misdiagnosis and the resultant treatment can lead to catastrophic consequences (7). For instance, if primary polydipsia is misdiagnosed as central DI and desmopressin treatment is initiated, severe hyponatremia can occur (14, 15). There have been several tests that have been developed over the past century to diagnose the presence of DI and to differentiate the various types of polyuria-polydipsia syndromes. This chapter specifically comprises the description of different diagnostic tests utilized in the diagnosis of these conditions.

DIAGNOSING DIABETES INSIPIDUS

The major challenge with diagnosing and classifying the type of DI arises from the fact that the various forms of polyuria-polydipsia syndromes can show overlapping features on diagnostic testing. The water deprivation test, also known as the indirect water deprivation test should potentially be able to distinguish the various forms of DI. Deprivation of water intake should allow patients with primary polydipsia to concentrate their urine while those with central or nephrogenic DI continue to excrete dilute urine. Administration of desmopressin (Deamino-8-D-arginine vasopressin), the synthetic analogue of AVP, after water deprivation should help with differentiating patients with central DI from those with nephrogenic DI as the former should be able to concentrate the urine once the deficient action of AVP is replaced with desmopressin, while the latter should not show a significant response due to end-organ resistance to the action of AVP or its analogues. While water deprivation followed by desmopressin administration should be theoretically sufficient to identify the type of DI, in reality, the interpretation of these tests is more complicated, especially if a patient has partial central DI, partial nephrogenic DI or chronic primary polydipsia (4). Patients with partial central or nephrogenic DI retain some amount of response to water deprivation and desmopressin administration (4, 16). In the case of chronic primary polydipsia, long-standing water diuresis blunts the renal medullary concentration gradient and causes down-regulation of the aquaporin-2 channels in the proximal tubule and the collecting duct due to suppressed endogenous AVP, thus creating a state mimicking nephrogenic DI (4, 17).

The basic algorithmic approach for diagnosing any suspected case of DI usually involves the following steps: 1. Confirmation of hypotonic polyuria 2. Diagnosis of the type of polyuria-polydipsia syndrome and 3. Identification of the underlying etiology (6). Performing diagnostic testing in this order can potentially aide with establishing the appropriate diagnosis and with choosing the most relevant biochemical and imaging tests.

Confirmation Of Hypotonic Polyuria

Polyuria is defined as excretion of a urinary volume >150 ml/Kg/24 hours at birth, >100-110 ml/Kg/24 hours up to the age of 2 years, and >50 ml/Kg/24 hours in older children or adults. A hypotonic urine is typically defined as a urine with an osmolality of <300 mOsm/Kg. The primary objective in this step involves differentiating between conditions that give rise to polyuria resulting from osmotic diuresis (such as in hyperglycemia) and DI/primary polydipsia, in which polyuria predominantly involves water diuresis.

Conformation Of Polyuria

The first step is to confirm if a patient indeed has polyuria. Complaints of ‘polyuria’ can often be a misrepresentation of the actual symptoms of urinary urgency, nocturia, urinary incontinence, urinary tract infection, or prostatic hypertrophy (2). Once these symptoms have been ruled out, a 24-hour urine collection should be obtained. A 24-hour urine volume of <2.5 L could be reassuring and there is no concern for osmoregulatory disruption. Those patients without polyuria but with other above-mentioned urinary symptoms must be referred for urological evaluation. Alternatively, individuals can keep a diary for 24 hours making a note of the amount of their urine output (3). A 24-hour urine creatinine will help ensure an appropriate sample collection. Patients need not hold any medications that can cause polyuria, such as diuretics or sodium-glucose co-transporter-2 (SGLT-2) inhibitors for this step as the goal of this step is to establish the presence of polyuria.

Excluding Other Causes of Polyuria

Once polyuria is confirmed, the next step would be to assess for urinary osmolality. The urine in cases of DI/primary polydipsia is hypotonic. A urine osmolality of >800 mOsm/Kg indicates optimal plasma AVP levels and appropriate renal response to AVP, therefore ruling out any form of DI (7, 18). In most cases, polyuria with isotonic/hypertonic urine is driven by glucose, sodium, urea, or medications such as diuretics or mannitol (19). Glycosuria can result either from uncontrolled diabetes mellitus (DM), general hyperglycemia (from steroid administration, tube-feeding/parenteral nutrition), or by the use of SGLT-2 inhibitors (20). Administration of normal saline in large volumes for intravascular volume expansion can lead to sodium-induced polyuria (19). Sodium-induced polyuria is also seen with release of bilateral bladder obstruction (21). Urea-induced solute diuresis can be seen with high amounts of protein intake, tissue catabolism, steroid administration, all of which result in production of urea from breakdown of proteins (19). Administration of urea for treatment of hyponatremia, and recovery from azotemia can also result in urea-solute diuresis (19). Mannitol-induced diuresis can result from treatment of increased intracranial pressure with mannitol (19).

Initial Serum/Plasma and Urine Investigations

In individuals with established hypotonic polyuria or in individuals with urine osmolality of ≥300 mOsm/Kg and <800 mOsm/Kg, further evaluation must be undertaken through laboratory investigations. Serum sodium and plasma osmolality measurements could assist with indicating the type of the underlying polyuric state. A high serum sodium (>146 mmol/L) could point towards central or nephrogenic DI while a low normal or low sodium (<135 mmol/L) could indicate primary polydipsia as the underlying disorder (5, 22, 23). Similarly, a high plasma osmolality (≥300 mOsm/Kg) is typically seen in DI while a normal or low plasma osmolality (≤280 mOsm/Kg) is usually seen in primary polydipsia (4, 24).

As an alternative to urine osmolality, urine specific gravity is also useful in identifying a hypotonic polyuric disorder. For normal plasma osmolality, the urine specific gravity is between 1.003 to 1.030 (25). The specific gravity value depends on the number and the size of particles in the urine, unlike urine osmolality which solely depends on the number of particles in the urine. Because of this, although urine specific gravity and urine osmolality generally correlate well, presence of large molecules might elevate urine specific gravity despite the chance of the urine osmolality actually being low (26). Unlike falsely elevated urine specific gravity, false low values of urine specific gravity are uncommon and a low urine specific gravity suggests DI or primary polydipsia. Both urine osmolality and specific gravity are easily measured on a urine specimen, but urine osmolality is more widely utilized in management of DI as osmolality is not affected by the size of the particles in the urine. Situations where urine specific gravity is suggested to be useful is when facilities for urine osmolality measurement are not available or if a rapid results are required, especially in managing neurosurgical patients, and on rare occasions where DI co-exists with DM with hyperglycemia (as in craniopharyngioma, Wolfram syndrome, described later) (27). With uncontrolled DM, the urine osmolality and specific gravity should be high. But with co-existent DI and DM, the urine osmolality and the urine specific gravity can be inappropriately low (28). Some studies have also noted low urine specific gravity but normal urine osmolality with concomitant DI and DM (27). In clinical practice, some physicians rely solely on urine osmolality while others prefer to assess both urine osmolality and urine specific gravity for managing DI.

Any underlying renal dysfunction must be ruled out by measuring urine sodium, blood urea nitrogen and serum creatinine (2). Electrolyte abnormalities including hypokalemia and hypercalcemia can give rise to polyuria due to down-regulation of aquaporin-2 channels giving rise to a clinical picture of nephrogenic DI (29). Therefore, any serum potassium or calcium abnormalities must be appropriately corrected.

DIAGNOSIS OF THE TYPE OF DIABETES INSIPIDUS

Probably the most challenging step in the work-up of a suspected case of DI is to assign an accurate diagnosis: central DI vs. nephrogenic DI vs. primary polydipsia. These polyuric-polydipsic states can demonstrate substantial overlap, both in their clinical presentation and in their response to diagnostic testing. It is also possible for one or more forms of polyuria-polydipsia syndrome to co-exist in a single individual. An algorithm for the diagnostic approach for DI is described in Figure 1.

Figure 1. Algorithm for Diagnosis of the Various Types of Polyuria Polydipsia Syndromes.

Under some circumstances, a diagnosis of DI or primary polydipsia is established based on clear evidence of hypotonic polyuria and based on serum sodium and plasma osmolality values, with high serum sodium/plasma osmolality being consistent with DI and a low serum sodium/plasma osmolality being consistent with primary polydipsia (see Figure 1). But in most situations, the diagnosis is still unclear. Such situations, where the initial diagnostic testing is indeterminate, include a urine osmolality value of 300 – 800 mOsm/Kg or a normal serum sodium/plasma osmolality. In these circumstances, there is a need to establish the diagnosis with more accuracy, and further testing must be considered. In this section, the various diagnostic tests specifically utilized to diagnose and identify the type of polyuria-polydipsia syndrome are discussed, along with their advantages and limitations.

Water Deprivation Test

The water deprivation test is also known by the terms ‘indirect water deprivation test’ and ‘dehydration test’. The term ‘indirect’ is utilized as this test generally does not involve ‘direct’ measurements of plasma AVP to diagnose and differentiate the various forms of DI. The water deprivation test is almost always followed with desmopressin administration to further characterize the type of polyuric polydipsic state. The basic principle behind the water deprivation test is that in individuals with normal posterior pituitary and renal function (or those with primary polydipsia), an increase in plasma osmolality from dehydration stimulates AVP release from the posterior pituitary which then leads to water reabsorption in the nephrons, thus resulting in concentration of urine and an increase in urine osmolality. In central or nephrogenic DI, the urine fails to optimally concentrate with water deprivation and there is persistent excretion of hypotonic urine. Once the diagnosis of DI is established, desmopressin administration can distinguish between central and nephrogenic DI. In central DI, once the deficient action of AVP is substituted with desmopressin administration, the urine osmolality should increase while in nephrogenic DI, as the desmopressin is ineffective due to lack of renal response to its actions, the low urine osmolality persists.

TESTING PROTOCOL

The test is performed either as an out-patient or preferably after admitting the patient to an in-patient ward in a controlled setting. For those individuals with milder polyuria (50 – 70 ml/Kg/24 hours), the test can be initiated in the evening and the dehydration can be performed overnight as an out-patient. However, for those who experience significant polyuria or nocturia, the test is better performed during the day so that the patient can be supervised (3). The individual undergoing the test must not have undergone thirsting prior to the test. Any electrolyte abnormalities (potassium, calcium) must be corrected prior to the test. The patient has to discontinue any medications that can affect urine output (diuretics, SGLT-2 inhibitors, desmopressin, carbamazepine, chlorpropamide, glucocorticoids, non-steroidal anti-inflammatory drugs) 24 hours prior to initiation of dehydration, and refrain from activities such as smoking and caffeine intake that might affect AVP release or urine output (6, 30). Baseline plasma osmolality, serum sodium, urine osmolality (and plasma AVP or plasma copeptin where available) are obtained. In case of an out-patient overnight water deprivation, these baseline measures are obtained on the morning preceding the overnight water deprivation test (31).

The dehydration phase is initiated overnight among those patients in whom this phase is performed as an out-patient. The timing of initiation of overnight water deprivation for out-patient testing is determined based on the 24-hour urine output of the patient (which would have been previously determined in order to establish the diagnosis of a polyuria-polydipsia syndrome), with a goal of achieving approximately 3% loss of body weight.

The duration of overnight water deprivation (in hours) can be determined by using the following formula: patient’s weight (Kg) x 0.03 x 1000 (ml) / urine output (ml/hour) (31). Clear, written instructions must be provided to the patient about terminating the dehydration phase in case of unseen adverse events such as nausea, diarrhea, dizziness, or syncope. Once the overnight dehydration phase is completed, the patient arrives at the hospital the following day around 07:00 – 08:00 am to obtain serum/plasma and urine studies. Patients are advised to bring along an accompanying person for their hospital visit following the overnight water deprivation. In case the overnight dehydration is prematurely terminated for any reason (dizziness, or intractable thirst leading to consumption of water), then in these patients, in-patient water deprivation must be undertaken.

The dehydration phase for in-patient testing usually begins at 08:00 am. The patient voids prior to beginning the test and the baseline weight, blood pressure, and heart rate is measured prior to initiation of dehydration. Following the initiation of the test, patient should have nothing by mouth. Weight, blood pressure and heart rate must be recorded hourly as a cautionary measure due to the risk of severe water loss and dehydration in the setting of lack of access to drinking water. Adequate supervision is advised to watch for any undisclosed drinking of water. Every voided urine is recorded and the osmolality of the urine is measured. Alternatively, for the convenience of measurement, urine output and urine osmolality can be measured once every 2 hours. Serum sodium and plasma osmolality are also obtained every 2 hours along with urine measurements. The dehydration phase should be discontinued if one of the following occur: loss of more than 3% of body weight, elevation of serum sodium to above normal limits (≥146 – 150 mmol/L as per most literature), orthostatic hypotension or orthostatic symptoms (dizziness), or intractable thirst (4).

The next phase of the test is the desmopressin phase, which involves administration of desmopressin following dehydration. This phase can be initiated if either one of the following end-points are achieved: 1. Dehydration phase is completed for 8 hours (except in those who need longer periods of dehydration), 2. Two consecutive urine osmolality measures do not differ by >10% and loss of 2% body weight, 3. Premature termination of dehydration phase due to loss of more than 3% of body weight, elevation of serum sodium to above normal limits, or intractable thirst (3). An injection of 2 µg desmopressin is administered either through intravenous or intramuscular route and the above-mentioned urine and serum/plasma measures are obtained hourly for 1 – 2 hours after injection. Administration of desmopressin through oral or intranasal route does not result in predictable absorption, and especially where assigning an accurate diagnosis is crucial, optimal desmopressin concentrations in the blood has to be ensured. So, oral or intranasal desmopressin administration is not recommended to be used as a part of water deprivation test. During this phase, the patient can eat and drink, even up to 1.5 – 2 times the volume of urine passed during the dehydration phase. The total duration of the test can vary based on the clinical presentation. In those patients with complete forms of DI, the test can be performed in less than 8 hours while in those with partial DI or a non-DI condition, the test could last longer, sometimes even over 18 hours (3).

INTERPRETATION OF RESULTS

In normal individuals, with dehydration, the urine osmolality usually increases up to 800 – 1200 mOsm/Kg (3). If the dehydration phase is begun at 12:00 am, a morning (8:00 - 9:00 am) urine osmolality of 800 – 1200 mOsm/Kg excludes DI (3). A urine osmolality of <300 mOsm/Kg with a concomitant plasma osmolality of >300 mOsm/Kg or a sodium level above upper limit of normal following dehydration (>146 mmol/L) is suggestive of either central or nephrogenic DI (3, 4, 6). Desmopressin could be administered at this time (8.00 - 9:00 am). An increase of at least >50% in urine osmolality after desmopressin administration suggests complete central DI (the increase can be up to 200% to 400%) while a <50% increase points towards complete nephrogenic DI (3). An increase in urine osmolality over 300 mOsm/Kg prior to an increase in serum sodium/plasma osmolality suggests preservation of some endogenous AVP activity/renal response to AVP, suggesting either partial central or partial nephrogenic DI (5). Thus, in patients with partial DI (central or nephrogenic) the urine osmolality after water deprivation is usually between 300 – 800 mOsm/Kg and there can be <50% increase in urine osmolality following desmopressin administration.

In primary polydipsia, water deprivation results in an increase in urine osmolality, anywhere between 300 – 800 mOsm/Kg (usually up to 600 – 700 mOsm/Kg), without a substantial increase in plasma osmolality, but the increase in urine osmolality is not as substantial as in a normal response (3, 4, 6). Following desmopressin administration, an increase of <9% in urine osmolality is usually associated with primary polydipsia with a concomitant plasma osmolality of 300 – 800 mOsm/Kg as per some literature (4). However, this amount of increase in the urine osmolality when the urine osmolality is between 300 – 800 mOsm/Kg can also be seen in partial nephrogenic DI and adds to the diagnostic conundrum, and these criteria alone should not be utilized to diagnose primary polydipsia. A graphical representation of the response of each of the polyuria-polydipsia disorders to the water deprivation test is provided in Figure 2.

Figure 2. Graphical representation of the water deprivation test. Image courtesy: Sriram Gubbi, NIDDK, NIH.

LIMITATIONS

Although widely regarded as the gold standard in literature for diagnosing DI, the water deprivation test does have its limitations (3, 22). The most common scenario being partial central or partial nephrogenic DI. Patients with partial central DI retain some degree of AVP secretory capacity that can be stimulated with dehydration leading to concentration of urine. In partial nephrogenic DI, maximal AVP secretion from water deprivation can overcome renal resistance to AVP’s action and enable water reabsorption. In addition, long-standing central DI and chronic primary polydipsia can also give rise to false results on water deprivation test. With long standing central DI, due to chronic deficiency of AVP, the aquaporin-2 channels are down-regulated as AVP is required for the synthesis and membrane translocation of these channels (32). Therefore, administration of desmopressin after water deprivation in long-standing central DI might not result in an adequate increase in urine osmolality due to lack of adequate water reabsorption from insufficient aquaporin-2 channel availability, which could misleadingly suggest a picture of nephrogenic DI. On the other hand, chronic primary polydipsia can suppress the release of endogenous AVP due to increased intravascular volume and decreased plasma osmolality from high volumes of water intake. In this situation, water deprivation might not lead to an adequate rise in plasma osmolality high enough to stimulate the release of endogenous AVP. This can result in a sub-optimal increase in urine osmolality, thus simulating a clinical picture of central/nephrogenic DI. Moreover, high volumes of water intake from chronic primary polydipsia can create a ‘wash out’ of the renal medullary osmotic gradient and also a down-regulation of the aquaporin-2 channels, and administering desmopressin in this situation may not result in adequate increase in urine osmolality (4, 17). Also, the water deprivation test must be performed in children only under the supervision of a pediatrician and the test should not be performed in infants (33). Adjunctive measurements of plasma AVP or copeptin levels can improve the diagnostic yield of the water deprivation test and are described in detail in the upcoming sections below. The protocol for the water deprivation test, interpretation of the results, and limitations are represented in Table 2.

Table 2. Summary of the Indirect Water Deprivation Test

Protocol

Preparation phase:

· Test can begin either overnight (for out-patient testing) or at 08:00 am (for in-patient testing).

· No thirsting prior to the test. Smoking and caffeine intake are avoided.

· Any electrolyte abnormalities (potassium, calcium) are corrected.

· Drugs that can affect urine output (diuretics, sodium-glucose co-transporter-2 inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs) must be held 24 hours prior to dehydration.

· Baseline weight, blood pressure and heart rate are measured prior to dehydration.

· Baseline plasma osmolality, serum sodium, urine osmolality (and plasma AVP or plasma copeptin where available) are obtained (these measures can be obtained on the morning prior to overnight dehydration in cases of out-patient water deprivation test).

Dehydration phase:

· This phase usually lasts for 8 hours (can last longer in certain cases).

· For out-patient testing, the duration (hours) of overnight dehydration can be determined using the formula: patient’s weight (Kg) x 0.03 x 1000 (ml) / urine output (ml/hour).

· Patient is allowed to have nothing by mouth.

· Adequate supervision is necessary to watch for any undisclosed drinking (in case of in-patient testing).

· Weight, blood pressure and heart rate are measured every 1 hour (in case of in-patient testing).

· Urine output, urine osmolality, serum sodium and plasma osmolality are measured every 2 hours (for in-patient testing). Plasma copeptin is measured towards the end of the dehydration phase. (In case of out-patient overnight dehydration, serum/plasma and urine measures are obtained around 07:00 – 08:00 am the following morning)

· Dehydration phase is discontinued if one of the following occurs:

o Dehydration is completed for 8 hours (not applicable for those who might need longer periods of dehydration).

o Two consecutive urine osmolality measures do not differ by >10% and loss of 2% body weight.

o The total body weight reduces by more than 3%.

o Serum sodium increases to above upper limit of normal (preferably >150 mmol/L).

o Orthostatic hypotension or orthostatic symptoms.

o Intractable thirst (or if patient admits to drinking water overnight in case of out-patient testing).

Desmopressin phase:

· An injection of 2 µg desmopressin is administered either through intravenous or intramuscular route (use of oral or intranasal desmopressin is not preferred due to unpredictable absorption).

· The patient is allowed to eat and drink, even up to 1.5 – 2 times the volume of urine passed during the dehydration phase.

· Urine output, urine osmolality, serum sodium and plasma osmolality are measured hourly for 1 – 2 hours after desmopressin administration.

Interpretation

Central DI:

o Baseline urine osmolality of <300 mOsm/Kg and an increase in urine osmolality by >50% from baseline following desmopressin administration (complete central DI).

o Baseline plasma copeptin of <2.6 pmol/L without prior dehydration (complete central DI).

o The ratio of Δ copeptin from start till the completion of dehydration phase to serum sodium at the end of the dehydration phase of <0.02 pmol/L (indicates partial central DI).

Nephrogenic DI:

o Urine osmolality fails to rise above 300 mOsm/Kg or by <50% after desmopressin administration (complete nephrogenic DI).

o Baseline plasma copeptin of ≥21.4 pmol/L without prior dehydration (complete and partial nephrogenic DI).

o Baseline plasma AVP of ≥3 pg/ml without prior dehydration (complete and partial nephrogenic DI).

Primary Polydipsia:

o Urine osmolality usually increases (300 – 800 mOsm/Kg, usually up to 600 – 700 mOsm/Kg) without significant changes in plasma osmolality following dehydration.

o The ratio of Δ copeptin from start till the completion of dehydration phase to serum sodium at the end of the dehydration phase of ≥0.02 pmol/L

Advantages

· Most extensively utilized and validated test.

· No risks of hypertonic saline administration (thrombophlebitis, need for central line).

· Plasma copeptin or AVP measurements are not necessary if the center does not have the facilities/assays to measure these peptides.

Disadvantages

· More time consuming than hypertonic saline infusion test.

· Can give overlapping results in cases of partial central DI, partial nephrogenic DI, or chronic primary polydipsia.

· Majority of the interpretation relies on urinary measurements, which can be affected by any of the above conditions due to their modulation of aquaporin-2 channel synthesis and expression.

· More burdensome for the patients and less convenient when compared with hypertonic saline infusion test.

Measurement Of Plasma AVP

AVP is encoded by the arginine vasopressin gene (AVP, 20p13), which also encodes prepro-AVP (a single peptide), neurophysin II (NPII), and a glycoprotein, copeptin. The pro-hormone of AVP is synthesized in the magnocellular neurons of the hypothalamus (Figure 3). Due to the above mentioned limitations of the indirect water deprivation test, measurement of plasma AVP levels was suggested for its potential to be used as a ‘direct’ test in conjunction with water deprivation test to distinguish the various polyuria-polydipsia syndromes (34). In nephrogenic DI, as there is no deficiency of AVP, the plasma AVP levels is high, in order to overcome the resistance posed by the kidneys to the action of AVP. On the other hand, plasma AVP should be low or relatively low in central DI for the elevated plasma osmolality (4). In primary polydipsia, plasma AVP levels may be normal or can be suppressed in long-standing cases. Subnormal AVP levels in cases of central DI could be due to osmoreceptor dysfunction in the hypothalamus or due to defective AVP release from the neurohypophysis (35). Despite the initial promising results of its potential utility, plasma AVP is seldom utilized for diagnosing DI in clinical practice.

Figure 3. Anatomy of the pituitary gland and the hypothalamus. The pituitary gland comprises of two developmentally and functionally distinct parts: the anterior pituitary (adenohypophysis), derived from the Rathke’s cleft and the posterior pituitary (neurohypophysis). The gland is attached to the hypothalamus through the posterior pituitary via a stalk. The posterior pituitary along with the hypothalamus and the stalk forms the functional unit of infundibuloneurohypophysis. This diagram emphasizes on the neuronal network that supplies the posterior pituitary. The vasopressinergic neurons (magnocellular neurons) are present in the supraoptic and paraventricular nuclei of the hypothalamus. These neurons synthesize arginine vasopressin (AVP), its precursors and co-peptides, and their axons project into the posterior pituitary where the AVP is stored along with oxytocin (produced by the oxytotic neurons present in the same nuclei) in the axonal terminals. These hormones are transported to the posterior pituitary through the supraoptic-hypophyseal tract. Another set of neurons of the paraventricular nucleus (also called parvocellular neurons) project into other areas of the brain and spinal cord, some of which secrete AVP. Image courtesy: Sriram Gubbi, NIDDK, NIH.

There are several limitations to measuring plasma AVP levels. AVP is rapidly cleared from the plasma, with a half-life of around 16 minutes (36). In addition, the pre-analytical instability of AVP in the plasma is high. A large amount of circulating AVP is bound to platelets through V1 receptors and failure to adequately segregate platelets from the plasma after blood sampling or prolonged storage of unprocessed blood samples can lead to wide fluctuations or even an increase in the measured plasma AVP levels (22, 37). For the AVP to be detectable by the currently available assays, an additional step of extraction/concentration of plasma is necessary, and this requires at least 1 ml of blood sample (22). Additionally, the current laboratory method employed in AVP measurement is radioimmunoassay (RIA), which has several limitations and analytical errors. Plasma concentration of AVP (measured in pg/ml) is one of the lowest among all hormones, and as AVP is a small peptide, sandwich assays cannot be effectively utilized to measure plasma AVP levels (5). The AVP levels in the plasma can be stabilized up to 2 - 4 hours after blood sampling by storing the sample at 4ºC (22, 38, 39). However, the average turn-around time for the measurement of AVP is 3-7 days in most laboratories which would make maintenance of hormonal stability even more challenging (4, 40). For these reasons, plasma AVP measurements are not routinely utilized. A related peptide to AVP, copeptin, is now emerging as a new, more stable marker to diagnose the various hypotonic polyuric states.

Measurement Of Plasma Copeptin

Copeptin (Carboxy-Terminal-Pro-vasopressin) is the C-terminal peptide of pro-vasopressin that is co-secreted with AVP in stoichiometric amounts from the posterior pituitary (4, 40). Both of these peptides are derived from a precursor molecule synthesized in the magnocellular neurons of the hypothalamus (Figure 3) (22). The post-transcription processing of AVP, copeptin, and related peptides are diagrammatically represented in Figure 4. Unlike plasma AVP measurement, which is technically challenging, copeptin measurement in the plasma is relatively less cumbersome and has several advantages: copeptin can remain stable for days after sampling of blood and can be measured relatively quickly (40). Plasma levels of copeptin strongly correlate with plasma AVP levels over a wide range of osmolalities, both in healthy individuals and those with DI or primary polydipsia (22, 41). Moreover, plasma copeptin demonstrates the same response to changes in plasma osmolality and plasma volume as does plasma AVP (5,22). Over the past 12 years, several studies have been conducted to validate the utility of plasma copeptin in the diagnosis of hypotonic polyuric states and to distinguish one form from the other (4, 5, 7, 22, 30).

Figure 4. Post-transcription processing of vasopressin and related peptides. The pre-pro-vasopressin is a peptide consisting of 164 amino acids. This pre-pro-hormone is then converted to pro-vasopressin after the removal of the signal peptide and N-linked glycosylation of copeptin. Further processing of pro-vasopressin gives rise to the individual peptides: arginine vasopressin (AVP), neurophysin II (NP II), and copeptin. Image courtesy: Sriram Gubbi, NIDDK, NIH.

The next question logically is whether plasma copeptin is a better test when compared to plasma AVP with regards to diagnosing the various forms of polyuria-polydipsia syndromes. Without prior thirsting (without water deprivation/hypertonic saline infusion), baseline plasma copeptin of ≥21.4 pmol/L or a plasma AVP level of ≥3 pg/ml have been shown to distinguish nephrogenic DI (partial and complete) from other types of polyuria-polydipsia syndromes with 100% sensitivity and specificity (5, 42). Similarly, a single baseline plasma copeptin measurement of ≥2.9 pmol/L without prior water deprivation has demonstrated the ability to differentiate primary polydipsia from central DI with a sensitivity of 82% and specificity of 78%, while plasma AVP of ≥1.8 pg/ml can distinguish primary polydipsia from central DI with 54% sensitivity and 89% specificity (5).

Prior data has also shown that a plasma copeptin of <2.6 pmol/L can accurately distinguish between complete central DI from primary polydipsia with 95% sensitivity and 100% specificity (4, 22). Some studies have utilized a combined water deprivation/hypertonic saline infusion test to stimulate endogenous release of AVP and copeptin, and based on the results, plasma copeptin levels of ≥4.9 pmol/L can differentiate primary polydipsia from central DI (partial or complete) with 94% sensitivity and 96% specificity, while a plasma AVP of ≥1.8 pg/ml can do the same with 83% sensitivity and 96% specificity (5). In addition, the ratio of Δ copeptin from start till the completion of water deprivation (08:00 am to 04:00 pm) to the serum sodium at the end of water deprivation test has been shown to discern partial central DI (<0.02 pmol/L) from primary polydipsia (≥0.02 pmol/L) with a sensitivity of 83% and a 100% specificity, although newer data has demonstrated that the ratio of Δ copeptin to serum sodium following water deprivation to be surprisingly of lower diagnostic accuracy than water deprivation alone (4, 30). Recent data suggests plasma copeptin measurement coupled with hypertonic saline infusion test provides more accurate results with regards to differential diagnosis of DI when compared with plasma copeptin measurement with water deprivation test (30)(see below, Hypertonic saline infusion test’). There is also emerging evidence for plasma copeptin as a valuable marker to predict post-operative risk for DI after pituitary surgeries, with plasma copeptin of <2.5 pmol/L predicting the risk of DI with a positive predictive value of 81% and plasma levels of >30 pmol/L on postoperative day 1 demonstrating a negative predictive value of 95% to rule out DI (43).

Based on these data, plasma copeptin measurement appears to be overall a better test when compared with plasma AVP measurement. However, it is the technical aspects of better pre-analytical stability, less cumbersome sample handling, and quicker reporting of results that make plasma copeptin a more attractive test. As of now, copeptin assays are not yet available worldwide and its utilization is limited to a few centers. Once the test becomes more widely available in the future, plasma copeptin level measurements are likely to be frequently pursued to diagnose and distinguish the various forms of polyuria-polydipsia syndromes.

Hypertonic Saline Infusion Test

The indirect water deprivation test is the most well tested and widely utilized as the standard diagnostic test for DI. Adjunctive measurements of plasma AVP or copeptin levels can enrich the diagnostic yield of the water deprivation test. But it is quite evident that the test has several limitations and, in many situations, just does not provide an accurate diagnosis. Although relatively newer than the water deprivation test, some of the earliest reports on the potential utility of hypertonic saline infusion in the differential diagnosis of DI dates to the 1940s (44). Hypertonic saline (3% saline, 513 mOsm/L) infusion coupled with plasma copeptin measurement is an alternative test that is now being recommended by many experts in the field of DI as the preferred test to be used in place of water deprivation test.

TESTING PROTOCOL

The test overall lasts for about 3 hours and can be initiated at 08:00 am. Medications that have diuretic or anti-diuretic effects (diuretics, SGLT-2 inhibitors, desmopressin, carbamazepine, chlorpropamide, glucocorticoids, non-steroidal anti-inflammatory drugs) need to be discontinued 24 hours prior to testing (30). The patient lies in a supine position. Two intravenous cannulas are inserted, one for infusion and the other for blood sampling. Before commencing the intravenous infusion, venous sampling is performed to obtain plasma copeptin, serum sodium, glucose, urea, and plasma osmolality (30). Hypertonic saline infusion is then commenced, initially with a bolus dose of 250 ml given over 10 – 15 minutes, followed by a slower infusion rate of 0.15 ml/Kg/min. Serum sodium and osmolality are measured every 30 minutes. The infusion is terminated once the serum sodium is ≥150 mOsm/L (30). Typically, protocols allow up to a maximum of 3 hours of infusion (30,45). At this point, a plasma copeptin is measured and the patient is asked to drink water at 30ml/Kg within 30 minutes (30, 45). This is followed by intravenous infusion of 5% glucose (dextrose) at 500 ml/hour for one hour (30, 45). Serum sodium is measured once more after completing the 5% glucose infusion to ensure its return to normal values. Vital parameters, including blood pressure and heart rate must be constantly assessed, preferably on a monitor.

TEST INTERPRETATION

A plasma copeptin level of <4.9 pmol/L after hypertonic saline infusion indicates central DI (partial and complete) while a level of ≥4.9 pmol/L indicates primary polydipsia (5, 30). It is likely that this cut-off value might be changed to 6.5 pmol/L in the future due to its higher diagnostic accuracy, based on more recent data (described below) (30). Baseline copeptin value of >21.4 pmol/L is indicative of nephrogenic DI (partial and complete) and <2.6 pmol/L indicates complete central DI (4, 5, 30).

The major advantage with the hypertonic saline infusion test is that it basically excludes the renal component out of the equation and any potential down-regulation of aquaporin-2 channels will not affect the diagnosis. This test depends only on two aspects: 1. Increasing the plasma osmolality, which is a strong stimulus for endogenous AVP synthesis and release and 2. Measurement of endogenous AVP secretory capacity by measuring plasma copeptin, which is secreted in equimolar proportions from the posterior pituitary along with AVP (3, 4). Thus, unlike water deprivation test which is mainly a test based on urine output and osmolality, the hypertonic saline infusion test is free from any requirements to measure urinary indices. In fact, when compared with water deprivation test with desmopressin administration with/without plasma copeptin measurement, the hypertonic saline infusion test with plasma copeptin measurements has been demonstrated to be the superior test for diagnosing the various forms of polyuria-polydipsia syndromes (30). Hypertonic saline infusion test also takes less time to perform, causes less patient burden, and is more convenient and tolerable when compared with indirect water deprivation test (30).

A modified hypertonic saline infusion test followed by water deprivation has also been previously described when the diagnosis after water deprivation is inconclusive (46). In this scenario, administration of hypertonic saline (as described in the protocol) followed by measurements of plasma AVP, serum sodium, plasma and urine osmolality can potentially differentiate the types of DI when the plasma AVP levels are plotted on a nomogram relating plasma AVP values to either urine osmolality or serum sodium/plasma osmolality (normal plasma AVP for urine osmolality or serum sodium/plasma osmolality suggests primary polydipsia, low plasma AVP for serum sodium/plasma osmolality suggests central DI, and low urine osmolality for plasma AVP levels suggests nephrogenic DI). However, this test is more cumbersome than either indirect water deprivation test or hypertonic saline infusion test alone and requires measurements of plasma AVP levels which is also challenging (as described above. Measurement of plasma AVP’). Therefore, this combined method is not utilized in routine clinical practice. With the advent of plasma copeptin assays and with the ability to eliminate urine measurements out of the equation for the regular hypertonic saline infusion test, the plasma AVP measurement-based combined water deprivation and hypertonic saline infusion test is likely going to be obsolete in the future.

Recent data by Fenske et al. have demonstrated the diagnostic accuracy of hypertonic saline infusion test combined with plasma copeptin measurement to be higher (96.5%) than that of water deprivation test (76.6%) in correctly identifying the type of polyuria-polydipsia syndrome (30). Even when it came to differentiating those with partial central DI from patients with primary polydipsia, the hypertonic saline infusion test with plasma copeptin measurement had a higher accuracy (95.2%) as compared to water deprivation test without plasma copeptin measurement (73.3%) (30). Data from the same study has also shown that a plasma copeptin value of 6.5 pmol/L provides the best diagnostic accuracy (97.9%) for differentiating central DI (partial and complete) from primary polydipsia (vs. 96.5% for the plasma copeptin cut-off level of 4.9 pmol/L), with a value of >6.5 pmol/L being consistent with primary polydipsia. Surprisingly, in the same study, water deprivation test followed by plasma copeptin measurement demonstrated a lower diagnostic accuracy when compared to water deprivation test alone without plasma copeptin measurement (44% vs. 76.6%). These results show that combining hypertonic saline infusion and plasma copeptin measurements currently offers the best diagnostic capability with distinguishing the various forms of hypotonic polyuric states. The protocol for the hypertonic saline infusion test, interpretation of the results, and limitations are presented in Table 3.

Table 3. Hypertonic Saline Infusion Test (with plasma copeptin measurement)

Protocol

Preparation phase:

· Test can begin at 08:00AM.

· Drugs that can affect urine output (diuretics, sodium-glucose co-transporter-2 inhibitors, glucocorticoids, non-steroidal anti-inflammatory drugs) must be held 24 hours prior to dehydration.

· Patient lies in a supine position. Two intravenous cannulas are placed: one for blood sampling and the other for infusion.

· Baseline serum sodium, glucose, urea, plasma osmolality and plasma copeptin are obtained prior to infusion.

Hypertonic saline infusion phase:

· Hypertonic saline infusion is commenced: Bolus dose of 250ml given over 10 – 15 minutes, followed by 0.15 ml/Kg/min.

· Serum sodium and osmolality are measured every 30 minutes.

· The infusion is stopped if:

o The serum sodium raises to >150 mmol/L.

o The infusion is completed for 3 hours.

· Plasma copeptin is measured after the infusion is stopped.

· Heart rate and blood pressure are continuously monitored throughout the phase.

Hypotonic fluid administration phase:

· Patient is asked to drink water at 30ml/Kg within 30 minutes.

· This is followed by intravenous infusion of 5% glucose at 500ml/hour for 1 hour.

· Serum sodium is measured after the completion of 5% glucose infusion to ensure its return to normal values.

· Heart rate and blood pressure are continuously monitored throughout the phase.

Interpretation

Central DI:

o Baseline plasma copeptin <2.6 pmol/L prior to hypertonic saline infusion (complete central DI).

o Plasma copeptin level of ≤4.9 pmol/L after hypertonic saline infusion (partial or complete). *

Nephrogenic DI:

o Baseline plasma copeptin ≥21.4 pmol/L prior to hypertonic saline infusion (partial or complete).

Primary polydipsia:

o Plasma copeptin level of >4.9 pmol/L after hypertonic saline infusion. *

Advantages

· Takes less time to perform than water deprivation test.

· Eliminates the need to obtain urine studies which can be affected by aquaporin-2 channel availability, thus reducing the chances of confounding results.

· Hypertonic saline is a stronger osmotic stimulus when compared to dehydration and is therefore more potent with causing AVP release in cases of partial central DI or chronic primary polydipsia. So, this is likely to be a better test to utilize in cases of partial central DI and chronic primary polydipsia.

· Distinguishes central DI (complete and partial) from primary polydipsia with a higher accuracy when compared with water deprivation (with/without plasma copeptin) test.

· Less burdensome and more convenient for the patients.

Disadvantages

· Several centers mandate the use of central lines for hypertonic saline administration.

· Hypertonic saline infusion has the theoretical risk of causing superficial thrombophlebitis.

· Higher risk for hypernatremia when compared to water deprivation test.

· Plasma copeptin assays are currently of limited availability.

*A higher cut-off value of 6.5 pmol/L distinguishes central DI from primary polydipsia with increased accuracy based on newer data, with a value of >6.5 pmol/L being consistent with primary polydipsia (Fenske et al., 2018).

LIMITATIONS

The challenges with utilizing hypertonic saline infusion test are as follows: 1. Hypertonic saline has been claimed to cause thrombophlebitis when administered through peripheral intravenous line. However, this risk is overestimated and is rarely observed in practice (47), 2. Several institutions require placement of a central venous catheter and admission to an intensive care unit for administration of hypertonic saline. Central line insertion is a more invasive and time-consuming procedure when compared to a peripheral intravenous line and can cause more bleeding or infections, and an admission to an intensive care unit exerts substantial expenditure on the patient and the institute, and 3. Copeptin assays are not yet commercially available in several countries.

IDENTIFICATION OF THE UNDERLYING CAUSE OF THE DI

Once the type of polyuria-polydipsia syndrome is identified, efforts must be undertaken to diagnose the underlying pathology responsible for this clinical presentation. A detailed list of the potential causes for each type of DI and primary polydipsia is provided in Table 1. In cases of central DI, a thorough clinical history should be obtained, and a detailed physical exam needs to be performed to evaluate for the signs and symptoms of hormonal deficiencies (or excess in cases of hyperprolactinemia) from other pituitary axes (9, 48). Biochemical evaluation must include a morning plasma measurement of pituitary hormones (growth hormone, prolactin, ACTH, TSH, FSH, and LH), and the hormones from their target organs (insulin-like growth factor 1, cortisol, free thyroxine, total and free testosterone, estradiol). An MRI of the sella and suprasellar regions with gadolinium needs to be obtained to evaluate for any anatomical disruptions of the pituitary or hypothalamic anatomy (macroadenomas, empty sella, infiltrative diseases). The normal posterior pituitary demonstrates hyperintensity on T1 images (also known as the ‘bright spot’), suggested to be due to phospholipid-rich granules storing AVP and oxytocin (Figure 5) (49). The absence of this bright spot could indicate an absence of posterior pituitary function. However, this should not be used as a sole criterion to attribute a pituitary etiology as causing the DI, because absence of the bright spot on the pituitary MRI is also seen in up to 25% of normal individuals and may disappear with aging (50). Also, an enlarged/thickened pituitary infundibular stalk can be found on the MRI, which may be seen in cases of hypophysitis, granulomatous disorders, tuberculosis, craniopharyngioma, germinoma or a metastasis to the sella or suprasellar region (3, 9, 51). The hypothalamus is the site of AVP synthesis (Figure 3) and any pathological involvement of the region (inflammatory, infectious, vascular, or neoplastic process) can certainly result in the destruction of the hypothalamus leading to deficiency of AVP synthesis (3). This is specifically important among patients with adipsic DI as the thirst center might be disrupted due to the above-mentioned etiologies that can involve the hypothalamus. Any history of cranial trauma or intra-cranial surgery could also give rise to central DI.

Figure 5. Magnetic resonance imaging (MRI) of the pituitary gland. The above image is a non-contrast T1 MRI image of a normal pituitary gland. The white arrow point towards the ‘bright spot’ seen in the posterior pituitary. This finding is a result of phospholipid-rich granules that store arginine vasopressin (AVP) and oxytocin. Image courtesy: NIDDK, NIH.

In cases of DI with onset during infancy or early childhood, genetic/congenital causes must be suspected. Some of the genetic conditions causing DI include AVP-neurophysin II gene alterations (AVP-NP II, autosomal dominant), Wolfram syndrome, an autosomal recessive disorder caused by alterations in WFS1(4p16.1) associated with diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (OMIM 22300, DIDMOAD syndrome), and other autosomal recessive disorders due to production of mutant, weaker forms of AVP, and X-linked recessive disorders resulting in sub-normal plasma AVP levels (52-55). Congenital malformations, such as septo-optic dysplasia (HESX1, OMIM 601802, 3p14), Schinzel-Giedion midface retraction syndrome (SETBP1, OMIM 611060, 18q12), Culler-Jones syndrome (GLI2,OMIM 165230, 2q14), Alstrom syndrome (ALMS1, OMIM 606844, 2p13), Hartsfield syndrome (FGFR, OMIM615465, 8p11), Webb-Dattani syndrome (ARNT2, OMIM 606036, 15q25), amongst others, can also give rise to childhood-onset DI (56-60).

Nephrogenic DI in most cases is acquired, usually in the setting of intake of certain drugs like lithium, demeclocycline, pemetrexed, cisplatin, and others (6, 7, 61). Therefore, a review of the patient’s medication intake history can lead to the identification of the potential culprit medication, which can then be discontinued. Use of osmotic diuretics or electrolyte abnormalities such as hypercalcemia or hypokalemia must be investigated in cases of nephrogenic DI (6, 29). Any underlying acute or chronic renal disease (vascular, inflammatory, or neoplastic processes, polycystic kidney disease), obstructive uropathy, and systemic diseases such as amyloidosis or sickle cell disease can also give rise to nephrogenic DI and prompt evaluation for these disorders is necessary (6, 7). Congenital causes for nephrogenic DI include mutations in the gene for aquaporin-2 receptor (autosomal recessive) and the gene for V-2 receptor (X-linked recessive inheritance), and must be suspected in childhood-onset nephrogenic DI (7). Other genetic causes of nephrogenic DI include; polyhydramnios, megalencephaly, and symptomatic epilepsy (STRADA, OMIM 608626, 17q23) and some Barter syndrome subtypes, including Bartter syndrome, type 4b, digenic forms (CLCNKA, OMIM 602024, 1p36.13) (62, 63).

Primary polydipsia or dipsogenic DI is often seen in individuals on treatment for mood disorders or schizophrenia (64). The dry mouth caused by intake of medications with strong anticholinergic properties to treat these disorders is most likely the cause for excessive water intake (64). Hypothalamic disease (sarcoidosis, tuberculosis, trauma, neoplasms) can alter the thirst response by lowering the thirst threshold, either by disruption of the thirst center or through osmoceptor dysfunction, which leads to polydipsia (3, 7). Primary polydipsia which results from this subset of cases with low thirst threshold in hypothalamus, is also referred to as ‘dipsogenic DI’. In other cases, individuals are just habitual compulsive drinkers of large volumes of water without any underlying organic or pharmacologic cause (6).

A unique presentation of DI which is worthy of note, is the one that occurs during pregnancy. Also known as gestational diabetes insipidus, this disorder occurs during pregnancy due to the enzymatic breakdown of the endogenous AVP by a placental cysteine aminopeptidase (6, 7). In women with borderline-low plasma AVP levels, pregnancy can unmask sub-clinical DI from enzymatic degradation of AVP (8). However, work-up for other etiologies of DI must be considered when appropriate in this situation.

If none of the modalities succeed in establishing an appropriate diagnosis of DI, a therapeutic trial with desmopressin can be undertaken (3, 46). The therapeutic trial can be provided in the form of desmopressin tablets of 100 µg by mouth every 8 hours for 48 hours. If this treatment abolishes polyuria, normalizes serum sodium/plasma osmolality or at least brings down serum sodium to near normal levels, resulting in elimination of thirst, the diagnosis is most certainly central DI. If there is cessation of polyuria and there is slight normalization of serum sodium/plasma osmolality without reduction in polydipsia/thirst, the most likely diagnosis is primary polydipsia. In nephrogenic DI, therapeutic trial with desmopressin does not result in any significant changes in serum sodium or plasma and urine osmolality (46). This method can distinguish between various forms of DI with 90% accuracy (46). However, caution is advised when utilizing desmopressin for diagnostic testing due to the risk of hyponatremia, which can sometimes be severe in patients with primary polydipsia and the test is preferably done in a monitored in-patient setting (46).

FUTURE DIRECTIONS

A major forthcoming change that could be anticipated is the renaming of the term, “diabetes insipidus/DI”. An expert panel comprising physicians from various endocrine societies around the globe, ‘The Working Group for Renaming Diabetes Insipidus’, has recommended to rename ‘central diabetes insipidus’ as ‘arginine vasopressin deficiency (AVP-D)’, and ‘nephrogenic diabetes insipidus’ as ‘arginine vasopressin resistance (AVP-R)’ (65). While gestational DI has not been specifically brought up in this proposal, it may be apt to rename the condition as ‘gestational AVP-D.’ Similarly, ‘adipsic DI’ could be renamed as ‘adipsic AVP-D.’ There are two main reasons for this proposal. One reason is that the current name (DI) is not reflective of the underlying pathophysiologic mechanism as this term was coined in the 18^th century when AVP was not yet discovered (66). The second reason is that both healthcare workers (especially those in non-endocrine-related fields) as well as patients tend to confuse the term ‘diabetes insipidus’ with the more common ‘diabetes mellitus’. This confusion has led to mismanagement of DI resulting in catastrophic consequences (67, 68). In fact, a large survey with 1034 patients with central DI showed that 80% participants encountered a scenario where a healthcare professional confused DI with DM, and 85% of participants supported renaming DI. The proposed plan for the upcoming years is to start transitioning to the new terms while retaining the old terms in parenthesis till the medical community and patients get used to the new terminologies.

With regards to diagnostic testing, further refinements are implemented to the copeptin-based testing, such as utilization of non-osmotic neurohypophyseal secretagogues, to minimize adverse effects or discomfort related to iatrogenic hypernatremia induced by hypertonic saline. Arginine is a non-osmotic stimulus to the posterior pituitary, and it increases plasma copeptin levels in healthy individuals (69). Arginine-stimulated copeptin measurement is being evaluated as a potential diagnostic test for central DI and primary polydipsia. In a recent prospective study, intravenous arginine-stimulation was able to distinguish patients with central DI, and primary polydipsia, and healthy controls using the 60-minute post-stimulation plasma copeptin levels, with high diagnostic accuracy (69). Glucagon, another neurohypophyseal stimulant, was also recently evaluated in a double-blind, placebo-controlled, randomized trial for differential diagnosis of DI (70). Glucagon injection led to significant increase in plasma copeptin levels in healthy controls compared to placebo, while there was a minimal increase in plasma copeptin in central DI patients, there was a substantial increase in patients with primary polydipsia. Further studies performing head-to-head prospective comparisons with hypertonic saline infusion test are required to identify the best copeptin-based diagnostic test.

CONCLUSIONS

Making an accurate diagnosis of DI and ascertaining its type and the underlying etiology poses a significant challenge to this day. In clinical practice, under some circumstances, a diagnosis of DI is established based on serum/plasma and urine studies alone and based on the history and clinical presentation, investigations to identify the underlying etiology are pursued. However, an accurate diagnosis of the type of DI or any polyuria-polydipsia syndrome in general, is difficult to establish as there can be a significant overlap in the results among the various forms of polyuria-polydipsia syndromes on diagnostic testing. Specific testing protocols, such as the indirect water deprivation test or the hypertonic saline infusion test can assist with providing a diagnosis with increased accuracy. Measurement of plasma AVP levels is not routinely performed due to several pre-analytical considerations and lack of widespread availability of assays. With the promising potential of the utility of plasma copeptin levels, diagnosing DI might get less cumbersome in the future once this assay is standardized across laboratories worldwide and once the test becomes commercially available on the global market. The combination of hypertonic saline infusion coupled with plasma copeptin level measurement has achieved diagnostic accuracies that have not been previously attained by any other testing modalities with regards to differential diagnosis of polyuria-polydipsia syndromes (30). But the newer non-osmotic, neurohypophyseal secretagogue (arginine and glucagon)-based plasma copeptin measurements hold the promise of being the safer, less cumbersome testing modalities. However, randomized trials comparing these modalities with hypertonic saline infusion are necessary, with cost and regional availability of these agents also taken into consideration. Therefore, hypertonic saline infusion and plasma copeptin-based approach could potentially become the standard of practice in the future to accurately establish the diagnosis of DI and related polyuria-polydipsia syndromes.

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Hypopituitarism

ABSTRACT

Hypopituitarism is described as partial or complete loss of isolated or combined deficits of pituitary hormones. The clinical presentation of this condition varies depending on the age of onset, underlying cause of the disease, severity of deficiency and number of affected hormones. Investigation to confirm the diagnosis is based on basal hormone status and dynamic stimulatory tests. In this review, we will provide a concise background knowledge regarding underlying etiologies, clinical manifestations, hormonal investigations, and related treatments which necessitate close lifelong monitoring.

INTRODUCTION

Hypopituitarism refers to complete or partial failure of secretion of anterior and/or posterior pituitary hormones. It may arise as a result of congenital defects in the development of individual anterior pituitary cell types or hypothalamic function, acquired disease of the pituitary or hypothalamus, or from infundibular lesions which interfere with the hypothalamic control of the pituitary. The multiple aspects of normal pituitary function serve to predict the wide range of clinical manifestations of hypopituitarism which are determined by the severity, extent and duration of the condition. Onset may occur during childhood or adult life and is generally permanent, requiring one or more specific hormone replacements.

ETIOLOGY OF HYPOPITUITARISM

The major causes of hypopituitarism are shown in Table 1.

Table 1. Causes of Hypopituitarism
Congenital	Isolated pituitary hormone deficiency	· Receptor mutation: GHRH, CRH, GnRH, TRH receptor mutations · Transcription factor defect: PITX2, TBX19, DAX1, NR5A1, NR0B1 · Hormone mutation: GH1, bio-inactive GH, FSHb, LHb, TSHb, POMC, POMC processing defect · Prader-Willi syndrome · Bardet-Biedl syndrome · Kallmann syndrome
Congenital	Multiple pituitary hormone deficiency	· Transcription factor defect: HESX1, SOX 2/3, LHX3/4, PROP1, POU1F1, IGSF1 mutations · Prohormone convertase enzyme mutation: PC1
Neoplastic	Pituitary adenoma	· Functioning and Non-functioning
Neoplastic	Peri-pituitary tumors	· Parasellar lesion: craniopharyngioma, Rathke's cleft cyst · Non-adenomatous neoplasm: meningioma, glioma, germ cell tumor · Metastases, especially breast, renal, bronchus
Vascular	Infarction/ hemorrhage	· Sheehan's syndrome · Pituitary apoplexy · Aneurysms
Inflammatory / Infiltrative / Immunological		· Hypophysitis: lymphocytic, granulomatous, xanthomatous, necrotizing, IgG4-related, immunotherapy-induced (CTLA-4 inhibitors), other immune-associated · Hemochromatosis · Sarcoidosis · Wegener's granulomatosis · Giant cell granuloma · Langerhans cell histiocytosis
Infectious		· Bacterial: tuberculosis, syphilis, leptospirosis · Fungal: candidiasis, aspergillosis · Viral: Herpes/Varicella infection, SARS-CoV-2 virus
Post-irradiation		· Pituitary · Nasopharyngeal · Cranial
Miscellaneous		· Empty sella · Traumatic brain injury · Medications

Congenital

Formation of the normal pituitary during embryonic development depends on the juxtaposition of cells of neuroectodermal origin, which form the posterior pituitary, and endodermal cells derived from the primitive stomodeum, which form the anterior pituitary. Congenital forms of hypopituitarism are best considered as being derived either from hypothalamic or pituitary origin. Defects of RPX/HESX1, PROP1, and PIT1 are associated with varying degrees of inherited hypopituitarism in humans (1). Autosomal dominant mutations of the arginine vasopressin-neurophysin II gene give rise to familial antidiuretic hormone (ADH) deficiency (central diabetes insipidus, CDI). Congenital combined pituitary hormone deficiency (CPHD) (or multiple pituitary hormone deficiency (MPHD)) may be associated with hypoplasia of the anterior pituitary and ectopic siting of the posterior pituitary in a superior position. The underlying mechanism for this condition, once thought to be a consequence of birth trauma, remains undetermined but a congenital molecular defect is likely. Furthermore, since some genetic mutations affect early development, they may include extra-pituitary features or midline anomalies as a part of a syndrome. For instance, the transcription factors, including HESX1, SOX2, SOX3, and OTX2, are the most common genes involved in the etiology of septo-optic dysplasia. Hypogonadotropic hypogonadism is a recognized feature of both Prader-Willi and Bardet-Biedl syndromes. The salient features of the currently described transcription factor defects are indicated below.

HESX1 MUTATIONS

HESX1 is a member of the homeobox gene family. It is one of the earliest markers of the pituitary primodium expressed as a prelude to the development of Rathke's pouch. Mutations of the HESX1 gene in humans are associated with septo-optic dysplasia and evolving hypopituitarism. Septo-optic dysplasia is characterized by the classical triad of optic nerve hypoplasia, midline neuroradiological abnormalities such as agenesis of the corpus callosum, and pituitary hypoplasia with consequent panhypopituitarism (2). Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and its consequences are more extensive.

PROP1 DEFECT

The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Individuals with a single inactivating mutation in PROP1 have deficiencies of luteinizing hormone (LH), follicle stimulating hormone (FSH), GH, prolactin, and TSH (3). Their pituitary glands may be small, normally sized, or large with extrasellar extension. Pituitary degeneration may produce acquired deficiency of adrenocorticotropic hormone (ACTH). Growth restriction and GH responses to GHRH stimulation are more variable in children with PROP1 mutation. This variability does not seem to be dependent on the type of PROP1 mutation because variability exists within sibships. Recessive mutation in PROP1 is the most frequent genetic cause of CPHD and appears to be much more common than POU1F1 mutation (4,5). Clinical suspicion of these mutations should be high in any individual with early onset CPHD, even in those with an intrasellar or suprasellar mass lesion. However, the clinical manifestations of each hormone may vary in onset and severity. GHD is usually present in the first years of life; whereas TSH and gonadotropin deficiency might manifest at birth or later in life. Most patients do not experience adrenal insufficiency during the first years of life although this can evolve later.

PIT1 DEFECT

PIT1, also known as POU1F1, is a pituitary specific transcription factor found in somatotrophs, lactotrophs, and thyrotrophs in the anterior pituitary gland from early fetal development and throughout life. Autosomal recessive defects of PIT1 are associated with combined deficiencies of growth hormone (GH), prolactin, and thyrotropin stimulating hormone (TSH) (6,7). Patients with PIT1 mutations (coded by POU1F1 gene) tend to firstly manifest with GHD and prolactin deficiency in the first year of life and generally do not release detectable amounts of GH after GHRH stimulation. TSH deficiency typically develops later in life (8).

SOX2 MUTATIONS

SOX2 belongs to the SRY-related HMG box (SOX) family of transcription factors which are expressed in various stages of embryonic development and cell differentiation, and play critical roles from the earliest stages of development, in particular expression of anterior neuroectoderm. De novo truncating mutations of SOX2 are found in individuals with bilateral anophthalmia/microphthalmia, small corpus callosum, hippocampal abnormalities, variable mental retardation, anterior pituitary hypoplasia and often hypopituitarism (9-11)

SOX3 MUTATIONS

SOX3 also belongs to the SRY-related HMG box (SOX) family of transcription factors. This dosage sensitive gene is essential for normal hypothalamic-pituitary development (12). Patients manifest with variable hypopituitarism, infundibular hypoplasia, abnormal corpus callosum, hypoplasia of anterior pituitary, absent stalk, ectopic/undescended posterior pituitary, and intellectual disability (13).

OTX2 MUTATION

OTX2 belongs to the orthodenticle homeobox (OTX) and plays an essential role in brain, eyes, and pituitary development. Patients with OTX2 mutations manifest with variable degrees of pituitary dysfunction, intellectual disability, ocular abnormalities including microphthalmia, anophthalmia, and abnormal peripapillary pigmentation (14).

DAX1 MUTATIONS

Mutations in DAX1 (coded by NR0B1), which is another X chromosome gene, cause hypogonadotropic hypogonadism in association with congenital adrenal hypoplasia in males. DAX1 encodes an orphan nuclear hormone receptor with a novel DNA-binding domain, that has a critical role in the development of the hypothalamus, pituitary, adrenal, and gonads (15). DAX1 appears to influence the maintenance of testicular epithelial integrity and spermatogenesis.

KAL1 MUTATION

Isolated gonadotropin deficiency with hyposmia (Kallmann syndrome) may be inherited as an autosomal X-linked disorder or occur sporadically. The X-linked disorder occurs in approximately 1/10,000 to 60,000 live births (16) and a mutation in the KAL1 gene is the basis for this disease. Gonadotropin-releasing hormone (GnRH) neurons develop in the medial olfactory placode and migrate to the hypothalamus during embryonic development; the KAL protein is essential for the initiation and maintenance of this process. Patients with the disorder usually present in late adolescence with delayed pubertal initiation or progression and characteristically exhibit hyposmia. They may develop eunuchoid proportions and usually demonstrate small testes and a short penis; other features which are specific to X-linked Kallmann syndrome include unilateral renal agenesis and synkinesis (mirror movements), whereas midline facial defects, choanal atresia, short metacarpals, malrotation of the gut, and ocular coloboma are more common in autosomal and sporadic forms of the condition.

GH1 MUTATIONS

Mutations in the major GH gene, GH1, can occur as deletions of different size, resulting in severe isolated GH deficiency and may be familial with either a dominant or recessive pattern of inheritance.

TBX19 MUTATION

TBX19, also known as TPIT, is essential for both pro-opiomelanocortin (POMC) transcription and terminal differentiation of POMC-expressing cells. A mutation of TBX19 results in congenital isolated ACTH deficiency and this mutation is discovered in 65% of patients with neonatal-onset congenital isolated ACTH deficiency (17).

Other single gene mutations affecting individual anterior pituitary hormones or pituitary receptors for hypothalamic peptides are indicated in Table 1.

Tumors

Pituitary adenomas are the most common cause of adult-onset hypopituitarism (18,19). Craniopharyngiomas are numerically the next most prevalent and may present as sellar and/or suprasellar masses.

PITUITARY ADENOMA

Pituitary tumors can be either non-functioning or secrete one or more anterior pituitary hormones. They are classified according to size and the presence of extrasellar extension; magnetic resonance imaging (MRI) permits accurate measurement of maximum diameter and tumors are conventionally classified as microadenomas (less than 10mm), and macroadenomas (more than 20mm in diameter) (20). The reported prevalence of pituitary tumors is approximately 10 per million with an average annual incidence of approximately 30 per million depending on tumor type, age, and gender with the highest incidence occurring in pre-menopausal women. Pituitary adenomas may cause typical clinical syndromes resulting from hypersecretion of one or more anterior pituitary hormones. Approximately 25-30% will not present with these symptoms, so they will only be detected when tumor expansion and local compression of the surrounding structures emerges. Post-mortem studies reveal a prevalence of incidental intrasellar adenomas of 10-20%.

Clinically non-functioning pituitary adenomas (NFPA) are non- or low grade-secreting tumors which frequently synthesize and secrete free alpha or beta glycoprotein subunits but do not cause clinically recognized symptomatology. Hence, the treatment of choice is surgical debulking, and sometimes followed by external radiation therapy. In contrast to patients with secreting pituitary tumor who present with clinical syndromes of acromegaly, hyperprolactinemia, Cushing's disease, and secondary hyperthyroidism non-functioning tumors present as an incidental finding, hypopituitarism, or due to mass effects of the tumor. The mainstream treatment in these diseases must be surgical therapy, except prolactinoma which is typically responsive to dopamine agonists.

CRANIOPHARYNGIOMAS

Craniopharyngiomas are benign neoplastic lesions which are presumed to originate from the embryological remnants of Rathke's pouch. They may be located in the suprasellar region, within the sella, or both. Due to the infiltration of surrounding structures, they may have extensive adverse consequences. This tumor accounts for 1% of all intracranial tumors in adults and 6-13% of intracranial tumors in children (21). Peak incidence is in the first decade with a subsequent increase in incidence between 50-60 years of age. Adamantinomatous craniopharyngiomas commonly occur in children and typically contain both solid and cystic components containing a lipid rich secretion. By contrast, squamous papillary craniopharyngiomas develop in adults and are rare in younger patients. This type of tumor is predominantly solid tumor with small well-defined cavities containing less lipid rich fluid. Craniopharyngiomas in adults have a generally better prognosis in respect to endocrine, visual, and other neurological deficits than those in children (22).

RATHKE’S CYSTS

Epithelial cysts known as Rathke's cysts are thought to originate from the remnants of Rathke's pouch. Their location can be intrasellar, with or without suprasellar extension and, rarely purely suprasellar. Compared to craniopharyngioma, they are more common in adults (23). Possible symptoms of Rathke's cleft cysts include pituitary hypofunction, hyperprolactinemia brought on by stalk disruption, visual disturbance, and headache. Due to the difficulty in differentiation between craniopharyngiomas and Rathke's cysts from both a clinical and radiologic standpoint, the diagnosis of an intrasellar Rathke’s cyst cannot always be made with absolute certainty. Definitive diagnosis is often only made histologically. Only 6.4% of asymptomatic patients in a recent retrospective cohort showed cystic enlargement over 41 months follow-up and no pituitary deficiency, confirming the safety of conservative management in these patients (24).

PERI-PITUITARY TUMOURS

Peri-sellar meningiomas are well-circumscribed masses, originating from the sphenoid ridge and are associated with varying degrees of hyperostosis. Meningiomas are typically single, but they can occasionally be multiple. However, females are more likely to develop multiple meningiomas.

Primary intracranial germ cell tumors are neoplasms that arise from aberrantly migrated primordial germ cells. According to histology, the tumor types are similar to those occurring in the gonads and are classified into two categories: germinomas, which are comparable to seminomas and dysgerminomas, and non-germinomatous germ cell tumors, which can include teratomas, yolk sac tumors, embryonal carcinomas, and choriocarcinomas. Geographically, their incidence varies; it accounts for 2-3% of all childhood primary CNS tumors in Western countries, whereas the incidence is higher at 4-15% in Japan (25-27). Patients commonly present in the first two decades of life with diabetes insipidus, visual failure, and hypopituitarism. A recent report highlighted the favorable response to a combined chemotherapy-radiotherapy protocol (27).

Metastasis to the pituitary gland may present as an intrasellar mass resulting in hypopituitarism, diabetes insipidus, or pressure effects. In a published series of 500 consecutive autopsy examinations of cancer patients in whom the pituitary fossa and gland were examined, pituitary metastases were found in 3.6% (28). Pituitary metastases have been described in patients with primary malignant tumors of breast, lung, kidney, thyroid, bladder, uterus, pancreas, and colon.

Vascular Causes

The term pituitary apoplexy denotes the clinical consequences of hemorrhage or infarction in a pre-existing adenoma. The expansion of pituitary mass to neighboring cranial nerves, cavernous sinus, optic pathways, or diencephalon results in localizing signs or altered conscious state. It frequently manifests as a sudden onset of severe headache, a visual disturbance, or ophthalmoplegia as a result of cranial nerve III IV or VI palsies (29). The clinical syndrome of pituitary apoplexy usually evolves fully within only hours to two days. The incidence of surgically treated pituitary adenomas ranges from 0.6 to 9.1%, (30) with a broad age range of occurrence from the first to the ninth decade. Although apoplexy usually occurs spontaneously, systolic and/or diastolic hypertension was seen in 26% of patient in a retrospective analysis, making it an important predisposing factor. (31). Other reported predisposing factors include diabetes mellitus, radiation therapy, anticoagulant therapy, bleeding disorders, head trauma, sudden changes in arterial or intracranial pressure, such as during carotid angiography, the use of bromocriptine, and postpartum hemorrhage (Sheehan's syndrome). Sheehan's syndrome, which denotes pituitary necrosis after postpartum hemorrhage and hypovolemia, may result in hypopituitarism, that is either immediate or delayed over several years depending on the extent of tissue destruction. However, this condition is rare with modern obstetric care (32).

Suprasellar or intrasellar aneurysms of the carotid arteries, as well as suprasellar aneurysms of the anterior or posterior communicating arteries can manifest as an expanding mass inside the fossa. Hypopituitarism may result directly from intrasellar aneurysms. Histologically, typical degenerative sclerotic changes of the arterial wall are found along with chronic inflammatory changes. An arteriovenous fistula may occasionally develop in the cavernous sinus as a result of an aneurysm.

Immunological/Inflammatory Disease

Inflammatory lesions of the pituitary gland can clinically and radiologically mimic pituitary tumors, with varying degrees of hypopituitarism and mass effects like headaches and visual field impairment.

Lymphocytic hypophysitis classically presents as partial hypopituitarism associated with a pituitary mass lesion, particularly in relation to pregnancy (33). It has been speculated that many cases of postpartum pituitary failure, previously attributed to Sheehan's syndrome, may in fact have been caused by lymphocytic hypophysitis (34,35). In this condition, the interstitial tissue of the adenohypophysis is more or less densely infiltrated by lymphocytes and plasma cells. As a result, the anterior pituitary exhibits necrosis and subsequent fibrosis of hormone-producing cells. Since the histological findings are similar to those of other organ-specific autoimmune diseases, the primary etiology is considered to be autoimmune (36,37). Partial hypopituitarism, commonly with isolated ACTH deficiency, or with TSH deficiency, is seen in approximately 80% of patients, with LH and FSH generally remaining unaffected.

Sarcoidosis is a multisystem disorder, which is characterized by the presence of non-caseating granulomas. It most often affects lungs and reticuloendothelial system but can involve any organ. Central nervous system sarcoidosis occurs with an incidence of 5-15% (38,39) and less than 1% of patients have symptoms of a hypothalamic-pituitary disease (40). Neurosarcoidosis affects primarily the leptomeninges of the brain base and posterior fossa; the hypothalamus and/or pituitary subsequently experience local granulomatous infiltration. Coexisting optic nerve infiltration occurs in many patients causing central and peripheral visual defects. In terms of anterior pituitary function, LH, FSH and GH deficiency are frequent, but TSH and ACTH are relatively preserved.

Langerhans cell histiocytosis is a rare disease characterized by aberrant proliferation of a specific dendritic (Langerhans) cell belonging to the monocyte-macrophage system (41). Deposits occur at multiple sites within the body, and frequently involve the hypothalamo-pituitary axis (42). Diabetes insipidus is a well-recognized and common feature of this condition but anterior pituitary dysfunction also occurs frequently.

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitor, a IgG1 monoclonal antibody against CTLA-4 is used for metastatic melanoma and other neoplasms. As a consequence of immune activation, secondary hypophysitis has been reported in 10-15% of ipilimumab treated patients (36). In severe hypophysitis, immune reactions induced extensive necrosis of the adenohypophyseal architecture. Pituitary autoantibodies against thyrotrophs, corticotrophs, and gonadotrophs were identified in patients with ipilimumab induced hypophysitis (37). Patients usually present with headache and fatigue, the diagnosis of hypopituitarism with multiple deficiencies is made 2-3 months or later after initiation of ipilimumab. Central hypothyroidism is the most frequent deficit, followed by central hypocortisolism, and hypogonadism.

Infections

Bacterial pituitary sepsis is a rare phenomenon which may arise as a consequence of hematogenous spread or by extension from sinus or meningeal sepsis with subsequent pituitary abscess formation. Small pituitary abscesses have been described at postmortem in patients dying from septicemia. Chronic infection predisposes to necrosis of pituitary tissue with subsequent hypopituitarism. Pituitary tuberculomas may present as space occupying lesions (43)but this appears to be a very rare phenomenon; isolated manifestation of the disease is rare and it is more likely to occur in the context of generalized tuberculous infection with meningitis. Gumma formation, as a manifestation of tertiary syphilis, may occur in the sella region but is extremely rare.

Fungal pituitary infection may occur as a complication of AIDS (44) and pituitary necrosis with hypopituitarism has been described in toxoplasmosis (45).

Radiation Therapy

Hypopituitarism is a common complication of irradiation administered for pituitary adenomas, head and neck tumors, intracranial malignancy, or as adjunctive cranial irradiation for acute lymphoblastic leukemia. Although fractionated radiotherapy with daily doses of <200cGy is well tolerated and safe in terms of neurological sequelae, the majority of patients who have undergone external irradiation will manifest some degree of pituitary failure during long term follow up. In general, this is a relatively late complication and is rarely evident in less than 3 years in patients with completely normal baseline pituitary function. Growth hormone reserve is particularly susceptible and is progressively more likely with time so that periodic assessment of residual pituitary function is mandatory; a five year follow up study after external pituitary irradiation therapy reported, 100% GH deficiency, 91% LH-FSH deficiency, 77% ACTH deficiency and 42% TSH deficiency (46).

Traumatic Brain Injury

Traumatic brain injury (TBI) is common, the prevalence of endocrine dysfunction in these patients ranges from 15-68% (47), with estimated annual incidence 30 patients per 100,000 population per year. Abnormal axes during the acute phase of injury may recover over time, but other pituitary hormone deficits may evolve later even at 6 months after the initial insult. A prospective longitudinal study looking at patients with severe TBI, found that 6% of their patients had proven severe GHD when evaluated after 12 months (48). Multiple pituitary deficiencies or isolated pituitary deficiencies (TSH or ACTH) were very rare.

Empty Sella Syndrome

An enlarged empty sella may be primary (due to a congenital diaphragmatic defect) or secondary to surgery, radiation, or pituitary infarction. The majority of patients with congenital empty sella have normal pituitary function. Hypopituitarism (and/or hyperprolactinemia) may be found in instances due to previous pituitary disease. Occasionally, a cystic pituitary mass may simulate an empty sella on CT or MRI, necessitating a cisternogram for precise delineation.

CLINICAL MANIFESTATION OF HYPOPITUITARISM

The clinical impact of pituitary insufficiency is dependent on the extent and severity of hormone deficiencies (Table 2), the duration of the disease and the age of onset; childhood onset hypopituitarism has consequences for all aspects of somatic development in addition to the pathophysiological effects of specific hormonal deficiencies. In addition, there may be clinical features relating to the mass effect of causative lesion or specific consequences attributable to hypersecretion of prolactin, GH, ACTH or TSH from individual tumor types. Hypopituitarism classically develops in sequential order with the secretion of growth hormone, then gonadotrophins being affected first, subsequently followed by TSH and ACTH. Prolactin deficiency is rarely seen, except in Sheehan's syndrome which is associated with failure of lactation. ADH deficiency is almost never seen as a primary feature of pituitary adenomas but is a usual presenting manifestation of germ cell tumors, pituitary metastases, and granulomatous disorders.

Table 2. Summary of Clinical Features of Hypopituitarism
Hormone deficiency	Presentation	Symptoms and signs
Adrenocorticotrophic hormone	Acute	Fatigue, weakness, dizziness, nausea, vomiting, circulatory failure. As in Addison's disease, except lack of hyperpigmentation, absence of hyperkalemia
Adrenocorticotrophic hormone	Chronic	Tiredness, pallor, anorexia, nausea, weight loss, myalgia, hypoglycemia
Gonadotropins	Children	Delayed puberty
	Men	Impaired fertility, impotence, reduced libido, decreased muscle mass and strength, decreased bone mass, decreased erythropoiesis and hair growth, fine wrinkles, testicular hypotrophy
	Women	Amenorrhea, oligomenorrhea, infertility, loss of libido, dyspareunia, fine wrinkles, breast atrophy, osteoporosis, premature atherosclerosis
Thyroid-stimulating hormone	Children	Growth retardation
Thyroid-stimulating hormone	Adult	Fatigue, cold intolerance, constipation, weight gain, dry skin, slow relaxing reflexes
Growth hormone	Children	Growth retardation, short stature, increased adiposity
Growth hormone	Adult	Reduced exercise capacity, impaired psychological wellbeing, increased cardiovascular risk, increased central obesity, reduced lean body mass
Prolactin	Decreased	Failure of lactation
Prolactin	Increased	Galactorrhea, oligo/amenorrhea, loss of libido
Antidiuretic hormone		Polyuria, polydipsia including nocturnal

The clinical features of anterior pituitary hypofunction described in this chapter will concentrate on adult-onset hypopituitarism. Many of the symptoms and signs of a specific pituitary hormone deficiency are similar to those that occur in patients with a primary deficiency of the target gland, but there are exceptions.

Growth Hormone Deficiency

GH deficiency (GHD) in adults is characterized by decreased exercise tolerance, decreased mood and general well-being, decreased quality of life, central adiposity, hyperlipidemia, increased predisposition to atherogenesis, and reduced bone remodeling activity. Fine facial wrinkles may result from a deficiency of growth hormone in addition to hypogonadism. The patient will usually have dry thin skin which contrasts with the thickened skin and increased sweating found in acromegaly. Adults with long-standing GHD are often overweight, have reduced lean body mass, increased fat mass, especially visceral fat, relative insulin resistance, and reduced total bone mass. There is a reduction in cardiac and physical performance. GHD is associated with increases in total cholesterol, LDL-cholesterol and apolipoprotein B (49-51). Studies in Sweden and the UK in patients with hypopituitarism receiving controlled thyroid and steroid hormone replacement but without growth hormone replacement, have demonstrated an approximately two fold increase in cardiovascular mortality compared to the general population. (51) and the increase in standardized mortality ratio is more striking in females (52).

The accumulating evidence suggests that the cardiovascular morbidity cannot be explained solely by suboptimal glucocorticoid, gonadal steroid, or thyroid hormone replacement and unsubstituted growth hormone deficiency is probably an important contributing factor. The decreased bone mineral density found in GHD is associated with increased fracture risk (53).

Adrenocorticotrophic Hormone Deficiency

Cortisol and adrenal androgen secretion are ACTH-dependent and are variably decreased in hypopituitarism. Patients with combined ACTH and LH deficiency are completely androgen deficient, a phenomenon that may be particularly important when considering optimum regimens for gonadal steroid replacement in females. Since aldosterone secretion is largely determined by activation of the renin-angiotensin-aldosterone system, it is relatively preserved in the hypopituitary patients. Major symptoms of ACTH deficiency are non-specific, including fatigue, weakness, headache, anorexia, weight loss, nausea, vomiting, abdominal pain, myalgia, and decreased concentration. Hypoglycemia may be present at diagnosis. Hyponatremia is common and is attributable to reduced renal free water clearance as a result of cortisol deficiency with an additional contribution from TSH deficiency if present. Hyperkalemia, which is a frequent finding in primary adrenal failure, does not occur in patients with hypopituitarism due to the relative preservation of aldosterone secretion. Over and above the effects of secondary hypogonadism, reduced adrenal androgen production further exacerbates loss of body hair, particularly in women.

Gonadotrophin Deficiency

Gonadotrophins are responsible for gonadal sex-steroid production, secondary sexual development, maintenance of secondary sexual characteristics, and fertility.

Gonadotrophin deficiency in males’ results in secondary hypogonadism with consequent testosterone deficiency. Clinical features include loss of libido, erectile impotence, oligospermia, reduced erythropoiesis, and decreased lean body mass. Testosterone, via estradiol derived by aromatization, plays an important role in the regulation of bone mineralization, therefore, hypogonadism results in decreased bone mineral density.

Clinical features of secondary hypogonadism in females include oligo/amenorrhea, breast atrophy, diminished secondary sexual hair especially when combined with ACTH deficiency, and a predisposition to osteoporosis.

Thyroid Stimulating Hormone Deficiency

The clinical features of secondary thyroid failure are similar to those of primary thyroid failure with the exception that weight gain is less likely to be a feature if ACTH deficiency is also present. Classical features include cold intolerance, fatigue, and myalgia. Physical examination may demonstrate periorbital edema and delayed reflex relaxation. Additionally, hyponatremia and normochromic normocytic anemia are observed, with the former being exacerbated by cortisol deficit while the latter is aggravated by secondary hypogonadism and GH deficiency.

Prolactin Deficiency

Prolactin deficiency in females result in puerperal alactogenesis, However, isolated prolactin deficiency is uncommon because the great majority of prolactin deficiency states develop as a result of general anterior pituitary problems or as a result of treatment intervention. On the other hand, hyperprolactinemia, which is caused by stalk disruption and results in a lack of dopaminergic regulation from the hypothalamus, is more frequent and can manifest as galactorrhea, abnormal menstruation, loss of libido, and decreased bone density.

Antidiuretic Hormone Deficiency

Antidiuretic Hormone Deficiency (AHD) previously called central diabetes insipidus (CDI) is characterized by polyuria and polydipsia due to decreased secretion of antidiuretic hormone by the neurosecretory cells terminating in the posterior pituitary. If excessive water excretion exceeds intake, patients will progressively become water-depleted and eventually have a reduction in circulating volume. It occurs more frequently in associated with tumors of the hypothalamus, pituitary metastases, lymphocytic hypophysitis, sarcoidosis, Langerhans cell histiocytosis, craniopharyngiomas, and Rathke's cleft cysts. (54). Additionally, it is commonly observed following neurosurgery and head injury. As cortisol is an essential prerequisite for normal glomerular filtration and free water clearance, it should be highlighted that AHD is masked by co-existing ACTH deficiency and only becomes clinically evident after commencement of glucocorticoid replacement. However, it is extremely unusual for AHD to be a primary manifestation of pituitary adenoma.

INVESTIGATION OF SUSPECTED HYPOPITUITARISM

In hypopituitarism, basal serum hormone measurements are required to confirm the insufficiency; however, dynamic testing is mandatory for the diagnosis of some deficiencies. Pituitary function testing is required for all patients presenting with pituitary disease or in whom an evolving endocrine deficiency is anticipated, e.g., those who have undergone pituitary or cranial radiotherapy.

Baseline Investigations

Basal concentrations of the anterior pituitary hormones and hormones produced by their respective target glands should be measured. Serum samples should be taken unstressed, with no physiological or pharmacological manipulation, between 7 and 9 AM when serum cortisol and testosterone levels are highest. This is important given that the decision to proceed to

testing is based on these levels. The pituitary hormones may remain within the normal range despite low levels of the target hormones indicating that target gland failure is a consequence of inadequate pituitary stimulation. Baseline investigations (Table 3) are sufficient for the diagnosis of secondary hypothyroidism and hypogonadism and will also confirm virtually complete ACTH deficiency (55).

Table 3. Baseline investigation of pituitary function

1. Adrenocortical axis: serum cortisol (09.00 AM), ACTH
2. Thyroid axis: free T4, TSH
3. Gonadal axis:

Men - testosterone (09.00 AM), SHBG, LH, FSH;

Women - estradiol, LH, FSH, progesterone (Day 21 if menstruating)
4. Prolactin
5. Insulin-like growth factor-1, growth hormone
6. Paired plasma and urine osmolality

ACTH, adrenocorticotropic hormone, FSH, follicle stimulating hormone; LH, luteinizing hormone; SHBG, sex hormone binding globulin

It should be mentioned that the serum total testosterone measurement should be performed in the morning by a reliable assay and the test should be repeated before verifying the diagnosis of testosterone deficiency. Measuring the level of free or bioavailable testosterone level should be performed in those whose total testosterone levels are close to the lower limit of the normal range or whose SHBG is suspected to have altered (56).

Dynamic Testing

Stimulation tests are used when hypofunction is suspected and are designed to assess the reserve capacity to form and secrete hormone. In contrast, suppression tests are used when endocrine hyperfunction is suspected and are designed to determine whether negative-feedback control is intact, as in the administration of glucocorticoids to inhibit corticotrophin secretion in patients with suspected Cushing’s syndrome or in the administration of glucose in patients with suspected acromegaly. Dynamic pituitary function tests may assess the hypothalamic-pituitary unit (e.g., insulin tolerance test, glucagon, and arginine tests) or directly stimulate the anterior pituitary with pharmacological doses of synthetic hypothalamic peptides and the pituitary hormone response measured (e.g., TRH, GnRH, GHRH tests).

HYPOTHALAMIC-PITUITARY-ADRENAL AXIS

Baseline and dynamic tests are valuable in the diagnosis of ACTH deficiency. With virtually complete ACTH deficiency, the 0900h serum cortisol is less than 100 nmol/L (3.625 µg/dL). In contrast, if the serum cortisol is 400-500nmol/L (14.5 – 18.1µg/dL) or more, ACTH deficiency is unlikely. (57-59). Therefore, dynamic testing of ACTH reserve is required if the basal serum cortisol measured lies between 100 and 400-500nmol/L. The insulin tolerance test (ITT) or glucagon test may be used to assess the adequacy of the hypothalamic-pituitary-adrenal axis. If the patient is taking hydrocortisone this should be discontinued for 18-24 hours; while prednisolone cross reacts in the cortisol assay and therefore should not be administered within 24 hours of investigation of adrenocortical reserve. The rationale of the ITT is to produce physiological stress in a controlled environment by inducing hypoglycemia with intravenous insulin. Hypoglycemia is a powerful stimulus which stimulates GH and ACTH release and a rise in serum cortisol levels in the presence of an intact hypothalamic-pituitary axis. Although the safety of ITT has been questioned, the only absolute contraindications are ischemic heart disease, epilepsy or unexplained loss of consciousness, untreated hypothyroidism or hypoadrenalism, and glycogen storage disease (58). A decrement in plasma glucose to less than 2.2 mmol/L (40 mg/dL) is required for the test to be valid. With normal ACTH reserve the serum cortisol should rise to at least 550 nmol/L (20 µg/dL). Patients who show a normal cortisol response can withstand major surgery without corticosteroid replacement; while patients with subnormal responses but satisfactory basal values (> 250 nmol/L (9 µg/dL) may not require regular replacement therapy but should be fully informed and carry a steroid card. The ITT should only be performed by fully trained staff in designated units. Hypoglycemia may occasionally require reversal with intravenous glucose to avoid the risk of cerebral edema, which affects 50% of adults but limited to 10% of children.

The glucagon stimulation test (GST) may be used for the assessment of ACTH/cortisol and GH reserve simultaneously when the ITT is contraindicated (60). The subcutaneous injection of glucagon causes a transient rise in plasma glucose. During the subsequent fall in plasma glucose, ACTH and GH are released and measured. Serum cortisol cut-off values are similar to those described for the ITT. Glucagon is less reliable than the ITT as a test of ACTH/ cortisol reserve; it is a less powerful stress stimulus and hence false positive results are a recognized problem.

The short synacthen (Cortrosyn in the USA) test (SST) was originally introduced as a test for primary adrenal failure. It involves the intramuscular or intravenous injection of a pharmacological dose (250μg) of synthetic ACTH, with measurement of the serum cortisol response at 30 and 60 minutes later. The test does not distinguish primary from secondary adrenal insufficiency, and it cannot directly assess pituitary ACTH reserve. The 30-minute serum cortisol response is advocated as a surrogate test of ACTH reserve and has been widely used because of its simplicity. However, there is no study showing that a normal SST indicates that the hypothalamic-pituitary-adrenal axis is capable of responding normally to major illness or stress. Demonstrations of good correlations between peak serum cortisol responses on ITT and 30-minute responses on SST have been published and are intuitively predictable. However, false negatives with the SST are well recognized although this may be partially obviated by the use of lower synacthen doses (1μg). Nonetheless, the concern about false negative results and the fact that assessment of GH reserve is also frequently required serve to limit the use of the SST in the investigation of pituitary function.

A recent study comparing the ITT, low dose ACTH, and glucagon stimulation test in the evaluation of the HPA axis and GH-IGF-1 axis in patients with pituitary disorders concluded that all three tests were well correlated in terms of peak cortisol and GH response (61). However, low dose ACTH stimulation gave a higher peak cortisol response. Therefore, the cut-off level for the diagnosis of insufficiency of the HPA axis needs to be individualized for each test. See Chapter Adrenal Insufficiency

GROWTH HORMONE

Normal GH secretion is pulsatile, with four or six pulses per 24 hours, mostly at night in association with REM sleep. A single measurement of serum GH is rarely useful although the fortuitous coincidence of blood sampling at the time of a GH secretory peak may exclude GHD. A stimulatory test is therefore usually required to assess somatotroph reserve. Most, actions of GH are mediated through hepatically or locally-derived insulin-like growth factor-1 (IGF-1). Measurement of baseline serum IGF-1 is a specific but insensitive test of GHD in patients with pituitary disease. Importantly, IGF-1 declines with normal aging. In adult onset GHD, 30% of patients may have serum IGF-1 levels in the lower half of the age-related reference range, with the percentage increasing with age (62). Therefore, a low serum IGF-1, in an adequately nourished patient without liver dysfunction, strongly supports a diagnosis of GHD, but a normal serum IGF-1 cannot exclude it.

A rigid and precise biochemical diagnosis of GHD is required in the context of justification of growth hormone replacement. The ITT is one of the most reliable provocative tests of GH secretion and currently remains the test of choice for this purpose since it has the advantage of assessing ACTH reserve simultaneously. Severe GHD is defined as a peak GH response to insulin induced hypoglycemia of less than 3ng/mL (9mU/L) (63). Obesity may blunt the GH response to dynamic testing so that the diagnosis of GHD should only be made in the context of structural pituitary disease or previous cranial irradiation and/or additional pituitary hormone deficits.

If the ITT is contraindicated, an alternative test for assessment of GH reserve is required. Glucagon, arginine, a combination of arginine and GHRH or growth hormone-releasing peptides may all be used for this purpose. The combination of GHRH plus arginine is the most powerful provocative test of GH secretion but appropriate normative data are required (64-66). Recently, the FDA approved macimorelin, which is a non-peptidyl agonist of GH secretagogue receptor 1a as a diagnostic agent for diagnosing GH deficiency, and macimorelin stimulation test was known to have good sensitivity and specificity in comparison with ITT (67). However, this oral agent is not widely used due to its cost and limited availability.

PITUITARY-THYROID AXIS

In the appropriate clinical context, secondary hypothyroidism can be diagnosed on the basis of a low serum thyroxine (T4, total or free) in the presence of low or low normal TSH (TSH is rarely undetectable in hypopituitarism). However, it should be borne in mind that any systemic illness may produce a reversible reduction in serum T4 (sick euthyroid state).

Dynamic testing using thyrotrophin releasing hormone (TRH) has no diagnostic value for secondary hypothyroidism or predicting a risk of developing TSH deficiency. Although patients with hypothalamic disease may show a delayed response to TRH, this test is seldom used in pituitary reserve assessment. Furthermore, intravenous TRH may precipitate hemorrhagic infarction of pituitary adenomas.

PITUITARY-GONADAL AXIS

Symptoms of sex steroid deficiency, menstrual disturbance, low serum estradiol or low serum testosterone levels in the presence of normal or low concentrations of FSH/LH are the mainstay of diagnosis of hypogonadotropic hypogonadism. The gonadotrophin releasing hormone (GnRH) test has virtually no diagnostic value but is used to confirm gonadotroph reserve in the setting of pulsatile GnRH therapy for infertility.

POSTERIOR PITUITARY FUNCTION

Basal investigations include plasma and urine osmolalities obtained simultaneously. In overt AHD, plasma osmolality is usually raised in the presence of inappropriately diluted urine. The diagnosis of partial AHD is confirmed by means of a water deprivation test followed by demonstration of a response to desmopressin. Copeptin is the most recent clinical diagnostic marker for AHD due to its strong correlation with plasma arginine vasopressin (AVP) (68). Copeptin is secreted in equimolar ratio to AVP, mirroring AVP concentrations in the circulation. A copeptin level of 4.9pmol/L stimulated with hypertonic saline infusion differentiates between AHD and primary polydipsia with a high diagnostic accuracy and is superior to the water deprivation test (69). See Chapters on Posterior Pituitary.

Imaging the Pituitary

Magnetic resonance imaging (MRI) is the optimum method of imaging, with computerized tomography (CT) as an acceptable alternative. The only disadvantage to MRI is its insensitivity in defining pathological calcification and lack of signal from corticated bone. CT may be required to demonstrate calcification in craniopharyngiomas and hyperostosis; it is also used by many surgeons to define skeletal anatomy prior to surgery.

MANAGEMENT OF HYPOPITUITARISM

Hypopituitarism, once established, is usually permanent. However, resection of pituitary tumors may, on occasion, result in resumption of normal pituitary function.

Although a seemingly straightforward clinical exercise, hormone replacement therapy cannot simulate normal physiology precisely. With the exception of GH replacement and treatment of infertility, replacement therapy is achieved by administering target hormones. The aim of hormone replacement is to safely eliminate or minimize the symptoms and clinical signs of specific hormone deficiencies. The Endocrine Society Clinical Practice Guidelines committee have issued recommended treatment option of each target hormone deficiency either primary or secondary for the management of hypopituitarism (70).

Hypocortisolism

Hydrocortisone is now the most widely used form of glucocorticoid replacement in patients with primary and secondary adrenal insufficiency. There is no universal agreement regarding the appropriate dose, timing, and monitoring of hydrocortisone replacement. Normal individuals demonstrate undetectable serum cortisol and ACTH when asleep at midnight, with a rise during the early hours of the morning to reach a peak at 0800-0900h, followed by a steady decline throughout the rest of the waking day. There are variable peaks of cortisol secretion due to other factors such as stress, meals, and exercise. Since cortisone acetate requires conversion into hydrocortisone under the influence of 11ß-hydroxysteroid dehydrogenase type 1 (11β-HSD1), and the fact that the enzyme activity is altered in patients with GHD; therefore, cortisone renders intuitively less satisfactory than hydrocortisone for replacement purposes in this condition. Prednisolone has been advocated by some on account of its longer duration of action than hydrocortisone. However, because it cannot be routinely measured, it is impossible to fine tune the replacement dose. Dexamethasone, the alternative synthetic glucocorticoid replacement, is even less satisfactory because of wide interindividual variations in metabolic clearance rates.

Traditional hydrocortisone replacement utilized twice daily dosing but many patients report fatigue or headache in the afternoon on this regimen. There is evidence that many patients 'feel better' on thrice daily regimes (71). Recent cross-sectional studies did not demonstrate any superiority between three times a day versus twice daily replacement in terms of quality of life (72). The average daily requirement is approximately 20mg of hydrocortisone. This should be given as 10mg on waking, 5mg at lunchtime and 5mg in the early evening. Enzyme-inducing drugs, especially, phenytoin, carbamazepine, and rifampicin can increase the metabolism of corticosteroids and should prompt an increment in replacement doses. Doses are therefore fine-tuned according to patient’s well-being and multiple serum cortisol levels (hydrocortisone day curve, HCDC) taken during the day in many centers. (55,73). Some centers have attempted to utilize urine free cortisol measurements to adjust the hydrocortisone dose; however, this is unreliable because saturation of cortisol binding globulin (CBG) following oral hydrocortisone results in supraphysiological urine free cortisol excretion (74). The HCDC should demonstrate adequate levels of cortisol throughout the day, without excess peak (cortisol e.g., >1000 nmol/L (36.25 µg/dL)) or trough (e.g., <100 nmol/L (3.625 µg/dL)) levels before or after doses.

In clinical practice, there is no reliable measure to be certain if patients are receiving optimal glucocorticoid replacement therapy. As a result, patients may be over- or under- treated with resultant morbidity (65). There is a significant increase in 11β-HSD1 activity resulting in abnormalities in corticosteroid metabolism in patients with ACTH deficiency treated with conventional doses of hydrocortisone (66). In ACTH-deficient patients daily hydrocortisone dose exceeding 20mg/day is associated with increase waist to hip ratio. The induction of 11β-HSD1 is associated with central adiposity and has an important role in the development of the metabolically adverse hypopituitary phenotype.

Conventional hydrocortisone cannot mimic the circadian rhythms of cortisol release; in particular the early morning rise in cortisol which slowly declines throughout the day. This has led to the development of a modified release formulation of hydrocortisone (MR-HC, Chronocort®) which can be taken late at night thus allowing a delayed and sustained release (75). A study in healthy men demonstrated that MR-HC 20mg and 10mg, given at 2300 and 0700 hours respectively, could achieve a near normal cortisol circadian rhythm (76). A subsequent phase II study demonstrated that the MR-HC mimics the normal circadian pattern closer to physiological baseline (77); however, the studied preparation is no longer available. An alternative modified formulation (Plenadren®) incorporates an immediate release and delayed release components, which displays diurnal plasma cortisol levels similar to physiological profile. Once daily Plenadren® has been shown to reduce weight, blood pressure and improve glucose metabolism when compared with thrice daily dosing (78).

Another newer oral immediate-release granule formulation of HC (Alkindi®) was approved in EU in 2018. This is an immediate-release hydrocortisone preparation specifically designed for pediatric dosing, which is available in four doses: 0.5mg, 1mg, 2mg and 5mg (79).

Table 5 summarizes the glucocorticoid compounds currently available for adrenal insufficiency.

Table 5. Compounds Available for Adrenal Insufficiency
Glucocorticoid	Timing	Dose
Hydrocortisone	2-3 times/day; waking, early PM, and at least 6 hours from bedtime	10-20mg
Prednisolone	Once in morning	3-5mg
Modified release hydrocortisone (Chronocort®)	2times/day; waking and before bed	10mg in AM and 20mg at bedtime
Modified release hydrocortisone (Plenadren®)	Once daily in early morning	Equal to standard HC dose or a 20% increase
Oral immediate release granules (Alkindi®)	Once daily in early morning	Equal to appropriate pediatric hydrocortisone dosing

The effects of continuous subcutaneous hydrocortisone infusion (CSHI) have been compared with conventional oral hydrocortisone in patients with Addison’s disease (80). CSHI produced a more physiological circadian rhythm, normalization of morning ACTH and restoration of nocturnal serum cortisol levels. These infusions are cumbersome and impractical outside of expert centers. Despite a more circadian pattern of cortisol exposure, subcutaneous hydrocortisone infusion was not associated with improved quality of life scores, casting doubt on the potential quality of life effects of circadian cortisol delivery (81).

There is a push to design orally active delayed or sustained release formulations of hydrocortisone to aid the physiological replacement of hydrocortisone and ultimately improve quality of life and side effect profiles in patients requiring lifelong glucocorticoid replacement.

Patients with ACTH deficiency receiving hydrocortisone therapy are unable to respond to surgery, trauma, infections, and severe illnesses by increasing their cortisol concentrations. They therefore require supplemental hydrocortisone therapy with increased oral doses during minor illness, or administration of intramuscular hydrocortisone 100mg four times per day with more severe disease or if oral intake is compromised. When intramuscular injections are contraindicated, a continuous intravenous infusion of hydrocortisone at a rate of 1-3mg per hour provides satisfactory replacement for the severely ill patient. Maintenance mineralocorticoid is not required since aldosterone secretion is usually preserved.

Patients should carry a 'steroid card' and wear a 'medic-alert' bracelet to indicate their requirement for supplemental hydrocortisone in the event of severe illness or trauma. They and their families should understand the importance of life-long compliance, be taught to double the hydrocortisone dose in the event of pyrexial illness, and understand the need for parenteral glucocorticoid replacement if vomiting or diarrhea occurs. An 'emergency' ampoule of hydrocortisone should be provided for domiciliary emergency intramuscular injection and the patient instructed on its use.

Dehydroepiandrosterone (DHEA) is an androgen produced by the adrenal cortex and is also under the regulation of corticotrophin, and is thus deficient in hypopituitarism. In hypopituitarism, DHEA supplementation (25-50mg per day) has shown benefit with respect to well-being and sexual function (82,83). Furthermore, in patients who are replaced with GH, DHEA can augment the IGF-1 response hence leading to a reduction of GH dose in females (84). Currently, there is no licensed preparation of DHEA available, and it is considered to be a food supplement rather than a bioactive drug. However, not all patients respond. Moreover, the androgenic side effects such as greasy skin, acne, and increased body hair may be a limiting factor although generally responsive to dose reduction.

Secondary Hypothyroidism

Synthetic levothyroxine sodium (for example, Synthroid) is the preferred form of replacement. It has a long half-life, allowing a once daily dose. Liothyronine (T3) displays superior gastrointestinal absorption, but its short half-life requires two to three daily doses. Use of T3 is largely restricted to thyroid cancer patients undergoing frequent isotopic imaging or treatment, and occasionally the initiation of thyroid hormone replacement when a gradual increase is desired. Although some experts consider using a T4/T3 combination as an experimental treatment for patients with persistent hypothyroid symptoms on LT4 since patients with normal thyrotropin levels may have lower T3 levels, the evidence is primarily limited to athyreotic patients.(85,86)

Commencing thyroid hormone replacement in patients with severe, untreated ACTH deficiency may result in hypoadrenal crisis. In the situation of combined deficiency, hydrocortisone should always be commenced before thyroxine. The duration of hypopituitarism and presence of co-morbidities, especially ischemic heart disease, should be considered. Young patients with a short history of hypopituitarism and TSH deficiency may commence an initial thyroxine dose of 100µg daily. On the other hand, in patients with a long history of hypopituitarism or elderly patients, a low dose of 25-50µg daily should be commenced, in order to minimize the risk of precipitation of cardiac events.

Alteration of the hypothalamic-pituitary-thyroid axis following growth hormone replacement is well documented (87-89). GHD will mask central hypothyroidism in a significant proportion of hypopituitary patients both in children and adults. It has been observed that apparently euthyroid hypopituitary patients will require commencement or an increase in thyroxine replacement following initiation of GH replacement. A higher target serum free T4 in the upper half of reference range is appropriate in the GH deficient patient who is not on GH replacement.

Unlike primary hypothyroidism, in which serum TSH is a sensitive marker of under-or over-replacement, there is no biochemical marker to indicate the optimum level of replacement in TSH deficiency. The serum free T4 is the best marker for assessing replacement adequacy in hypopituitarism. By analogy with serum T4 levels in adequately replaced primary hypothyroidism, a conventional recommendation is to maintain serum free T4 in the upper part of the reference range for normal individuals (90). Serum total T4 levels are elevated artefactually by conditions which increase serum thyronine binding globulin, especially estrogen administration.

Gonadotrophin Deficiency

Choice of replacement ranges from oral, transdermal, intramuscular, or subcutaneous administration of gonadal steroids to gonadotropin or gonadotropin-releasing hormone therapy if and when fertility is desired.

WOMEN

Estrogen replacement should be offered to all women with secondary hypogonadism under the age of 50 years in order to avoid immediate symptoms of estrogen deficiency and prevent premature reduction in bone mineral density. The addition of progesterone is mandatory if the uterus is intact in order to avoid unopposed estrogen stimulation of the endometrium with the attendant risk of hyperplasia and neoplasia. The standard regimen for replacement involves the daily administration of estrogen with progesterone co-administrated for 12-14 consecutive days during a 4-week cycle; menses occur cyclically after progesterone withdrawal. Alternatively, a continuous regimen may be employed in which estrogen and progesterone are combined. The latter may be preferred by older patients and there are no adverse effects described apart from unpredictable menstrual bleeding during the initial few months of therapy in a minority of patients. However, since the risk-benefit profile of estrogen therapy is influenced by numerous factors, such as age, treatment-onset since menopause, and existing comorbidities, shared decision making is essential when determining what and route to administer as well as when to stop this treatment.

Estrogen replacement can be administrated via the oral, transdermal, and subcutaneous routes. Oral estrogens undergo extensive hepatic first-pass metabolism necessitating average doses of 1-2mg estradiol per day, or equivalent.

Transdermal preparations are usually applied twice weekly and provide 50-100µg mcg of estradiol per 24 hours in a cyclical combination with a progestogen. Skin irritation may occur but transdermal therapy is the first choice in patients with complex pituitary disease since it avoids the effects of oral estrogen on other hormone binding proteins. Furthermore, in women on concomitant GH replacement, IGF-1 generation is greater when transdermal rather than oral estrogen is used, therefore decreasing the dose of GH required. Subcutaneous implants are inserted every six months, but tachyphylaxis is a frequent problem and limits the value of this regimen.

In addition to relieve vasomotor symptoms and reduce risk of bone loss, hormone therapy may be prescribed on the market to seeking for cardioprotection. According to recent research from the combined Women’s Health Initiative (WHI) studies, women who start hormone therapy closer to the period of natural menopause have a decreased risk of coronary heart disease than women who wait longer after menopause. However, this replacement is not advised for either primary or secondary cardiovascular disease prevention (91).

Concerning the negative effects of estrogen replacement, it has been well established that estrogen, particularly oral estrogen, increases the risk of venous thromboembolism; although transdermal hormone therapy does not seem to enhance this risk (92). In terms of breast cancer, risk varies depending on hormonal formulation and length of treatment. In light of this, selective estrogen receptor modulators (SERMs) have an important role and have obtained licensing for this purpose. Raloxifene has very little effect on the vasomotor symptoms of estrogen deficiency, but is beneficial in prevention and treatment of osteoporosis as well as chemoprevention of breast cancer. Tibolone, an agent with estrogenic, progestogenic, and weak androgenic activity, can provide an alternative treatment for postmenopausal symptoms and also exerts favorable effects on bone.

In some patients with combined LH/FSH and ACTH deficiency, low libido may persist despite conventional estrogen and progesterone replacement. This could be a consequence of complete androgen deficiency and some studies have shown that low dose testosterone replacement, e.g., 50-100mg subcutaneous implants every 6 months, is effective for treating hypoactive sexual desire disorder. The guideline also recommends considering a 6-month therapeutic trial of transdermal testosterone for women who fit this diagnosis (93). Oral combinations of estradiol and testosterone are also available. FSH and LH injections are necessary for fertility; recombinant forms of both are now available and this treatment is usually supervised through fertility clinics.

MEN

Apart from relief of the symptoms of hypogonadism, androgen replacement is also important in maintaining bone integrity, muscle mass, and normal erythropoiesis (94).

The most common method of androgen replacement is as an intramuscular depot injection of testosterone ester (e.g., testosterone enanthate, 250mg intramuscularly three weekly). The ensuing supraphysiological peaks and troughs of serum testosterone may lead to fluctuations in mood, libido, and energy levels but treatment is generally very well tolerated. Depot testosterone injection has become available; testosterone undecanoate (Nebido), can be administrated intramuscularly every three months (95), and maintains physiological testosterone levels without major fluctuation.

Oral testosterone undecanoate is administered two or three times a day. It is extensively metabolized to dihydrotestosterone in the intestine and is absorbed via the lymphatic system. It is generally well tolerated and is most useful in patients with partial hypogonadism or in those who are unable to tolerate depot injections.

Testosterone pellets, implanted subcutaneously at a dose between 400-600mg, will provide normal testosterone levels as well as physiological levels of estradiol and dihydrotestosterone for up to six months (96). Peak serum testosterone levels are seen 2-4 weeks after placement with a gradual decline thereafter. The main disadvantage is the need for a skin incision and the occasional complication of local infection and extrusion of the pellets.

Several other systems for testosterone delivery are available and include patches (97) and gels (98) applied to the skin and buccal bioadhesive tablets (99). Transdermal gel (Testogel, Testim) has become very popular; it must be applied daily to maintain desirable serum testosterone levels. Patches can cause skin irritation in approximately 50% of patients. Buccal testosterone (Striant) is placed on the buccal mucosa above the incisor tooth; testosterone is slowly released over 12 hours. However, gum irritation and inconvenience have been reported.

Induction of spermatogenesis requires injections of FSH and LH; our own practice is to administer FSH 300 units three times weekly and LH 1500 units twice weekly; increases in sperm density are not evident for at least 4 months.

Monitoring Testosterone Replacement

Testosterone levels can be measured in blood as a guide of the adequacy of replacement. With intramuscular depot injections, serum testosterone usually peaks at approximately one week after injection with a nadir prior to the next injection. The nadir serum testosterone concentration should approximate the lower end of the normal reference range and this may require adjustment of the frequency of injections. Random serum testosterone is often low or low normal on oral testosterone undecanoate, but the additional measurement of serum dihydrotestosterone is useful.

Published guidelines (100) addressed the concerns of the risk for either benign or malignant prostate disease. There is no evidence that the incidence of prostate carcinoma in patients on testosterone replacement is greater than background population risk. Referral to a urologist is recommended if the patient has prostatic symptoms or an abnormal digital rectal examination or elevated serum prostate specific antigen (PSA) increasing by more than 1.4ng/mL over 12 months. With the increasing use of long-acting testosterone, it is important to monitor the hematocrit to detect polycythemia. The hematocrit should return to normal before testosterone may be reinstituted at a lower dose.

The available evidence suggests that testosterone replacement should be offered cautiously in patients with a low risk of recurrence for prostatic neoplasm who have been treated with radical prostatectomy and in whom the PSA has normalized (101). Similarly, a retrospective study of patients treated with prostatectomy as primary management with a low PSA at baseline of testosterone therapy and concurrent use of 5α reductase reported that there was no increase in PSA at 15 months (56). However, patients with no definitive surgery treated with brachytherapy or external beam radiation and a raised PSA at baseline should not be offered this treatment.

Growth Hormone Deficiency

The rationale and protocol for growth hormone replacement in adults is discussed in detail in the "Adult Growth Hormone Deficiency" chapter.

Diabetes Insipidus

1-desamino-8-D-arginine-vasopressin (DDAVP) is a synthetic analogue of arginine vasopressin which produces prolonged antidiuresis after intravenous, intranasal or oral administration in patients with AHD. A therapeutic trial of DDAVP, 10-20 mcg intranasally should control polyuria for up to 16 hours. Patients with AHD must have instant improvement in symptoms. In the acute clinical setting, for example where diabetes insipidus follows pituitary surgery, DDAVP is best administrated via the subcutaneous route at a dose of 0.5-1μg. Doses of DDAVP that are too high can lead to hyponatremia if patients continue to drink inappropriately despite antidiuresis. Patient education is required to achieve optimum symptom control particularly at night and to maintain a normal serum osmolality and sodium concentration (102). Slight undertreatment, with normal water homeostasis being maintained by thirst mechanisms, is the preferred approach. Many patients demonstrate adequate control of the condition with a single bedtime dose of intranasal DDAVP but an additional morning dose may be required. Mild degrees of AHD may be treated with oral DDAVP up to 600µg daily in divided doses. If ADH deficiency is accompanied by a reduced thirst threshold, it is most important to monitor body weight and urine output on a fixed dose of DDAVP and adjust fluid intake accordingly.

A recent guideline has been developed for inpatient treatment of diabetes insipidus highlighting that this can develop into a life-threatening situation if inappropriately managed (103). This again highlights that there is an increased risk of mortality and adverse outcomes in patient with hypopituitarism admitted for acute medical conditions especially ones with diabetes insipidus (104).

GUIDELINES

Wierman ME, Arlt W, Basson R, Davis SR, Miller KK, Murad MH, Rosner W, Santoro N. Androgen therapy in women: a reappraisal: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2014 Oct;99(10):3489-510.

Fleseriu M, Hashim IA, Karavitaki N, Melmed S, Murad MH, Salvatori R, Samuels MH. Hormonal Replacement in Hypopituitarism in Adults: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016 Nov;101(11):3888-3921.

Bhasin S, Brito JP, Cunningham GR, Hayes FJ, Hodis HN, Matsumoto AM, Snyder PJ, Swerdloff RS, Wu FC, Yialamas MA. Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018 May 1;103(5):1715-1744

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The Neurohypophysis: Endocrinology of Vasopressin and Oxytocin

ABSTRACT

The neurohypophysis is the structural foundation of a neuro-humoral system coordinating fluid balance and reproductive function through the action of two peptide hormones: vasopressin and oxytocin. Vasopressin is the principle endocrine regulator of renal water excretion, facilitating adaptive physiological responses to maintain plasma volume and plasma osmolality. Oxytocin is important in parturition and lactation. Data support a wider role for both peptides in the neuro-regulation of complex behavior. Clinically, deficits in the production or action of vasopressin manifest as diabetes insipidus. An understanding of the physiology and pathophysiology of vasopressin is also critical in approaching the diagnosis and management of hyponatremia, the most common electrolyte disturbance in clinical practice. This chapter explores the anatomy, physiology, and pathophysiology of the neurohypophysis, vasopressin and oxytocin: highlighting developments in the neural basis of osmo-sensing; the mechanism of action of vasopressin and oxytocin; together with a description of the cell and molecular biology underpinning some of the disease processes in which both the structure and functions of the two hormones are involved.

INTRODUCTION

The neurohypophysis consists of three parts: the supraoptic and paraventricular nuclei of the hypothalamus; the supraoptico-hypophyseal tract; and the posterior pituitary. The neurohypophysis is one component of a complex neurohumoral system coordinating physiological responses to changes in both the internal and external environment. This chapter will concentrate on the physiology and pathophysiology of two hormones made by the hypothalamus and posterior pituitary, vasopressin (AVP) and oxytocin (OT). These hormones have key roles in water balance and reproductive function.

ANATOMY, CELL BIOLOGY AND PHYSIOLOGY OF THE OF THE HYPOTHALAMO-POSTERIOR PITUITARY AXIS

Anatomy Of the Neurohypophysis

The posterior pituitary is derived from the forebrain during development and is composed predominantly of neural tissue. It lies below the hypothalamus, with which it forms a structural and functional unit: the neurohypophysis. The supraoptic nucleus (SON) is situated along the proximal part of the optic tract. It consists of the cell bodies of discrete vasopressinergic and oxytotic magnocellular neurons projecting to the posterior pituitary along the supraoptico-hypophyseal tract. The paraventricular nucleus (PVN) also contains discrete vasopressinergic and oxytotic magnocellular neurons, also projecting to the posterior pituitary along the supraoptico-hypophyseal tract. The PVN contains additional, smaller parvocellular neurons that project to the median eminence and additional extra-hypothalamic areas including forebrain, brain stem, and spinal cord. Some of these parvocellular neurons are vasopressinergic. A group of those projecting via the median eminence co-secrete VP and corticotrophin releasing hormone (CRH), and terminate in the hypophyseal-portal bed of the anterior pituitary. These and other vasopressinergic parvocellular neurons terminating in the hypophyseal-portal bed have a role in the regulation of adrenocorticotrophin (ACTH) release from the anterior pituitary gland, acting synergistically with CRH produced by other hypothalamic neurons. A schematic overview of the anatomy of the neurohypophysis and its major connections is shown in Figure 1.

Figure 1. Schematic representation of the anatomy of the neurohypophysis, and its major afferent and efferent connections.

The posterior pituitary receives an arterial blood supply from the inferior hypophyseal artery and the artery of the trabecula (a branch of the superior hypophyseal artery), derivatives of the internal carotid artery and its branches. The SON and PVN receive an arterial supply from the supra-hypophyseal, anterior communicating, anterior cerebral, posterior communicating and posterior cerebral arteries, all derived from the circle of Willis. Venous drainage of the neurohypophysis is via the dural, cavernous, and inferior petrosal sinuses.

Molecular-Cell Biology of Vasopressin and Oxytocin

AVP is a 9 amino acid peptide with a disulphide bridge between the cysteine residues at positions 1 and 6 (Figure 2). Most mammals have the amino-acid arginine at position 8, though in the Pig family arginine is substituted by lysine. The structure of OT differs from that of AVP by only 2 amino acids: isoleucine for phenylalanine at position 3; and leucine for arginine at position 8. Non-mammalian species have a variety of peptides very similar to AVP and OT, suggesting they derive from a common ancestral gene.

Figure 2. The structural and chemical characteristics of Vasopressin and Oxytocin. The cyclical peptides differ in only 2 amino acid positions. Both contain disulphide bridges between Cysteine residues at positions 1 and 6

THE VASOPRESSIN-NEUROPHYSIN AND OXYTOCIN-EUROPHYSIN GENES

The genes encoding AVP and OT are in a head-to-head tandem array on chromosome 20p13 in Man, separated by 12 Kb of DNA. Each has 3 exons, and encodes a polypeptide precursor with a modular structure: an amino-terminal signal peptide; the AVP or OT peptide; a hormone-specific mid-molecule peptide termed a neurophysin (NPI and NPII for OT and AVP respectively); and a carboxyl-terminal peptide known as copeptin (Figure 3). There is considerable homology between the NP sequences of the AVP-NP and OT-NP genes, positions 10-74 of the NP sequences being highly conserved at the amino acid level.

Figure 3. Structural organization of the Vasopressin-neurophysin II gene, and processing of its product. The AVP-NPII gene has 3 exons. Translation of the mRNA yields a larger preprohormone precursor, subsequently modified through substantial post-translational modification. The OT gene has a similar structure, and its product undergoes similar processing and post-translational modification. AVP: Vasopressin. NPII: Neurophysin II. OsRE: osmo-sensitive response element. GRE: glucocorticoid response element. ERE: estrogen response element. AP1-RE: AP1 response element.

Regulatory control of AVP gene expression is mediated through positive and negative elements in the proximal promoter. Several transcription factors bind to these elements. AP1, AP2 and CREB stimulate AVP gene expression. The glucocorticoid receptor (GR) represses expression (1, 2). The human, rat and mouse OT promoters contain half estrogen-response elements, and IL-6 response elements (3). The inter-genic region between the AVP and OT genes contains regulatory elements responsible for selective expression. The region -288 to -116 5′ upstream of the AVP gene promoter confers cell-type (magnocellular neuron) specific expression of the AVP gene (4). AVP gene expression can also be regulated at a post-transcriptional level. The length of the poly (A) tail of AVP mRNA increases in response to water deprivation, influencing mRNA stability (4). AVP mRNA also contains a dendritic localization sequence (DLS). Interaction of the DLS with a multifunctional poly(A) binding protein (PABP) may play key role in RNA stabilization, initiation of translation and translational silencing (5).

Synthesis And Release of Vasopressin and Oxytocin

Synthesis of the AVP and OT precursors occurs in the cell bodies of discrete vasopressinergic and oxytotic magnocellular neurosecretory neurons within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. Generation of the mature hormone entails post-translational modification of the large primary precursor (Figure 4). Following ribosomal translation of the respective mRNA, the carboxyl terminal domain of the precursor is glycosylated, and the product packaged in vesicles of the regulated secretory pathway. These migrate along the axons of the magnocellular neurons, during which the precursor is cleaved by basic endopeptidases into the mature hormone and the associated NP and copeptin. These are stored in secretory granules within the terminals of the magnocellular neurons in the posterior pituitary. Increased firing frequency of vasopressinergic and oxytotic neurons opens voltage-gated Ca2+ channels in these nerve terminals. This, in turn, leads to transient Ca2+ influx, fusion of the neurosecretory granules with the nerve terminal membrane, and release of the hormone and its NP and copeptin into the systemic circulation in equimolar quantities. NPs act as carrier proteins for AVP and OT during axonal migration, and appear to serve no other function. The function of copeptin remains unclear to date, it has been postulated to play a role as a prolactin-releasing factor, but never confirmed (6, 7). Another role as a chaperone-like molecule involved in the folding of the AVP precursor has been speculated (8) .In addition, it was reported to interact with the calnexin–calreticulin system (9), which prevents the export of misfolded, glucose-tagged proteins from the endoplasmic reticulum.

Figure 4. Schematic overview of the post-translational processing of the AVP-NP II gene product. Sequential modification of 164 amino-acid AVP-NPII preprohormone in endoplasmic reticulum and golgi lead to trafficking through the regulated secretory pathway and ultimately release from neurosecretory vesicles in the posterior pituitary. AVP-NPII precursor is complexed as tetramers or high oligomers during processing. A small amount of partially processed precursor is released through the constitutive secretory pathway. OT is processed in a similar manner.

AVP and OT circulate unbound to plasma proteins, though AVP does bind to specific receptors on platelets. AVP concentrations in platelet-rich plasma are 5-fold higher than in platelet-depleted plasma (10). AVP and OT have short circulating half-lives of 5-15 minutes. Several endothelial and circulating endo- and amino-peptidases degrade the peptides. A specific placental cysteine amino-peptidase degrades AVP and OT rapidly during pregnancy and the peri-partum period.

The Physiology of The Secretion of Vasopressin and Thirst

AVP is a key component in the regulation of fluid and electrolyte balance, through direct effects on renal water handling. However, the physiology of AVP has a wider context, encompassing roles in the integrated response to changes in cardiovascular status.

VASOPRESSIN RECEPTORS

There are three distinct AVP receptor (V-R) subtypes (Table 1). All have seven transmembrane spanning domains, and all are G protein coupled. They are encoded by different genes and differ in tissue distribution, down-stream signal transduction and function. The human V2-R gene maps to Xq28. Interestingly, the V2-R is up regulated by its ligand (11).

Table 1. Vasopressin Receptor Subtypes
	Vasopressin receptor
	V1a	V1b	V2
Expression	Vascular smooth muscle Liver Platelets CNS	Pituitary corticotroph	Basolateral membrane of distal nephron
Amino acid structure	418 amino acids (human)	424 amino acids (human)	370 amino acids (human)
Second messenger system	Gq/11mediated phospholipase C activation: Ca²⁺, inositol triphosphate & diacyl glycerol mobilization	As V1a	Gs mediated adenylate cyclase activation: cAMP production & protein kinase A stimulation
Physiological effects	●Smooth muscle contraction ●Stimulation of glycogenolysis. ●Enhanced platelet adhesion ●Neurotransmitter & neuromodulatory function	Enhanced ACTH release	Increased synthesis & assembly of aquaporin-2

VASOPRESSIN AND RENAL WATER HANDLING

Although AVP has multiple actions, its principle physiological effect is in the regulation of water resorption in the distal nephron, the structure and transport processes of which allow the kidney to both concentrate and dilute urine in response to the prevailing circulating AVP concentration. Active transport of solute out of the thick ascending loop of Henle generates an osmolar gradient in the renal interstitium, which increases from renal cortex to inner medulla, a gradient through which distal parts of the nephron pass end route to the collecting system. AVP stimulates the expression of a specific water channel protein (aquaporin) on the luminal surface of the interstitial cells lining the collecting duct. The presence of aquaporin (AQP) in the wall of the distal nephron allows resorption of water from the duct lumen along an osmotic gradient, and excretion of concentrated urine. To date, 13 different AQPs have been identified in Man, seven of which (AQP1-4, AQP6-8) are found in the kidney. AQPs act as passive pores for small substrates and are divided into 2 families: the water only channels; and the aquaglyceroporins that can conduct other small molecules such as glycerol and urea. Most substrates are neutral. However, this is not always the case. For example, AQP6 is a gated ion channel. AQPs are involved in a variety of cell processes: small molecule permeation; gas conduction and cell-cell interaction. As with other membrane channels, specific structural arrangements within the primary, secondary, and tertiary structure convey the three functional characteristics of permeation, selectivity, and gating. The structure of AQPs involves 2 tandem repeats, each formed from 3 transmembrane domains, together with 2 highly conserved loops containing the signature motif asparagine-proline-alanine (NPA). All AQPs form homotetramers in the membrane, providing 4 functionally independent pores with an additional central pore formed between the 4 monomers. Water can pass through all the 4 independent channels of water-permeable AQPs. There are data to suggest that the central pore may act as independent channel in some AQPs (12-14). AQP1 is constitutively expressed in the apical and basolateral membranes of the proximal tubule and descending loop of Henle, where it facilitates isotonic fluid movement. Loss of function mutations of AQP1 in man lead to defective renal water conservation (15). AQP2 is expressed on the luminal surface of collecting duct cells and is the water channel responsible for AVP-dependent water transport from the lumen of the nephron into the collecting duct cells. V2-R activation in collecting duct cells produces a biphasic increase in expression of AQP2. Ligand-receptor binding triggers an intracellular phosphorylation cascade ultimately resulting in phosphorylation of the nuclear transcription factor CREB and expression of c-Fos. In turn, these transcription factors stimulate AQP2 gene expression through CRE and AP-1 elements in the AQP2 gene promoter. In addition, AVP stimulates an immediate increase in AQP2 expression by accelerating trafficking and assembly of pre-synthesized protein into functional, homo-tetrameric water channels.

Maximum diuresis occurs at plasma AVP concentrations of 0.5 pmol/l or less. As AVP levels rise, there is a sigmoid relationship between plasma AVP concentration and urine osmolality, with maximum urine concentration achieved at plasma AVP concentrations of 3-4 pmol/L (Figure 5). Following persistent AVP secretion, antidiuresis may diminish. Down-regulation of both V2-R function and AQP2 expression may be responsible for this escape phenomenon.

Figure 5. The relationship of plasma AVP concentration to urine osmolality. Shaded area represents range of normal; single line indicates representative individual. AVP has additional effects at other sites in the nephron: decreasing medullary blood flow; stimulating active urea transport in the distal collecting duct; and stimulating active sodium transport into the renal interstitium. AVP up-regulates the bumetanide-sensitive sodium-potassium-chloride cotransporter (SLC12A1) in the thick ascending loop of Henle through both a rapid acceleration of post-translational processing/trafficking and an increase in SCLC12A1 gene expression. Together, these contribute to the generation and maintenance of a hypertonic medullary interstitium, and augment AVP-dependent water resorption (16).

REGULATION OF VASOPRESSIN RELEASE

Osmoregulation of Vasopressin

Plasma osmolality is the most important determinant of AVP secretion. The osmoregulatory systems for thirst and AVP secretion, and in turn the actions of AVP on renal water excretion, maintain plasma osmolality within narrow limits of 284 to 295 mOsmol/kg. The relationship between plasma osmolality and plasma AVP concentration has 3 characteristics.

The osmotic threshold or 'set point' for AVP release.
The shape of the line describing changes in plasma AVP concentration with changing plasma osmolality
The sensitivity of the osmoregulatory mechanism coupling plasma osmolality and AVP release.

Increases in plasma osmolality increase plasma AVP concentrations in a linear manner (Figure 6). The abscissal intercept of this line indicates the mean 'osmotic threshold' for AVP release (284 mOsmol/kg): the mean plasma osmolality above which plasma AVP increases in response to increases in plasma osmolality. AVP levels increase from a basal rate through activation of stimulatory osmoreceptor afferents, and decrease to minimal values when this drive is removed and synergistic inhibitory afferents are activated. The slope of the line relating plasma osmolality to plasma AVP concentration reflects the sensitivity of osmoregulated AVP release. There are considerable inter-individual variations in both the threshold and sensitivity of AVP release. However, they are remarkably reproducible within an individual over time (17).

Figure 6. The relationship of plasma AVP concentration to changes in plasma osmolality during controlled hypertonic stimulation. AVP concentration determined during progressive hypertonicity induced by infusion of 855 mmol/l saline in a group of healthy adults. Increases in plasma osmolality increase plasma AVP concentrations in a linear manner, defined by the function, plasma AVP = 0.43 (plasma osmolality - 284), r = +0.96. The abscissal intercept of this regression line indicates the mean 'osmotic threshold' for AVP release: the mean plasma osmolality above which plasma AVP starts to increase. The shaded area represents the range of normal response. LD represents the limit of detection of the assay, 0.3 pmol/l.

There are situations where the normal relationship between plasma osmolality and AVP concentration breaks down:

Rapid changes of plasma osmolality: rapid increases in plasma osmolality result in exaggerated AVP release.
During the act of drinking: drinking rapidly suppresses AVP release, through afferent pathways originating in the oropharynx.
Pregnancy: the osmotic threshold for AVP release is lowered in pregnancy.
Whether aging is accompanied by changes in AVP concentrations is controversial.

As befits its major function and physiological role, AVP production by the neurohypophysis is influenced by sensory signals reflecting osmotic status and blood pressure/circulating volume. The relationships of the SON and PVN with the autonomic afferents and central nervous system nuclei responsible for osmo- and baroregulation are key to the physiological regulation of AVP. Functional osmoreceptors are situated in anterior circumventricular structures: the subfornicular organ (SFO), and the organum vasculosum of the lamina terminalis (OVLT). Local fenestrations in the blood brain barrier at these sites allow neural tissue direct contact with the circulation. Subsequent sensory input to the SON and PVN is via glutaminergic afferents. Moreover, these neurons integrate osmolar status with additional endocrine signals reflecting circulating volume status through the action of angiotensin II (A-II), relaxin, and atrial natriuretic peptide (ANP). A-II and relaxin excite both OT and AVP magnocellular neurons. In contrast, ANP inhibits AVP neuron activity. AVP neurons themselves have independent osmo-sensing properties and V-Rs are present on vasopressinergic neurons of both the PVN and SON, highlighting the potential for auto-control of AVP release through direct osmoregulation and short loop feedback (18). AVP magnocellular neurons in the SON and PVN co-express the peptide Apelin and its G-protein coupled receptor. A ‘yin and yang’ relationship has been proposed between AVP and these 36 amino-acid peptides (and indeed it’s shorter active derivatives Apelin-17 and Apelin-13). Intra-cerebroventricular injection of Apelin-17 inhibits the phasic firing of AVP magnocellular neurons, reducing AVP release and stimulating aquaresis. Hypertonic stress and water loading have reciprocal effects on plasma AVP and Apelin concentrations. Apelin receptors are also co-expressed in AVP target cells in the renal collecting duct. AVP and Apelin are thus regulated in opposite directions to maintain volume and osmolar homeostasis (19-22).

Changes in the osmotic environment of osmo-sensitive neurons in the OVLT, SFO and vasopressinergic neurons of the SON and PVN result in altered cell volume. These physical changes alter the activity of the stretch-sensitive cationic channel TRPV1, expressed on the cell surface of these neurons. TRPVI thus acts as the transduction mechanism linking changes in osmolality to altered membrane potential and firing frequency. Osmoregulatory function is not lost in Trpv1−/−mice, indicating additional osmo-sensing pathways must be in operation (23).

A related, but distinct osmo-sensory input feeds additional data on peripheral osmolar status to the neurohypophysis. Hepatic portal blood vessels contain sensory neurons responsive to changes in the osmolality of peripheral blood. In contrast to central mechanisms, the key transducing element of the peripheral process is the stretch-sensitive ion channel, TRPV4. Plasma osmolality is frequently elevated in patients after liver transplant in which the donor organ is denervated, demonstrating the function of this peripheral pathway (24).

Osmosensitivity of AVP release is influenced by circadian rhythms. AVP release increases during sleep. This effect is mediated by clock neurons projecting from the suprachismatic nucleus, increasing the activity of osmosensory afferent input to the SON (25).

Baroregulation of Vasopressin

Reductions in circulating volume stimulate AVP release. Falls in arterial blood pressure of 5 to 10 per cent are necessary to increase circulating AVP concentrations in man. Progressive reduction in blood pressure produces an exponential increase in plasma AVP, in contrast to the linear increases of osmoregulated AVP release. Baroregulatory influences on neurohypophyseal AVP release derive from aortic arch, carotid sinus, cardiac atrial, and great vein stretch-sensitive afferents via cranial nerves IX and X. Ascending projections are via the nucleus tractus solitarius (NTS) in the brain stem. From the NTS, further afferents project to the SON and PVN, which also receive additional adrenergic afferents from other brain stem nuclei involved in cardiovascular control, such as the locus coeruleus. These nuclei integrate a number of afferent inputs that reflect volume status. Ascending baroregulatory pathways must affect some tonic inhibition of AVP release, as interruption increases plasma AVP levels (26, 27). Osmoregulated AVP responses can be modified by factors triggered as part of the coordinated neurohumoral response to changes in circulating volume and blood pressure. A-II amplifies the proportional relationship between osmolality and action potential firing in the SON. The peptide produces this effect through polymerization of intracellular actin filaments, resulting in altered cell shape, a mechanism that is synergistic with those mediating responses to changes in extracellular osmolality. A-II thus enhances osmosensitivity. This mechanism underpins the changes in osmo-regulated AVP release in hypo- and hypervolemia: the osmotic threshold and sensitivity of AVP release is lowered by hypovolemia; while the converse is found in hypervolemia and hypertension (19).

Additional Mechanisms Regulating Vasopressin Release

A number of other stimuli influence AVP release independent of osmotic and hemodynamic status.

Nausea and emesis
Unspecific stress
Pain
Manipulation of abdominal contents
Immune-response mediators and inflammatory triggers

These stimuli contribute to high plasma AVP values observed in acute illness and after surgery.

ADDITIONAL EFFECTS OF VASOPRESSIN

Cardiovascular Effects

AVP is a potent pressor agent; its effects mediated through a specific receptor (V1-R) expressed by vascular smooth muscle cells. Though systemic effects on arterial blood pressure are only apparent at high concentrations, AVP is important in maintaining blood pressure in mild volume depletion. The most striking vascular effects of AVP are in the regulation of regional blood flow. The sensitivity of vascular smooth muscle to the pressor effects of AVP varies according to the vascular bed. Vasoconstriction of splanchnic, hepatic and renal vessels occur at AVP concentrations close to the physiological range. Furthermore, there are differential pressor responses within a given vascular bed. Selective effects on intrarenal vessels lead to redistribution of renal blood flow from medulla to cortex. Baroregulated AVP release thus constitutes one of the key physiological mediators of an integrated hemodynamic response to volume depletion.4.2.

Effects on the Pituitary

AVP is an ACTH secretagogue, acting through pituitary corticotroph-specific V1b-Rs. Though the effect is weak in isolation, AVP and CRF act synergistically. AVP and CRF co-localize in neurohypophyseal parvocellular neurons projecting to the median eminence and the neurohypophyseal portal blood supply of the anterior pituitary. Levels of both AVP and CRF in these neurons are inversely related to glucocorticoid levels, consistent with a role in feedback regulation.

Effects of AVP on Regulation of Bone Mass

AVP exerts its action both on osteoblasts and osteoclasts thru AVPR1 and AVPR2 receptors (28). Mice studies showed that AVP upregulates osteoclast differentiation genes and inhibits osteoblast formation. Mice rendered deficient in AVPr1a (Avpr1a-/-) have a high bone mass, and they show an increase in osteoblastogenesis after additional inhibition of AVPR2 (29) .

The AVP effect is opposed by oxytocin, which stimulates osteoblast formation and inhibits mature osteoclast activation. Mice studies showed that both haploinsufficiency for oxytocin and deletion of the oxytocin receptor (Oxr-/-) result in osteopenia (30). The role of both hormones in the regulation of skeletal physiology remains to be further explored (31).

Behavioral Effects of Vasopressin

Vasopressinergic fibers and V-Rs are present in CNS neural networks anatomically and functionally independent of the neurohypophysis, including the cerebral cortex and limbic system. An increasing amount of data highlight the role of central vasopressinergic systems in mediating complex social behavior. Data in Man link V1a-R gene sequence variation with a range of normal and abnormal behavior patterns, including gender dimorphic behavior. Dysregulation of central AVP action may be a distal end point in complex conditions characterized by altered social and emotional behavior (32, 33).

Thirst

Renal free water clearance can be reduced to a minimum by the antidiuretic actions of vasopressin, but water loss is not completely eliminated, and insensible water loss from respiration and sweating is a continuous process. To maintain water homeostasis, water must also be consumed to replace the obligate urinary and insensible fluid losses. This is regulated by thirst. Thirst and drinking are key processes in the maintenance of fluid and electrolyte balance. Thirst perception and the regulation of water ingestion involve complex, integrated neural and neurohumoral pathways. As with those mediating AVP release, the osmoreceptors regulating thirst are situated in the OVLT, effectively outside the blood-brain barrier and distinct from those mediating AVP release. Neural activity in and around the OVLT remains active in hyperosmolar states following immediate satiety of thirst, indicating that other centers must be involved in thirst perception. The anterior cingulate cortex and insular cortex receive input from osmo-sensitive afferents and have been implicated as key higher centers in thirst pathways (20). There is a linear relationship between thirst and plasma osmolalities in the physiological range. The mean osmotic threshold for thirst perception is 281 mOsm/kg, similar to that for AVP release. Thirst occurs when plasma osmolality rises above this threshold. As with osmoregulated AVP release, the characteristics of osmoregulated thirst remain consistent within an individual on repeated testing, despite wide inter-individual variation.

As with AVP release, there are also specific physiological situations in which the relationship between plasma osmolality and thirst breaks down.

The act of drinking: reduces osmotically stimulated thirst.
Extracellular volume depletion: this stimulates thirst through volume-sensitive cardiac afferents and the generation of circulating and intra-cerebral A-II, a powerful dipsogen.
Pregnancy, the luteal phase of the menstrual cycle and super ovulation syndrome: these states reduce the osmolar threshold for thirst.
Aging: both thirst appreciation and fluid intake can be blunted in the elderly

The act of drinking reduces thirst perception before any change in plasma osmolality. This effect is produced through three mechanisms: oropharyngeal sensory afferents; gastro-intestinal stretch-sensitive afferents; and peripheral osmoreceptors in the hepatic portal vein. Recent data have highlighted how thirst-promoting neurons in the SFO integrate sensory inputs from the oropharynx (drinking and food composition) with central osmolar status to influence thirst perception. This complex mechanism effectively explains anticipatory changes in water consumption that precede changes in plasma osmolality (34). As with AVP release, hypovolemia resets the relationship between plasma osmolality and thirst. A-II is one of the key mediators of this physiological response. Peripheral A-II generation can act on central osmoreceptors, to increase both thirst and AVP release. An independent, intra-cerebral A-II system is activated in parallel. A-II is a powerful central dipsogen.

The Physiology of Oxytocin

OT binds to specific G-protein coupled cell surface receptors (OT-Rs) on target cells to mediate a variety of physiological effects, largely concerned with reproductive function. The classical physiological roles of OT are the regulation of lactation, parturition and reproductive behavior. Data from transgenic animals with targeted disruption of the oxytocin gene (and thus lacking OT) have forced a review of this dogma (35).

OXYTOCIN AND LACTATION

In the rat, stimulation of vagal sensory afferents in the nipple by the act of suckling triggers reflex synchronized firing of oxytotic magnocellular neurons in the neurohypophysis, and corresponding pulsatile OT release. OT acts on OT-Rs on smooth muscle cells lining the milk ducts of the breast, initiating milk ejection. OT is essential for completion of this milk ejection reflex in rodent. Mice lacking OT fail to transfer milk to their suckling young. This deficit is corrected by injection of OT. In contrast, women lacking posterior pituitary function can breast-feed normally, illustrating that OT is not necessary for lactation in man. Pituitary lactotrophs express OT-R mRNA, and OT released into the hypophyseal portal blood supply from the median eminence can stimulate prolactin release. However, the role of OT in the physiology of prolactin release remains unclear.

OXYTOCIN AND PARTURITION

OT is a uterotonic agent. In many mammals, there is both an increase in OT secretion and an increase in uterine responsiveness to OT during parturition (3). These data suggest a key role for the hormone in the initiation and progression of labor. Falling progesterone concentrations toward the end of pregnancy lead to up-regulation of uterine myometrial OT-Rs, enhanced contractility, and increased sensitivity to circulating OT. Stretching of the 'birth canal' during parturition leads to the stimulation of specific autonomic afferents, reflex firing of oxytotic neurons and OT release. A positive feedback loop is formed, OT stimulating uterine contraction further and enhancing the production of additional local uterotonic mediators such as prostaglandins. The difficulties of analyzing pulsatile release, and the short circulating half-life of the hormone (due to placental cysteine aminopeptidase), have made it difficult to demonstrate increased circulating OT levels in women during labor. Mice lacking OT have normal parturition. Moreover, women with absent posterior pituitary function can have a normal labor. However, the importance of OT in the birth process is highlighted by the effectiveness of OT antagonists in the management of pre-term labor (36). The role of OT in parturition is not limited to maternal responses. Maternal OT produces a switch to inhibitory GABAergic signaling in the fetal CNS. This, in turn, increases fetal neuronal resistance to damage that may occur during delivery. OT therefore mediates direct adaptive mother-fetal signaling during parturition in line with a wider-ranging role in maternal-fetal physiology (37).

OXYTOCIN AND BEHAVIOUR

OT-R expression is widespread in the CNS of many species, and OT has widespread roles as a neurotransmitter. The central oxytocinergic system and related limbic networks affect complex neural circuits of socio-emotional behavior and promote pro-social effects such as in-group favoritism and protection against social threats, interpersonal trust and attachment, empathy, and emotion recognition (38-41). In humans, brain regions such as the amygdala, hippocampus, cingulate cortex, and nucleus accumbens, which play a key role in human socio-emotional behavior, show a high density of OT-R expression. In some cases, these overlap those involving AVP (32, 33).

OT knockout (OT-KO) models have been used to identify aspects of dysfunctions in social behavior. Generally, an impairment in forming social memories and higher anxiety-related behaviors were observed (42-44). These results were substantiated by the fact that central administration of OT rescued OT-KO mice from these changes. In the context of behavior; OT facilitates both lordosis and the development of maternal behavior patterns in rat (3). However, mice lacking OT exhibit normal sexual and maternal behavior, suggesting the behavioral effects to be species-specific or the potential for considerable redundancy in neural pathways. In humans, lower endogenous OT or impaired signaling has been linked with mental disorders associated with social deficits, such as autism spectrum disorder (ASD), anxiety and depression disorder or borderline personality disorder (45-50). In these disorders, no OT deficiency per se has been proven; the evidence is largely based on observational studies with individual variations in social behavior associated with alteration in peripheral OT levels, in genes involved in OT signaling or OT-R polymorphisms (51-53). In ASD, recent data have highlighted associations with the single nucleotide polymorphisms (SNPs) rs7632287, rs237887, rs2268491 and rs2254298 (54-56). Intranasal OT has been investigated to ameliorate symptoms of these conditions. However, overall, the effect size is inconsistent, and the results of these studies are controversial. Central oxytocinergic transmission reduces anxiety behavior and hypothalamo-pituitary-adrenal stress responses in female rats. It may be that OT has a complex role in the stress response, with context-dependent differential effects. It buffers responses to social stress, reduces cortisol levels during conflict situations, improves self-representations in patients with anxiety disorder, and reduces amygdala response to emotional stimuli, consequently reducing fear reactivity and anxiety (57-63).

Only limited research has been devoted to the role of OT in patients with hypothalamic-pituitary dysfunction and focused primarily on patients with craniopharyngioma (CP). These data assume direct tumor-induced or post-surgical damage to the SON/PVN and consequent disruption of the oxytocinergic system (64-68). Results demonstrate personality changes and increased psycho-social comorbidities – including anxiety, depression, and social withdrawal (64, 69-71). This was confirmed by a systematic review showing behavioral dysfunctions in 57%, and social impairment and difficulties holding relationships in 40% (72). Interestingly, the age of onset appears to influence the type of socio-behavioral dysfunction: while adult-onset had higher levels of anxiety and depression, younger patients had more impact in the domains of social isolation. Research in patients with confirmed posterior pituitary dysfunction, i.e., CDI, demonstrated higher depression and anxiety levels, self-reported autistic traits, lower joy when socializing and worse scores on an emotional recognition task than healthy adults (73, 74).

Similar to other hormones, single basal OT levels are unreliable and insufficient in identifying a deficiency (75) with inconclusive results in patients with hypothalamic-pituitary dysfunction. Research still disagrees regarding the precise relationship between peripheral OT and centrally generated behavior. Cerebrospinal fluid (CSF) concentrations of OT are more likely to mirror the behavioral effects than plasma concentrations, and whether plasma levels may serve as a surrogate for CSF OT is controversially discussed (76, 77). Accordingly, peripheral levels of OT correlate to central levels only after stimulation but not at baseline (78). Established pituitary provocation tests do not show a consistent strong OT increase to test for a suspected deficiency (79). OT-R expression is widespread in the CNS of many species and OT has widespread roles as a neurotransmitter, including neural networks that mediate a range of complex behaviors. In some cases, these overlap those involving AVP (32, 33).

CLINICAL PROBLEMS SECONDARY TO DEFECTS IN THE HYPOTHALAMO-POSTERIOR PITUITARY AXIS

Defects in the production or action of AVP manifest as clinical problems in maintaining plasma sodium concentration and fluid balance, reflecting the key role of the hormone in these processes. A further group of related clinical conditions reflect primary defects in thirst. In some cases, the two may coincide, reflecting the close anatomical and functional relationship of both processes.

Diabetes Insipidus

CLASSIFICATION (see Figure 7)

Diabetes insipidus (DI) is characterized by production of dilute urine in excess of >50 ml/kg/24 hours in adults. DI arises through one of four mechanisms (Figure 7 and Table 2).

Deficiency of AVP: central diabetes insipidus (CDI). Also called Arginine Vasopressin Deficiency
Inappropriate, excessive water drinking: primary polydipsia.
Renal resistance to the antidiuretic action of AVP: nephrogenic diabetes insipidus (NDI). Also called Arginine Vasopressin Resistance
increased vasopressinase expression in pregnancy: Gestational diabetes insipidus

Figure 7. Different forms of hypotonic polyuria (80)

Table 2. Classification of Hypotonic Polyuria (80)
Type of hypotonic polyuria	Basic defect	Causes
Central DI	Deficiency in AVP synthesis or secretion	Acquired · Trauma (surgery, deceleration injury) Neoplastic (craniopharyngioma, meningioma, germinoma, metastases) · Vascular (cerebral/ hypothalamic hemorrhage, infarction or ligation of anterior communicating artery aneurysm) · Granulomatous (histiocytosis, sarcoidosis) · Infectious (meningitis, encephalitis, tuberculosis) · Inflammatory/autoimmune (lymphocytic infundibuloneurohypophysitis, IgG4 neurohypophysitis) · Drug/toxin-induced · Osmoreceptor dysfunction (adipsic DI) · Others (hydrocephalus, ventricular/suprasellar cyst, trauma, degenerative disease) · Idiopathic Congenital · Autosomal dominant: AVP gene mutation · Autosomal recessive: Wolfram Syndrome (DIDMOAD) · X-linked recessive
Primary Polydipsia	Excessive osmotically unregulated fluid intake	· Dipsogenic (downward resetting of the thirst threshold; idiopathic or similar lesions as with central DI) · Psychosis intermittent hyponatremia polydipsia (PIP syndrome) · Compulsive water drinking · Health enthusiasts
Nephrogenic DI	Reduced renal sensitivity to antidiuretic effect of physiological AVP levels	Acquired · Drug exposure (lithium, demeclocycline, cisplatin etc.) · Hypercalcemia, hypokalemia · Infiltrating lesions (sarcoidosis, amyloidosis, multiple myeloma etc.) · Vascular disorders (sickle cell anemia) · Mechanical (polycystic kidney disease, ureteral obstruction) Congenital · X-linked AVPR2 gene mutations · autosomal recessive or dominant AQP2 gene mutations
Gestational DI	Exaggerated enzymatic metabolism of circulating AVP hormone	Increased AVP metabolism · Pregnancy

Central Diabetes Insipidus (CDI) (Arginine Vasopressin Deficiency)

CDI (also known as neurogenic or cranial DI) is the result of partial or complete lack of osmoregulated AVP secretion. Plasma AVP concentrations are inappropriately low with respect to prevailing plasma osmolalities. Presentation with CDI implies destruction or loss of function of more than 80% of vasopressinergic magnocellular neurons. It is rare (estimated prevalence of 1: 25000), with an equal gender distribution. Though persistent polyuria can lead to dehydration, most patients can maintain water balance through appropriate polydipsia if given free access to water.

Most cases of CDI are acquired. Trauma (head injury or surgery) can produce CDI through damage to the hypothalamus, pituitary stalk, or posterior pituitary. Pituitary stalk trauma may lead to a triphasic disturbance in water balance, an immediate polyuric phase followed within days by a more prolonged period (up to several weeks) of antidiuresis suggestive of AVP excess. This second phase can be followed by reversion to CDI, or recovery. This characteristic 'triple response' reflects initial axonal damage; the subsequent unregulated release of large amounts of pre-synthesized AVP; and either recovery or development of permanent CDI (as determined by the magnitude of initial damage to vasopressinergic neurons). All phases of the response are not apparent in all cases.

Hypothalamic tumors (e.g., craniopharyngioma) or pituitary metastases (e.g., breast or bronchus) can present with CDI. However, primary pituitary tumors rarely cause CDI. In childhood, craniopharyngioma and germinoma/teratoma are a relatively common cause (81).

When obvious causes are not present, most cases of CDI will be “idiopathic”. However, the possibility of an autoimmune process should be considered, as many idiopathic cases are considered to be autoimmune in origin (82). A well-recognized cause of autoimmune CDI is lymphocytic infundibulohypophysitis (83).

Familial forms are rare, but are a recognized cause of CDI in childhood. Most reported cases are expressed as autosomal dominant and the genetic defect is usually in the biologically inactive neurophysin or in the signal peptide of the pre-prohormone. Lack of normal cleavage of the signal peptide from the prohormone and abnormal folding of the vasopressin/neurophysin precursor are thought to produce fibrillar aggregations in the endoplasmic reticulum, which is cytotoxic to the neuron, explaining the dominant phenotype (84, 85). Wolfram syndrome is a rare autosomal recessive disease with diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD). The genetic defect is for the protein wolframin that is found in the endoplasmic reticulum and is important for folding proteins (86). Wolframin is localized to chromosome 4. It is involved in beta-cell proliferation and intracellular protein processing and calcium homeostasis, producing a wide spectrum of endocrine and central nervous system (CNS) disorders. Diabetes insipidus is usually a late manifestation and is associated with decreased magnocellular neurons in the paraventricular and supraoptic nuclei (87, 88).

Primary Polydipsia (PP)

PP is a polyuric syndrome secondary to excess fluid intake. PP can be associated with organic structural brain lesions, e.g. sarcoidosis of the hypothalamus (89) and craniopharyngioma (90). It can also be produced by drugs that cause a dry mouth or by any peripheral disorder causing an elevation of renin and/or angiotensin. However, mostly there is no identifiable pathologic etiology; in this circumstance the disorder is often associated with psychiatric syndromes. Series of patients in psychiatric hospitals have shown that as many as 42% have some form of polydipsia and for over half of those there was no obvious explanation for the polydipsia (91). It also seems to be increasingly prevalent in health conscious people who voluntarily change their drinking habits with the aim to improve their well-being, in which case it is often called habitual polydipsia (92).

Nephrogenic Diabetes Insipidus (NDI)

NDI is due to renal resistance to the antidiuretic effects of AVP. Genetic variants of NDI usually present in infancy (93). In these forms, NDI can occur as a result of mutations in the V2 receptor and mutations of the aquaporin 2 water channels. Over 90% of cases are X-linked recessive in males, and over 200 different mutations of the V2 receptor have been reported affecting all aspects of receptor function: expression; ligand binding; and G-protein coupling. Most lead to complete loss of function, though a few are associated with a mild phenotype. 10% of kindreds with familial NDI have an autosomal recessive form, with normal V2-R function. Affected individuals harbor loss of function mutations of the AQP2 gene. Most mutations occur in the region coding for the transmembrane domain of the protein. Additional rare kindreds have been described harboring a mutation in the portion of the gene encoding the carboxyl-terminal intracellular tail of AQP2. The NDI of these kindreds is inherited as an autosomal dominant trait, mutant protein sequestering the product of the wild type AQP2 allele within mixed tetramers in a dominant-negative manner.

The development of NDI in an adult is less likely to reflect a genetic cause. The commonest cause of acquired NDI in clinical practice is lithium therapy, with other causes including hypokalemia, hypercalcemia, and release of bilateral urinary tract obstruction associated with downregulation of aquaporin 2 and decreased function of vasopressin (94, 95). NDI secondary to lithium is characterized by dysregulated AQP2 expression and trafficking along the whole collecting duct as well as dysregulated expression of the amiloride-sensitive epithelial sodium channel (ENaC) in the cortical collecting duct. Lithium enters collecting duct cells through ENaC expressed on the apical cell membrane and leads to inhibition of glycogen synthase kinase type 3 (GSK-3) signaling pathways. NDI secondary to lithium toxicity can persist after drug withdrawal, and may be irreversible. Demeclocycline is another commonly recognized drug to causes NDI and is sometimes used clinically to treat SIAD. The final common pathway producing NDI in many of these cases is down-regulation of AQP2 expression (96).

Gestational Diabetes Insipidus

In normal pregnancy, physiologic adaptations include expansion of blood volume and decreased plasma osmolality and serum sodium. Thirst and increased fluid intake are commonly reported in pregnancy, but in some patients the increased thirst is driven by marked polyuria, which may point to the presence of diabetes insipidus. Two types of transient diabetes insipidus must be differentiated in pregnancy, both caused by the placental enzyme cysteine aminopeptidase, named oxytocinase, which enzymatically degrades oxytocin (97). Because of the close structural homology between AVP and oxytocin, this enzyme also metabolizes AVP. In the first type of pregnancy-associated DI, the activity of oxytocinase is abnormally elevated. This syndrome has been referred to as vasopressin resistant diabetes insipidus of pregnancy (98) and has been reported to be associated with preeclampsia, acute fatty liver, and coagulopathies. Symptoms usually develop at the end of the second or early third trimester and it is more common during multiple pregnancy (99). In the second type of pregnancy-associated DI, the accelerated metabolic clearance of vasopressin produces DI in a patient with borderline pre-existing vasopressin function from a mild nephrogenic diabetes insipidus or partial central diabetes insipidus. Vasopressin is rapidly destroyed and the neurohypophysis is unable to keep up with the increased demand. Symptoms in this second type usually appear early in pregnancy (100).

INVESTIGATIONS FOR DIAGNOSIS

Investigations have the following three aims:

To confirm DI
To classify the DI into central or nephrogenic DI or PP (or gestational DI in case of pregnancy)
To establish the etiology of the specific form of DI

After establishing polyuria and polydipsia, and excluding hyperglycemia, hypokalemia, hypercalcemia, and significant renal insufficiency, attention should be focused on the AVP axis.

For many years, the indirect water deprivation test was the gold standard for differential diagnosis of diabetes insipidus. This test indirectly assesses AVP activity by measurement of the urine concentration capacity during a prolonged period of dehydration, and again after a subsequent injection of an exogenous synthetic AVP variant, desmopressin. Interpretation of the test results goes back to the publication of Miller et al (101). If upon thirsting, urinary osmolality remains <300mOsm/kg and does not increase >50% after desmopressin injection, complete nephrogenic DI is diagnosed. If the urinary osmolality increase after desmopressin injection is >50%, complete central DI is diagnosed. In partial central DI and primary polydipsia, urinary concentration increases to 300–800mosm/kg, with an increase of >9% (in partial central DI) and <9% (in primary polydipsia), respectively, after desmopressin injection. However, these published criteria are based on post hoc data from only 36 patients, who had a wide overlap in their urinary osmolalities. Furthermore, the diagnostic criteria for this test are derived from a single study with post-hoc assessment (101) and have not been prospectively validated. Consequently, the indirect water deprivation test has been shown to have considerable diagnostic limitations with an overall diagnostic accuracy of 70%, and an accuracy of only 41% in patients with primary polydipsia (102).

Several reasons exist for this limited diagnostic outcome of the indirect water deprivation test. First, chronic polyuria itself can affect renal concentration capacity, through renal washout (103) (104, 105) or downregulation of AQP2 expression in the kidneys (106). This may lead to a reduced renal response to osmotic stimulation or exogenous desmopressin in different forms of chronic polyuria. Second, in patients with AVP deficiency, urine concentration can be higher than expected (107, 108), especially in those patients with impaired glomerular function, or can result from a compensatory increase in AVPR2 expression in patients with chronic central DI (109). Finally, patients with acquired nephrogenic DI are often only partially resistant to AVP, resulting in a clinical presentation that is similar to partial central DI.

To overcome these limitations, direct measurement of AVP levels has been proposed. In a study published in 1981, patients with central DI were reported to have AVP levels below a calculated normal area (defining the normal relationship between plasma osmolality and AVP levels), whereas AVP levels were above the normal area in patients with nephrogenic DI and within the normal area in patients with primary polydipsia (110). However, despite these promising initial results, direct measurement of AVP levels failed to enter routine clinical use for various reasons. First, several technical limitations of the AVP assay result in a high preanalytical instability of AVP in samples (111). Second, the accuracy of diagnoses using commercially available AVP assays has been disappointing, with correct diagnoses in only 38% of patients with DI, and particularly poor differentiation between partial central DI and primary polydipsia (102, 112). Third, an accurate definition of the normal physiological area defining the relationship between plasma AVP levels and osmolality is still lacking, especially for commercially available assays (113, 114), which is a crucial prerequisite for the identification of AVP secretion outside the normal range in patients suspected of having DI (102).

Copeptin, the C-terminal segment of the AVP prohormone, is an easy-to- measure AVP surrogate that is very stable ex vivo (111), mirroring AVP concentrations. Two studies have shown that a basal copeptin level >21.4pmol/l without prior thirsting unequivocally identifies nephrogenic DI, rendering a further water deprivation test unnecessary in these patients (102, 115). For the differentiation between patients with primary polydipsia and central DI, results of a study including 144 patients (116) showed that an osmotically stimulated copeptin level >4.9pmol/l after infusion of 3% saline (aiming at a sodium level >150mmol/l) had an overall diagnostic accuracy of 96.5% (93.2% sensitivity and 100% specificity) in distinguishing between patients with primary polydipsia and those with central DI. The classic water deprivation test had an overall diagnostic accuracy of only 76.5%. Importantly, the hypertonic saline infusion test requires close monitoring of sodium levels to ascertain a diagnostically meaningful increase of plasma sodium within the hyperosmotic range while preventing a marked increase. Addition of copeptin measurement did not improve the diagnostic performance of the indirect water deprivation test, most likely due to the lack of osmotic stimulus by thirsting alone. Another study suggests that non-osmotically stimulated copeptin levels upon arginine infusion also provide a high diagnostic accuracy in differentiating central DI from PP (117). These data indicate that plasma copeptin is a promising biomarker to distinguish between different forms of polyuria–polydipsia syndrome. A possible diagnostic algorithm with and without the availability of copeptin measurement is shown in Figure 8.

Figure 8. Algorithm for the differential diagnosis of diabetes insipidus (Rev Christ-Crain, Nat Rev Primer)

In establishing the underlying mechanisms of central DI once the diagnosis is confirmed, imaging of the hypothalamus, pituitary and surrounding structures with MRI is essential. If no mass lesion is identified, imaging should be repeated after 6-12 months so that slow growing germ cell tumors are not missed. Idiopathic and familial central DI are often associated with loss of the normal hyper-intense signal of the posterior pituitary on T1-weighted images (Figure 9). Signal intensity is correlated strongly with AVP content of the gland (118). In the absence of appropriate history and diagnostic testing, the loss of a posterior pituitary bright spot does not make the diagnosis of central DI. Importantly, presence of an appropriate bright spot does not exclude the diagnosis of central DI.

Figure 9. Loss of the posterior pituitary 'bright spot' on T1 weighted MRI in hypothalamic diabetes insipidus. The normal posterior pituitary can be demonstrated as a 'bright spot' within the sella turcica on T1-weighted MRI (a). This increased signal intensity can be lost in HDI (b). An ectopic posterior pituitary 'bright-spot' can be seen some cases of childhood onset hypopituitarism, implying failure to complete normal developmental migration. Function can be normal despite the aberrant position

Evidence of anterior pituitary dysfunction should be looked for in central DI, though it is relatively uncommon in the adult population. Interestingly, evidence of organ-specific autoimmune disease is relatively common in adult patients with isolated central DI, consistent with an autoimmune basis for the condition (119).

TREATMENT OF DIABETES INSIPIDUS

Treatment of Central DI

The primary aim of treatment in patients with diagnosed central DI should be to reduce polyuria and polydipsia to levels that allow maintenance of a normal lifestyle. The treatment of choice for those with significant symptoms is the synthetic, long-acting AVP analogue DDAVP. The long half-life, selectivity for AVPR2 and availability of multiple preparations renders desmopressin an ideal treatment for central DI. Optimal dosage and dosing intervals should be determined for each patient. The available treatment options are the intranasal spray, oral tablets, sublingual tablets or a parenteral injection, in divided doses. Oral preparations provide greater convenience and are usually preferred by patients. However, starting with a nasal spray initially is preferable because of greater consistency of absorption and physiological effect, after which the patient can be switched to an oral preparation. After trying both preparations, the patient can then choose which they prefer for long-term treatment. A satisfactory schedule can generally be determined using modest doses of desmopressin. The maximum dose of desmopressin required rarely exceeds 0.2 mg orally, 120 µg sublingually or 10 µg (one nasal spray) given 2–3 times daily. These doses usually produce plasma desmopressin levels higher than those required to cause maximum antidiuresis but reduce the need for more frequent treatment (120).

Hyponatremia is the major complication of desmopressin therapy — a 27% incidence of mild hyponatremia (serum sodium 131–134 mmol/l) and a 15% incidence of more severe hyponatremia (serum sodium ≤130 mmol/lm) have been reported after long-term follow-up of patients with chronic central DI (121). Hyponatremia usually occurs if the patient is antidiuretic while continuing normal fluid intake. Severe hyponatremia in patients with central DI who are treated with desmopressin can be avoided first by monitoring serum electrolyte levels frequently during initiation of therapy and second, patients should be instructed to delay a scheduled dose of desmopressin once or twice weekly until polyuria recurs, thereby allowing excess retained fluid to be excreted. A recent publication showed that patients using this approach had a significantly lower the risk of hyponataemia compared to those who did not follow this approach (OR 0.4, 95%CI 0.3-0.7, p<0.01) (122).

Mostly, Desmopressin is a lifelong treatment. An exception is treatment of postsurgical central DI. If the patient is awake and responds to thirst, thirst is a sufficient guide for water replacement. If the duration of diabetes insipidus is transient, it is therefore acceptable to treat simply with fluid replacement, parenterally or orally. However, most patients who develop diabetes insipidus require desmopressin 0.5 to 2 μg subcutaneously, intramuscularly, or intravenously. Urine output will be reduced in 1 to 2 hours and the duration of effect is 6 to 24 hours. Care should be taken that hypotonic intravenous fluids are not given excessively after administering desmopressin, as the combination can lead to profound hyponatremia. Because there is always the possibility of developing the triphasic response due to pituitary stalk damage, it is recommended that polyuria should be recurrent before a decision to administer subsequent doses of desmopressin is made (123).

Patients with hypernatremia due to osmoreceptor dysfunction (adipsic central DI) should be treated acutely with the same treatment as any hyperosmolar patient. The long-term management of osmoreceptor dysfunction syndromes requires a thorough search for potentially treatable causes, combined with measures to prevent dehydration. Because hypodipsia cannot be cured, the focus of management is based on education of the patient and family about the importance of regulating their fluid intake according to their hydration status (124). This can be accomplished most efficaciously by establishing a daily schedule of fluid intake regardless of the patient's thirst, which can be adjusted in response to changes in body weight (125). As these patients will not drink spontaneously, daily fluid intake must be fixed and prescribed. If the patient has polyuria, desmopressin should also be prescribed, as in any patient with central DI. The success of the fluid prescription should be monitored periodically by measuring serum Na⁺concentration. In addition, periodic recalculation of the target weight (at which hydration status and serum Na⁺concentration are normal) might be required.

Treatment of NDI

Treatment of acquired nephrogenic DI should target the underlying cause (e.g., correction of hypercalcemia or hypokalemia), if possible. If not, different approaches are possible.

Low salt diet to minimize the osmotic load
For patients on long-term lithium therapy, amiloride prevents uptake of lithium in the collecting duct epithelial cells and thus the inhibitory effects of intracellular lithium on water transport (126).
Hydrochlorothiazide has been shown to reduce urine output in both central and nephrogenic DI (127, 128). Thiazides decrease salt reabsorption by inhibiting the thiazide-sensitive co-transporter SLC12A3 in the distal tubule. The loss of sodium reduces plasma volume, so that less water is presented to the collecting duct and lost in the urine.
In an animal model of nephrogenic DI, use of the NSAID indomethacin reduced water diuresis independently of AVP (129). A similar effect of prostaglandin synthesis inhibitors was later reported in patients with nephrogenic DI (130). Since these early studies, prostaglandin synthesis inhibitors have become an essential component of the treatment of nephrogenic DI
High dose DDAVP can produce a response in partial NDI

Treatment of PP

Treatment of primary polydipsia entails reduction of excessive fluid intakes, best done in a graded fashion to allow patients to slowly achieve a level of intake that reduced urine volume below polyuric levels (50 mL/kg BW). Measures to reduce mouth dryness (e.g., ice chips, hard candy to stimulate salivary flow) are useful adjuncts to reduce thirst. Pharmacologic therapies have been tried but without consistent evidence of success. A recent study suggests that GLP-1 analogues reduce fluid intake, urine output, and thirst perception(131).

Treatment of Gestational DI

Desmopressin is the only therapy recommended for treatment of diabetes insipidus during pregnancy. Desmopressin is reported to be safe for both the mother and the child (132, 133). During delivery, patients can maintain adequate oral intake and are therefore safe to continue administration of desmopressin. Physicians should be cautious about over administration of fluid parenterally during delivery because these patients will not be able to excrete the fluid and can develop water intoxication and hyponatremia. After delivery, plasma oxytocinase decreases and patients can recover completely or be asymptomatic with regard to fluid intake and urine excretion.

Syndrome Of Inappropriate Antidiuresis

HYPONATREMIA

Hyponatremia (serum sodium <135 mmol/l) is a clinical feature in some 15–20% of non-selected emergency admissions to hospital. It is associated with increased morbidity and mortality across a range of conditions. Moreover, data support the association of hyponatremia correction with improvements in clinical outcome. The relationship of serum sodium and outcome is not straightforward. Co-morbidity and disease severity, rather than hyponatremia per se, may make a significant contribution to adverse outcome in these patients. Further data are needed to clarify whether the relationship between sodium levels and outcome is causal or the association of two variables linked with disease severity (134-137).

Hyponatremia is not invariably associated with a low serum osmolality; high concentrations of other circulating osmolytes (e.g., glucose) can lead to a fall in plasma sodium that is appropriate to maintain normal osmolar status. A reduced plasma aqueous phase secondary to dyslipidemia can result in artefactual hyponatremia with normal plasma osmolality, even when using an ion-specific electrode. This is consequent to the use of a standard dilution step in most clinical biochemistry laboratories. This type of artefactual hyponatremia is not seen when a direct potentiometric method is used, such as when using a blood-gas analyzer. In many clinical situations, hyponatremia is multifactorial (Table 4).

Table 4. Causes of Hyponatremia
Pseudohyponatremia		Reduced renal free water clearance
Hyperglycemia, Hyperlipidemia, Non-physiological osmolyte, Elevated paraprotein		Hypovolemia Cardiac failure Nephrotic syndrome Hypothyroidism Hypoadrenalism SIAD Nephrogenic syndrome of antidiuresis	Drugs Renal failure Portal hypertension & ascites Hypoalbuminemia Sepsis Fluid sequestration
Sodium depletion
Renal loss	Diuretics Salt wasting nephropathy Hypoadrenalism Central salt wasting
Extra-renal loss	Gut loss
Excess water intake
Dipsogenic DI Sodium-free, hypo-osmolar irrigant solutions Dilute infant feeding formula Exercise-associated hyponatremia

AVP plays a key role in many pathophysiological situations of which hyponatremia is a feature. Importantly however, even when AVP plays a role in the development of hyponatremia, AVP production may not be inappropriate. Hyponatremia may reflect an appropriate physiological response to volume depletion. To maintain circulating volume in hypovolemia, baroregulated AVP release may proceed despite plasma osmolalities below the osmotic threshold for AVP release. This can result in hyponatremia, which can become persistent. Though clinical assessment can identify the extracellular volume status of some patients, it is unreliable and has poor sensitivity and specificity (138).

PATHOPHYSIOLOGY AND DIAGNOSIS OF SIAD

An individual with hypoosmolar plasma, a normal circulating volume, and a plasma AVP concentration high for the prevailing osmolality, has the syndrome of inappropriate antidiuresis (SIAD). The clinical criteria to diagnose SIAD go back to Schwartz and Bartter (139) in 1967 and are summarized in Table 5.

Excretion of urine that is not maximally dilute in the context of dilute plasma (i.e., urine concentration greater than 100mOsm/Kg) indicates the action of AVP on renal water resorption. Importantly however, it does not define whether this action is appropriate (for instance in the context of hypovolemia and baro-stimulated AVP release) or inappropriate. Measurement of urinary sodium concentration is key in the differential diagnosis of SIAD from hypovolemia. Renal sodium excretion should be above 30mol/L to make a diagnosis of SIAD. Below this value, volume depletion needs to be considered more likely and below 20 mmol/L, hypovolemia is the likely cause of hyponatremia. SIAD is often associated with urine sodium concentrations of 60 mmol/L or more. SIAD is a volume-expanded state and there is evidence of mild sodium loss as other homeostatic regulators of volume homeostasis attempt to minimize volume expansion. The utility of urinary sodium concentration in defining the etiology of hyponatremia is limited by concurrent use of drugs that produce a natriuresis: diuretics, angiotensin converting enzyme inhibitors, and angiotensin II antagonists. In this situation, a serum urate <4 mg/dl, or a fractional urate excretion >12% can help differentiate SIAD from mild hypovolemia (140, 141).

Cortisol deficiency is a key differential diagnosis as secondary adrenal deficiency can present a biochemical picture identical to SIAD. Therefore, formal biochemical exclusion of adrenal insufficiency is mandatory before diagnosing SIAD.

Four patterns of abnormal AVP secretion have been identified (table 6). The same pattern has also been shown for copeptin (142). Absolute plasma AVP or copeptin concentrations may not be strikingly high and in fact AVP and copeptin measurement is not helpful in establishing the diagnosis (143). The key feature is that they are inappropriate for the prevailing plasma osmolality (10).

● Table 5. Criteria for Diagnosis of SIAD

● Low serum osmolality <275 mOsmol/kg H₂0

● Elevated urine osmolality >100 mOsmol/kg H₂0 (inappropriately concentrated)

● Clinical euvolemia (absence of signs of hypovolemia or hypervolemia)

● Elevated urinary sodium excretion >30mEq/L with normal salt and water intake

● Absence of other potential causes of euvolemic hypo-osmolality (glucocorticoid insufficiency, severe hypothyroidism)

● Normal renal function and absence of diuretic use

Table 6. Classification of SIAD
	Characteristics	Prevalence
SIAD Type A	Wide fluctuations in plasma AVP concentration independent of plasma osmolality	35%
SIAD Type B	Osmotic threshold for AVP release subnormal Osmoregulation around subnormal osmolar set point	30%
SIAD Type C	Failure to suppress AVP release at low plasma osmolality Normal response to osmotic stimulation
SIAD Type D	Normal osmoregulated AVP release Unable to excrete water load.	<10%

Etiology of SIAD

Many conditions have been reported to cause SIAD, though the mechanism(s) of inappropriate AVP release are not clear in many cases (Table 7). SIAD is a non-metastatic manifestation of small cell lung cancer and other malignancies. Some tumors express AVP ectopically. However, excessive posterior pituitary AVP secretion also occurs in association with malignancy. The normal osmoregulated AVP release found in the Type D syndrome suggests an increase in renal sensitivity to AVP, or the action of an additional antidiuretic factor.

Table 7. Causes of SIAD
Neoplastic disease	Chest disorders
Carcinoma (bronchus, duodenum, pancreas, bladder, ureter, prostate) Thymoma Mesothelioma Lymphoma, leukemia Ewing's sarcoma Carcinoid Bronchial adenoma	Pneumonia Tuberculosis Empyema Cystic fibrosis Pneumothorax Aspergillosis
Neurological disorders	Drugs
Head injury, neurosurgery Brain abscess or tumor Meningitis, encephalitis Guillain-Barré syndrome Cerebral hemorrhage Cavernous sinus thrombosis Hydrocephalus Cerebellar and cerebral atrophy Shy-Drager syndrome Peripheral neuropathy Seizures Subdural hematoma Alcohol withdrawal	Sulphonylureas Alkylating agents & Vinca alkaloids Thiazide diuretics Dopamine antagonists Tricyclic antidepressants MAOIs SSRIs 3,4-MDMA ("Ecstasy") Anti-convulsants
Miscellaneous
Idiopathic Psychosis Porphyria Abdominal surgery Diverse non-osmotic stimuli (e.g., nausea, stress, pain)

SIAD is a common mechanism of drug-induced hyponatremia, and can reflect direct stimulation of AVP release from the hypothalamus; indirect action on the hypothalamus; or aberrant resetting of the hypothalamic osmostat (table 8). The prevalence of hyponatremia in patients taking high dose dopamine antagonists is greater than 25%, and is not restricted to one class of these drugs. Hyponatremia secondary to antidepressants is well recognized, occurring with most SSRIs, and the related drug Venlafaxine. It can arise in the first few weeks of treatment. Anticonvulsants are another common cause of SIAD and hyponatremia. The frequency in patients treated with carbamazepine (CBZ) ranges from 4.8 to 40%. Increased sensitivity of central osmoreceptors and increased renal responses to AVP have both been described with CBZ.

Table 8. Mechanisms of Drug Induced Hyponatremia
Reduction in free water clearance		Sodium depletion
SIAD	Dopamine antagonists Tricyclic antidepressants MAOIs SSRIs Venlafaxine Carbamazepine Oxcarbamazepine Sodium valproate 3,4-MDMA ('ecstasy') Clofibrate Cyclophosphamide Sulphonylureas	Diuretics	Spironolactone Thiazides Loop diuretics
AVP-like activity	DDAVP Oxytocin	ACE inhibitors/Angiotensin II receptor antagonists
Potentiation of AVP action	NSAIDS Carbamazepine Sulphonylureas Cyclophosphamide	Direct renal toxicity	Cyclophosphamide Ifosfamide Cisplatin Carboplatin Vincristine Vinblastine

Exercise Associated Hyponatremia

Extreme endurance exercise is a profound physiological stressor. While the magnitude of the physiological stress is likely to reflect a number of factors, duration of the event and the effort entailed are likely to be major contributors. Non-osmoregulated AVP release is a feature of extreme endurance exercise: a reflection of the stressed state. When combined with reduced renal blood flow, another feature of extreme endurance exercise, this can lead to a marked antidiuretic state. If endurance athletes maintain a fluid intake in excess of water loss, hyponatremia will ensue. This can be further complicated if there is aggressive fluid resuscitation in the event of collapse. There is a positive correlation between the odds ratio for developing hyponatremia during extreme endurance exercise and the length of time taken to complete the event. Athletes developing hyponatremia also demonstrate weight gain over the course of the event, clearly implicating water intake in excess of water and electrolyte loss as the cause. Occasional runners should be advised to follow their thirst as they run and avoid rigid, time-based fluid intake. Health professionals attending endurance events need to be aware of the problem of exercise-associated hyponatremia. In addition, they should avoid attempting resuscitation with large volumes of hypotonic fluid in the absence of appropriate indications and without biochemical monitoring (144).

Figure 10. Exercise associated hyponatremia in triathletes. 1089 triathletes were studied. The mean plasma sodium level at the finish was 140.5±4.2 mmol/L (range 111-152). Among athletes completing the study events, 10.6% had documented hyponatremia: 8.7% mild; 1.6% severe; and 0.3% critical (3 athletes in total with plasma sodium levels 120, 119, and 111 mmol/L respectively). Multivariate analysis showed a significant association between development of hyponatremia and the following factors: female gender; longer times to complete a race. Critical hyponatremia occurred in participants who finished in the 12th and 14th hours of the race (145).

Nephrogenic Syndrome of Inappropriate Antidiuresis

The G-protein-coupled V2-R mediates the action of AVP on renal water excretion. Rare kindreds have been found that harbor constitutively activating mutations in the V2-R that led to AVP-independent, V2-R mediated, antidiuresis associated with persistent hyponatremia (Figure 11). This nephrogenic syndrome of inappropriate antidiuresis (NSIAD) was initially described in male infants with persistent hyponatremia in keeping with the haploinsufficiency associated with the V2-Rgene being on the X chromosome. However, subsequent studies have found the condition is not limited to males, expression of the condition being clearly identified in heterozygous females. The true prevalence of NSIAD is not known. However, as some 10% of patients with SIAD have been described as having undetectable AVP, it seems likely that at least some of these cases may be due to activating mutations of the V2-R (146, 147).

Figure 11. In vitro bioactivity of different V2-R constructs relative to wild type (WT) in a cAMP-dependent luciferase reporter system in the absence of AVP. The R137H construct is the V2-R found in X-linked NDI. R137C and R137L are receptor variants found in NSIAD. Constructs differ only by the amino acid at position 137. R137C and R137L found in NSIAD demonstrate constitutive, AVP-independent activity. R: arginine. H: histidine. C: cysteine. L: leucine.

Cerebral Salt Wasting

This acquired, primary natriuretic state remains a subject of controversy. It has been characterized as a combination of hyponatremia with hypovolemia associated with neurological or (more often) neurosurgical pathologies. The underlying mechanism(s) remain unclear, but increased release of natriuretic peptides and/or reduced sympathetic drive have been proposed. The diagnosis of cerebral salt wasting (CSW) hinges on the natural history: the development of hyponatremia being preceded by natriuresis and diuresis with ensuing clinical and biochemical features of hypovolemia. In contrast to SIAD, urea and creatinine are elevated and there may be postural hypotension. The simple observation of weight loss over the period in question can be helpful.

SIAD can occur in the same group of patients in whom CSW has been reported. Both CSW and SIAD are associated with urine sodium concentrations greater than 40mmols/L. However, the natriuresis of CSW is much more profound than that of SIAD and precedes the development of hyponatremia. CSW is a particular concern for the neurosurgical patient in whom autoregulation of cerebral blood flow is disturbed and in whom small reductions in circulating volume can reduce cerebral perfusion. The management of CSW is volume replacement with 0.9% saline; while the cause-directed approach to SIAD would often involve restriction of fluid. The clinical and practice context, together with the opposed cause-directed management approaches to CSW and SIAD can lead to significant tension. A cause-independent approach to the management of the neurosurgical patient with hyponatremia is often the practical and pragmatic approach to take: balancing management of hyponatremia with the need to avoid threatening cerebral perfusion and avoidable vasospasm (148). Importantly, in a prospective, single center study of 100 patients developing hyponatremia after subarachnoid hemorrhage not a single case of CSW was identified: hyponatremia was attributable to SIAD, glucocorticoid deficiency, or inappropriate fluid administration (Figure 12). Therefore, CSW is probably a very rare condition.

Figure 12. Hyponatremia after subarachnoid hemorrhage. Prospective study of 100 patients. Demographics, outcomes and etiology of hyponatremia noted. SAH: subarachnoid hemorrhage (149).

MANAGEMENT OF HYPONATREMIA SECONDARY TO SIAD

The morbidity and mortality of hyponatremia secondary to SIAD are the result of disturbance in central nervous system (CNS) function: the combined impact of cerebral oedema and direct neuronal dysfunction (Table 9). While it is intuitive that the greater the derangement in serum sodium should be associated with the most profound neurological disturbance, the relationship between serum sodium and neurological function is not simple. Patients with marked biochemical disturbance may have mild symptoms if hyponatremia develops over a prolonged period. This reflects CNS adaptation: brain edema being limited by efflux of organic solutes. This adaptation can complicate the management of hyponatremia. Rapid correction of serum sodium following the gradual development of hyponatremia (which has resulted in a degree of CNS adaptation) can lead to significant changes in brain volume as the osmolar gradient across the blood-brain barrier alters. This can trigger CNS demyelination (osmotic demyelination syndrome, ODS). This is a rare but serious complication of hyponatremia and its treatment. ODS develops within 1-4 days of rapid correction of serum sodium, irrespective of the method employed to achieve it. It can even occur when sodium levels are corrected slowly. Risk factors for ODS are severe liver disease, potassium depletion, malnutrition and profound hyponatremia, especially if <105mmol/L. Neurological manifestations include quadriplegia, opthalmoplegia, pseudo-bulbar palsy and coma.

Table 9. Clinical Features of Hyponatremia Secondary to SIAD

• Headache

• Nausea

• Vomiting

• Muscle cramps

• Lethargy

• Disorientation

• Seizure

• Coma

• Brain-stem herniation

Management of Hyponatremia Associated with Severe or Moderately Severe Symptoms

Hyponatremia associated with severe or moderately severe symptoms requires urgent management as an emergency. Treatment is cause-independent. Treatment should be given priority over establishing the etiology of hyponatremia. The aim of treatment is to reduce immediate risk through increasing serum sodium to a level that decreases morbidity and mortality. Importantly, the rate of increase in serum sodium must be at a rate that does not result in harm through precipitating ODS (Figure 13). The immediate target should not be to normalize serum sodium. Once the patient is stable, investigations to establish the cause of hyponatremia can begin, the outcome of which subsequently guiding cause-directed therapy (140, 150, 151).

Figure 13. Immediate, cause-independent management of hyponatremia associated with severe or moderately severe symptoms.

Previous strategies for the emergency management of hyponatremia have stratified patients based on presumed duration of the electrolyte disturbance. Furthermore, some have advocated hypertonic fluid administration volume and rates based on calculated deficits in serum sodium using a standard formula (152). A more pragmatic and reliable approach to patient stratification is to base the decision on use of hypertonic fluid on patient symptoms and signs; while the determining hypertonic fluid prescription by calculated serum sodium deficit is associated with a significant risk of over-correction (153). Over-correction of hyponatremia (increase in serum sodium above the recommended rate) should prompt review of the fluid regimen and consideration of active management with hypotonic fluid, with or without concurrent use of DDAVP (140, 154).

Two recent studies have compared bolus versus infusion hypertonic saline, both in favor of a bolus regimen. An Irish observational study compared outcomes for 22 hyponatremic patients treated with bolus 100ml 3% NaCl, compared to a historical cohort of 28 patients managed with 20ml/hour 3% NaCl infusion (155). Bolus hypertonic saline was associated with a faster elevation of serum sodium at 6 hours and significantly greater improvement in Glasgow Coma Scale at 6 hours. There were no cases of ODS in either group. A larger randomized trial from Korea included 178 patients with hyponatremia who were randomized to either bolus or infusion NaCl 3% (156). The results showed that both therapies were effective and safe, with no significant difference in overcorrection risk, but there was less need for therapeutic relowering in the bolus group compared to the slow continuous infusion. Bolus therapy is therefore currently recommended in guidelines.

Management of SIAD Associated with Mild to Moderate Symptoms

Fluid restriction: Fluid restriction of 0.5–1L/day is the first-line treatment recommended by EU and US guidelines (157). It is a reasonable initial intervention when the clinical condition is not critical. All fluids need to be included in the restriction. As SIAD is associated with a degree of natriuresis, sodium intake should be maintained. Fluid restriction may need to be maintained for several days before sodium levels normalize and it is important that a negative fluid balance is confirmed during this period. As cause-directed therapy progresses (e.g., treatment of underlying infection or removal of drug causing SIAD), fluid restriction may be relaxed. A recent randomized controlled trial evaluated the efficacy of fluid restriction in comparison to no treatment showing a modest early rise in serum sodium of 3mmol/L in fluid restricted patients compared to 1mmol/L with no treatment. Similar results were seen in other recent trials (158).

Of note, fluid restriction is not always effective, and a clinically useful response is only seen in around half of hyponatremic patients. In a large hyponatremia registry fluid restriction was unsuccessful in 55% of cases (159). Factors predicting a poor response to fluid restriction include high urine osmolality (>500 mosm/L), and high urine sodium concentration (or high urine/serum electrolyte ratio i.e. (UNa + UK)/pNa > 1, i.e., ‘Furst equation’) (150, 160, 161).

Drug treatment in SIAD: If patients do not respond to fluid restriction, a second-line treatment has to be initiated. There are several different possibilities, which are discussed in the following.

Urea is a product of hepatic nitrogen metabolism which is renally excreted and has an osmotic effect to promote free water excretion. It is recommended as a second line treatment in the European and US guidelines(140, 150). The evidence base for the use of urea is limited due to the retrospective observational nature of most current studies. In fact, there are several observational studies of urea, but no randomized trials to date. A retrospective observational trial reported a mean increase in sodium levels of 7 mmol/L over 4 days, with no instances of rapid correction (162). Another retrospective study reported that the majority of patients normalized sodium levels within 48 hours despite fluid intake >2 L/d (163). Finally, in patients with SIAD in the context of subarachnoid hemorrhage urea treatment led to sodium normalization within a mean time of 3 days (164).

Although overcorrection has been described, there have been no published cases of ODS due to urea therapy. The main limitations are the difficult access and poor palatability due to bitter taste, which can be ameliorated by combining it with a small volume of orange juice. Urea therapy can cause an increase in serum urea concentration and blood urea nitrogen, but this does not necessarily represent deterioration in renal function or hypovolemia (162).

Tolvaptan is an oral vasopressin V2-receptor antagonist that blocks AVP action in the kidney, inducing water diuresis and thereby raising sodium levels. An intravenous alternative conivaptan is also approved, but its use is limited to a maximum duration of 4 days because of drug interaction effects with other agents metabolized by the CYP3A4 hepatic isoenzyme.

The efficacy of tolvaptan versus placebo was investigated in the randomized placebo-controlled SALT-1 and SALT-2 trials in patients with chronic eu- or hypervolemic hyponatremia (165). The results showed that tolvaptan increased sodium levels within 4 days by 4 mmol/L compared to only 1 mmol/L in the placebo group and an even higher increase within 30 days. The American expert panel recommends its use as a second-line treatment, whereas the European clinical practice guidelines recommend against the use of tolvaptan in moderate to profound hyponatremia, mainly due to lack of proven outcome benefit aside from increase in sodium levels and the concern of overcorrection. Importantly, discontinuing fluid restriction when vaptans are started, initiation of treatment in the hospital and regular sodium monitoring limit the risk for overcorrection. A lower initial dose of tolvaptan, 7.5 mg, which has been associated with lower rates of overcorrection, should also be considered (166). Common side effects of tolvaptan include dry mouth, thirst, and urinary frequency.

Sodium-glucose co-transporter 2 inhibitors are approved for treatment of diabetes and heart failure, but may also be beneficial in SIAD. The sodium-glucose co-transporter 2 in the proximal renal tubule is responsible for 90% of renal glucose resorption (167). Inhibition of SGLT2 leads to glycosuria, accompanied by an osmotic diuresis. A randomized trial of inpatients with SIAD compared 4 days of empagliflozin 25mg or placebo, in addition to fluid restriction (168). Plasma sodium increased by 10mmol/L over 4 days in the empagliflozin group, compared to 7mmol/L with placebo (p = 0.04). There was no hypotension or hypoglycemia observed. The relatively high sodium increment in the placebo group suggests that at least in some patients, reversible stimuli to AVP secretion may have been present. A second, randomized controlled cross-over study in 14 patients’ outpatients with chronic SIAD compared a 4-week treatment with empagliflozin 25mg/day to placebo, without concomitant fluid restriction. Empagliflozin treatment resulted in a serum sodium increase of 4.1mmol/L (p=0.004) under empagliflozin as compared to placebo. Treatment with empagliflozin was generally well tolerated with no events of sodium overcorrection, hypoglycemia, hypotension, urinary tract or genital infection occurring during the observation period (169). Future larger studies are needed to validate these findings.

Loop diuretics block the Na+-K+-2CL- cotransporter in the thick ascending limb of the loop of Henle and decrease delivery of sodium and chloride to the kidney medulla, decreasing the medullary gradient necessary for water reabsorption in the collecting duct. Salt tablets are added to replace loop diuretic-induced sodium losses. They are listed in the European practice guidelines as a second line therapy for SIAD in combination with salt tablets. However, a recent randomized controlled trial comparing the effect of FR alone versus FR plus loop diuretics versus FR plus loop diuretics plus salt tablets did not show a significant additive effect of loop diuretics or salt tablets (158). Acute kidney injury and hypokalemia were more common in patients receiving furosemide, therefore caution should be taken when adopting this strategy.

Demeclocycline has been used for many years in management of hyponatremia of SIAD. It produces a form of NDI and so increases renal water loss even in the presence of AVP. There is a lag time of some 3–4 days in onset of action and the effect is unpredictable occurring in 33-60% of patients. Photosensitive skin reactions are a significant additional adverse effect. There are very limited data to support long-term efficacy and a systematic review did not find good quality evidence for its use (170).

Lithium also induces nephrogenic diabetes insipidus and has therefore been tried as treatment for SIAD, however, due to adverse effects and questionable efficacy it is not recommended in European guidelines.

Adipsic And Hypodipsic Syndromes

Adipsic and hypodipsic disorders are characterized by inadequate spontaneous fluid intake due to defects in osmoregulated thirst. Patients deny thirst and do not drink, despite dehydration and hypovolemia. If the defect is mild, the resultant hypernatremia is often well tolerated. Severe disorders can lead to somnolence, seizures, and coma. Because of the close anatomical relationship of the osmoregulatory centers for thirst and AVP release, adipsic syndromes are often associated with defects in osmoregulated AVP release and HDI.

CLASSIFICATION AND ETIOLOGY

Four patterns of adipsic/hypodipsic syndrome are recognized (Table 10, Figure 14). Causes are outlined in Table 11. Patients with the Type A syndrome osmoregulate around a supra-normal osmolar set point and are protected from extreme hypernatremia, as are those with the Type B syndrome. In Type C adipsia, osmoregulated thirst and AVP release are absent. Patients present with adipsic diabetes insipidus. Precipitants include rupture and repair of anterior communicating artery (ACA) aneurysm, as the osmoreceptors mediating both thirst and AVP release receive a blood supply from perforating branches of the anterior cerebral artery and ACA. Some patients with the Type C syndrome have constitutive low level AVP release, and are at risk of dilutional hyponatremia.

Table 10. Classification of Adipsic and Hypodipsic Syndromes
Adipsia/hypodipsia Syndrome	Osmoregulated Thirst	Osmoregulated AVP release
Type A (essential hypernatremia)	Osmotic threshold increased Normal sensitivity	Osmotic threshold increased Normal sensitivity Normal non-osmotic stimulation
Type B	Normal osmotic threshold Reduced sensitivity	Normal osmotic threshold Reduced sensitivity Normal non-osmotic stimulation
Type C	No response to osmotic stimulation	Persistent low level AVP release No response to osmotic stimulation Normal non-osmotic stimulation
Type D	No response to osmotic stimulation	Normal

Table 11. Causes of Adipsic and Hypodipsic Syndromes
Neoplastic (50%)
Primary	Craniopharyngioma Germ cell tumor Meningioma
Secondary	Pituitary tumor Bronchial carcinoma Breast carcinoma
Granulomatous (20%) Histiocytosis Sarcoidosis
Miscellaneous (15%) Hydrocephalus Ventricular cyst Trauma Toluene poisoning
Vascular (15%) Internal carotid artery ligation Anterior communicating artery aneurysm Intra-hypothalamic hemorrhage

Figure 14. Patterns of plasma AVP and thirst responses to hypertonic stress in patients with adipsic syndromes. Normal range responses to osmolar stimulation are shown by the shaded areas. The 4 types of adipsic syndrome are demonstrated. Patients with the Type A syndrome osmoregulate around a higher osmolar set point, while those with the Type B syndrome mount AVP and thirst responses but with reduced sensitivity. Patients with the Type C syndrome have much reduced or absent AVP and thirst responses to osmolar stimulation. Those with the Type D syndrome demonstrate normal AVP responses to osmolar stimulation but much reduced thirst responses.

MANAGEMENT

As those with Type A and Type B adipsia are protected from extreme hypernatremia, treatment is to recommend an obligate fluid intake of about 2L/24 hours with appropriate adjustment for climate and season. If fluid balance cannot be maintained during intercurrent illness, hospital in-patient management may be required. The adipsic central diabetes insipidus of the Type C syndrome can be difficult to manage. The structural and vascular problems producing the syndrome often led to associated defects in short term memory and task organization, complicating long-term management. A pragmatic approach is to effectively dictate an acceptable urine output (1-2L/24 hours) with regular DDAVP (producing a fixed obligate antidiuresis). Together with an estimation of standard insensible fluid loss (approximately 0.4L), this can be set to create a fixed net fluid loss of some 2Ls. In turn, this can be balanced by a daily fluid intake that varies in response to depending on day-to-day fluctuations from a target weight at which the patient is euvolemic and normonatremic.

Daily fluid intake = 2L + (daily weight in Kg- target weight in Kg).

Plasma sodium should be checked weekly, to avoid the creeping development of hyper- and hyponatremia as dry weight changes. This approach can result in stable fluid balance and successful independent living (171).

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ABSTRACT

INTRODUCTION

OSM (calculated) = 2 x Na (mmol/l) + Glucose (mmol/l) + Urea (mmol/l)

CLINICAL PRESENTATION OF SIADH

Corrected (Na+) = Measured (Na+) + 2.4 x (glucose (mg/dl) - 100 mg/dl)/100mg/dl

HYPONATREMIA IN EUVOLEMIA: ASSESSMENT FOR SIADH

1st Step

2nd Step

3rd Step

4th Step

INTERPRETATION OF CLINICAL AND LABORATORY RESULTS

DIFFERENTIAL DIAGNOSIS OF HYPONATEMIA OTHER THAN SIADH

THERAPY OF SIADH

Severity of Hyponatremia-Related Symptoms

Drug-Induced SIADH

Primary and Secondary SIADH

REFERENCES

ABSTRACT

INTRODUCTION

EMBRYOLOGY

ETIOLOGY

CLINICAL PRESENTATION

ENDOCRINE EFFECTS OF CRYPTORCHIDISM

GERM CELL MATURATION IN CRYPTORCHIDISM

DIAGNOSIS

TREATMENT

Newborn and Infant

Prognosis

ACQUIRED CRYPTORCHIDISM

Retractile Testes

Ascending Testes

Management of Acquired Cryptorchidism

HYPOSPADIAS

REFERENCES

ABSTRACT

INTRODUCTION

Androgen Physiology

THE HYPOTHALAMIC-PITUITARY AXIS

CONSEQUENCES OF ANDROGEN DEFICIENCY

MEASUREMENT OF CIRCULATING ANDROGEN CONCENTRATIONS

Androgens and Male Ageing

DECLINE IN CIRCULATING ANDROGENS WITH MALE AGING

CONTROVERSIES OVER FUNCTIONAL HYPOGONADISM

Androgens and Risks of Cardiovascular Disease

AGE AND OBESITY AS COMMON RISK FACTORS

CONFLUENCE OF AGE, OBESITY, LOW TESTOTERONE AND CARDIOVASCULAR DISEASE

EPIDEMIOLOGICAL STUDIES

Associations of Androgens with Cardiovascular Events

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR DISEASE

Associations of Androgens with Cardiovascular Mortality

PROSPECTIVE COHORT STUDIES: IMMUNOASSAY RESULTS

PROSPECTIVE COHORT STUDIES: MASS SPECTROMETRY RESULTS

SUMMARY: ENDOGENOUS SEX HORMONES VS. CARDIOVASCULAR MORTALITY

MECHANISTIC STUDIES

Potential Mechanisms

Cholesterol Accumulation in Animal Models

Neointimal Formation and Vascular Smooth Muscle Proliferation

NEOINTIMAL RESPONSES TO INJURY

VASCULAR SMOOTH MUSCLE

Inflammation

INFLAMMATION AND ATHEROTHROMBOSIS

TESTOSTERONE EFFECTS ON IMMUNE CELLS

TESTOSTERONE EFFECTS ON CYTOKINES

CLINICAL TRIALS WITH SURROGATE ENDPOINTS

Testosterone Effects on Angina and Vascular Function

EFFECTS OF EXOGENOUS TESTOSTERONE ON ANGINA

EFFECTS OF EXOGENOUS TESTOSTERONE ON VASCULAR FUNCTION

Testosterone and Atherosclerosis

CIRCULATING CHOLESTEROL CONCENTRATION

CAROTID ATHEROSCLEROSIS

CORONARY ATHEROSCLEROSIS

TRIALS REPORTING ADVERSE EVENTS

Testosterone RCTs and Cardiovascular Adverse Events

Meta-analyses of Cardiovascular Adverse Events in Testosterone RCTs

RETROSPECTIVE CASE-CONTROL STUDIES

Retrospective Studies of Testosterone Prescriptions

Abuse of Androgenic Steroids

DISCUSSION

1^st Step

2^nd Step

3^rd Step

4^th Step