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.

 

 

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.

 

 

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.

 

 

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.

 

 

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.

 

 

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).

 

 

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

 

 

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.

 

 

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.

 

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).

 

 

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).

 

 

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.

 

 

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).

 

 

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

 

 

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

 

The pineal gland was described as the “Seat of the Soul” by Renee Descartes and it is located in the center of the brain. The main function of the pineal gland is to receive information about the state of the light-dark cycle from the environment and convey this information by the production and secretion of the hormone melatonin. Changing photoperiod is indicated by the duration of melatonin secretion and is used by photoperiodic species to time their seasonal physiology. The rhythmic production of melatonin, normally secreted only during the dark period of the day, is extensively used as a marker of the phase of the internal circadian clock. Melatonin itself is used as a therapy for certain sleep disorders related to circadian rhythm abnormalities such as delayed sleep phase syndrome, non-24h sleep wake disorder and jet lag. It might have more extensive therapeutic applications in the future, since multiple physiological roles have been attributed to melatonin. It exerts physiologic immediate effects during night or darkness and when suitably administered has prospective effects during daytime when melatonin levels are undetectable. In addition to its role in regulating seasonal physiology and influencing the circadian system and sleep patterns, melatonin is involved in cell protection, neuroprotection, and the reproductive system, among other possible functions. Pineal gland function and melatonin secretion can be impaired due to accidental and developmental conditions, such as pineal tumors, craniopharyngiomas, injuries affecting the sympathetic innervation of the pineal gland, and rare congenital disorders that alter melatonin secretion. This chapter summarizes the physiology and pathophysiology of the pineal gland and melatonin.

 

PINEAL PHYSIOLOGY

 

Pineal Anatomy and Structure

 

The pineal gland in humans is a small (100-150 mg), highly vascularized, and a secretory neuroendocrine organ (1). It is located in the mid-line of the brain, outside the blood-brain barrier and attached to the roof of the third ventricle by a short stalk. In humans, the pineal gland usually shows a degree of calcification with age providing a good imaging marker (for a speculative discussion of pineal calcification see reference(2)). The principal innervation is sympathetic, arising from the superior cervical ganglia (3). Arterial vascularization of the pineal gland is supplied by both the anterior and posterior circulation, being the main artery supplying the lateral pineal artery, which originates from the posterior circulation (4). In mammals, the main cell types are pinealocytes (95%) followed by scattered glial cells (astrocytic and phagocytic subtypes) (5). Pinealocytes are responsible for the synthesis and secretion of melatonin.

 

Main Function of the Pineal Gland

 

The main function of the pineal gland is to receive and convey information about the current light-dark cycle from the environment via the production and secretion of melatonin cyclically at night (dark period) (6, 7). Although in cold-blooded vertebrates (lower-vertebrate species), the pineal gland is photosensitive, this property is lost in higher vertebrates. In higher vertebrates, light is sensed by the inner retina (retinal ganglion cells) that send neural signals to the visual areas of the brain. However, a few retinal ganglion cells contain melanopsin and have an intrinsic photoreceptor capability that sends neural signals to non-image forming areas of the brain, including the pineal gland, through complex neuronal connections. The photic information from the retina is sent to the suprachiasmatic nucleus (SCN), the major rhythm-generating system or “clock” in mammals, and from there to other hypothalamic areas. When the light signal is positive, the SCN secretes gamma-amino butyric acid, responsible for the inhibition of the neurons that synapse in the paraventricular nucleus (PVN) of the hypothalamus, consequently the signal to the pineal gland is interrupted and melatonin is not synthesized. On the contrary, when there is no light (darkness), the SCN secretes glutamate, responsible for the PVN transmission of the signal along the pathway to the pineal gland. However, it is important to note that in continuous darkness the SCN continues to generate rhythmic output without light suppression since it functions as an endogenous oscillator (master pacemaker or clock). The rhythm deviates from 24h and ‘free-runs’ in the absence of the important light time cue. Light-dark cycles serve to synchronize the rhythm to 24h.

 

The PVN nucleus communicates with higher thoracic segments of the spinal column, conveying information to the superior cervical ganglion that transmits the final signal to the pineal gland through sympathetic postsynaptic fibers by releasing norepinephrine (NE).  NE is the trigger for the pinealocytes to produce melatonin by activating the transcription of the mRNA encoding the enzyme arylalkylamine N-acetyltransferase (AA-NAT), the first molecular step for melatonin synthesis (8) (Figure 1).

 

 

MELATONIN SYNTHESIS AND METABOLISM

 

Melatonin Synthesis

 

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized within the pinealocytes from tryptophan, mostly occurring during the dark phase of the day, when there is a major increase in the activity of serotonin-N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT), responsible for the transformation of 5-hydroxytryptamine (5HT, serotonin) to N-acetylserotonin (NAS) (Figure 1). Finally, N-acetylserotonin is converted to melatonin by acetylserotonin O-methyltransferase. The rapid decline in the synthesis with light treatment at night appears to depend on proteasomal proteolysis (9). Both AA-NAT and serotonin availability play a role limiting melatonin production. AA-NAT mRNA is expressed mainly in the pineal gland, retina, and to a lesser extent in some other brain areas, pituitary, and testis. Melatonin synthesis is also described in many other sites. AA-NAT activation is triggered by the activation of β1 and α1b adrenergic receptors by NE (9). NE is the major transmitter via β-1 adrenoceptors with potentiation by α-1 stimulation. NE levels are higher at night, approximately 180 degrees out of phase with the serotonin rhythm. Both availability of NE and serotonin are stimulatory for melatonin synthesis. Pathological, surgical or traumatic sympathetic denervation of the pineal gland or administration of β-adrenergic antagonists abolishes the rhythmic synthesis of melatonin and the light-dark control of its production.

 

There is evidence that melatonin can be synthesized in other sites of the body (skin, gastrointestinal tract, retina, bone marrow, placenta and others) acting in an autocrine or paracrine manner (10). Very recently it has been demonstrated that in the mouse brain melatonin is exclusively synthesized in the mitochondrial matrix. It is released to the cytoplasm, thereby activating a mitochondrial MT1 signal-transduction pathway which inhibits stress-mediated cytochrome c release and caspase activation: these are preludes to cell death and inflammation. This is a new mechanism whereby locally synthesized melatonin may protect against neurodegeneration. It is referred to as automitocrine signaling (11). Except for the pineal gland, other structures contribute little to circulating concentrations in mammals, since after pinealectomy, melatonin levels are mostly undetectable (12). Importantly several other factors, summarized in Table 1, have been related to the secretion and production of melatonin (13, 14).

 

Table 1. Factors Influencing Human Melatonin Secretion and Production (11),(12)
Factor	Effect(s) on melatonin	Comment
Light	Suppression	>30 lux white 460-480 nm most effective
Light	Phase-shift/ Synchronization	Short wavelengths most effective
Sleep timing	Phase-shift	Partly secondary to light exposure
Posture	­ standing (night)
Exercise	­ phase shifts	Hard exercise
ß-adrenoceptor-A	¯ synthesis	Anti-hypertensives
5HT UI	­ fluvoxamine	Metabolic effect
NE UI	­ change in timing	Antidepressants
MAOA I	­ may change phase	Antidepressants
α-adrenoceptor-A	¯ alpha-1, ­ alpha-2
Benzodiazepines	Variable¯ diazepam, alprazolam	GABA mechanisms
Testosterone	¯	Treatment
OC	­
Estradiol	¯? Not clear
Menstrual cycle	Inconsistent	­ amenorrhea
Smoking	Possible changes ­¯ ?
Alcohol	¯	Dose dependent
Caffeine	­	Delays clearance (exogenous)
Aspirin, Ibuprofen	¯
Chlorpromazine	­	Metabolic effect
Benserazide	Possible phase change, Parkinson patients	Aromatic amino-acid decarboxylase-I

Abbreviations: A: antagonist, U: uptake, I: inhibitor, MAO: monoamine oxidase, OC: oral contraceptives, 5HT: 5-hydroxytryptamine.

 

Control of Melatonin Synthesis: A Darkness Hormone

 

The rhythm of melatonin production is internally generated and controlled by interacting networks of clock genes in the bilateral SCN (15). Damage to the SCN leads to a loss of the majority of circadian rhythms. The SCN rhythm is synchronized to 24 hours mainly by the light-dark cycle acting via the retina and the retinohypothalamic projection to the SCN; the longer the night the longer the duration of secretion is, and the ocular light serves to synchronize the rhythm to 24h and to suppress secretion at the end of the dark phase, as explained above. Light exposure is the most important factor related to pineal gland function and melatonin secretion. A single daily light pulse of suitable intensity and duration in otherwise constant darkness is enough to phase shift and to synchronize the melatonin rhythm to 24h (16). The amount of light required at night to suppress melatonin secretion varies across species. In humans, intensities of 2500 lux full spectrum light (domestic light is around 100 to 500 lux) or light preferably in the blue range (460 to 480 nm) are required to completely suppress melatonin at night, but lower intensities < 200 lux can suppress secretion and shift the rhythm (17-21). Previous photic history influences the response: living in dim light increases sensitivity. Furthermore, the degree of light perception between individuals is related to the incidence of circadian desynchrony; along these lines, blind people with no conscious or unconscious light perception show free-running or abnormally synchronized melatonin and other circadian rhythms (22-24). Some blind subjects retain an intact retinohypothalamic tract and therefore a normal melatonin response despite a lack of conscious light perception (25, 26). It seems clear that an intact innervated pineal gland is necessary for the response to photoperiod change (27). Melatonin functions as a paracrine signal within the retina, it enhances retinal function in low intensity light by inducing photomechanical changes and provides a closed-loop to the pineal-retina-SCN system. All together, they are the basic structures to perceive and transduce non-visual effects of light, and to generate the melatonin rhythm by a closed-loop negative feedback of genes (Clock, “Circadian locomotor output cycles kaput” and Bmal, “Brain and muscle ARNT-like” genes), positive stimulatory elements (Per, “period” and Cry, “Cryptochrome” genes), and negative elements (CCG, clock-controlled genes) of clock gene expression in the SCN (Figure 2).

Melatonin Metabolism

 

Once synthesized, melatonin is released directly into the peripheral circulation (bound to albumin) and to the CSF without being stored. In humans, melatonin’s half-life in blood is around 40 minutes and it is metabolized within the liver, converted to 6-hydroxymelatonin mainly by CYP1A2 and conjugated to 6-sulfatoxymelatonin (aMT6s) for subsequent urinary excretion. The measurement of urinary aMT6s is a good marker of melatonin secretion, since it follows the same pattern with an approximate 2-hours offset (28, 29). Overall, women have slightly higher values of plasma melatonin at night than men (30). On average, the maximum levels of plasma melatonin in adults occur between 02.00 and 04.00 hours and are on average about 60 to 70 pg/mL when measured with high-specificity assays. Concentrations in saliva, like other hormones, are three times lower than in plasma. Minimum concentrations detected are below 1 pg/mL. The plasma melatonin rhythm (timing and amplitude) strongly correlates with urinary aMT6s. Although there is a large variability in amplitude of the rhythm between subjects, the normal human melatonin rhythm is reproducible from day to day intraindividually (Figure 3 and 4).

 

Melatonin Production During Development and Across Life

 

At birth, melatonin levels are almost undetectable, the only fetal source of melatonin being via the placental circulation. Melatonin levels in fetal umbilical circulation reflect the day-night difference as seen in the maternal circulation. Maternal melatonin sends a temporal circadian signal to the fetus (called “maternal photoperiodic adaptative programming”), preparing the CNS to properly deal with environmental day/night fluctuations after birth. A melatonin rhythm appears around 2 to 3 months of life (31), levels increasing exponentially until a lifetime peak on average in prepubertal children; melatonin concentrations in children are associated with Tanner stages of puberty (32). Thereafter, a steady decrease occurs reaching mean adult concentrations in late teens (33, 34). Values are stable until 35 to 40 years, followed by a decline in amplitude of melatonin rhythm and lower levels with ageing, associated with fragmented sleep-wake patterns (35). In people >90 years, melatonin levels are less than 20% of young adult concentrations (36). The decline in age-related melatonin production is attributed to different reasons; calcification of the pineal gland starting early in life and an impairment in the noradrenergic innervation to the gland or light detection capacity (ocular mydriases, cataracts) (2, 37). Interestingly, pinealectomy accelerates the aging process and several reports suggest that melatonin has anti-aging properties (38).

 

MELATONIN’S MECHANISMS OF ACTION

 

Melatonin Target Sites and Receptors

 

Melatonin’s target sites are both central and peripheral. Binding sites have been found in many areas of the brain, including the pars tuberalis and hypothalamus, but also in the cells of the immune system, gonads, kidney, and the cardiovascular system (39, 40). Melatonin binding sites in the brain might vary according to species. Melatonin exerts both non-receptor and receptor-mediated actions. Non-receptor-mediated actions are due to amphipathic properties (of both lipo- and hydrophilic structure) that allows melatonin to freely cross the cell and nuclear membranes. It can be easily detected in the nucleus of several cells in the brain and peripheral organs (41). Antioxidant properties are one example of non-receptor-mediated actions of melatonin. On the other hand, melatonin has receptor-mediated actions.

 

Two types of a new family of G protein coupled melatonin receptors (MT) have been cloned in mammals (42). Melatonin receptors are widely expressed, often with overlapping distributions. MT1 is primarily expressed in the pars tuberalis, the SCN together with other hypothalamic areas, pituitary, hippocampus, and adrenal glands, suggesting that circadian and reproductive effects are mediated through this receptor. MT2 is mainly expressed in the SCN, retina, pituitary and the other brain areas, and is associated with phase shifting (43). The affinity of melatonin is five-fold greater for MT1 than MT2. Melatonin administration induces (1) an acute suppression of neuronal firing in the SCN via the MT1 receptor, and (2) a phase-shifting of SCN activity through the MT2 receptor. However, agonists that exclusively act on one or another receptor have not been identified as yet, and the understanding of the role of each receptor in most of the tissues in which both receptors are present is difficult. Also, melatonin receptors are transiently expressed in neuroendocrine tissues during development, suggesting that melatonin has a role as a neuroendocrine synchronizer in developmental physiology (44).

 

Chronobiotic Effects of Melatonin

 

Phase shifting of circadian rhythms by melatonin (Figure 5) was first described in the 1980s (45). Melatonin has chronobiotic effects and is able to synchronize and reset biological oscillations. Melatonin acts on oscillators according to a well-defined phase-response curve (PRC). PRC is characterized by phase-advance zone (early in the evening before the beginning of the nocturnal melatonin production), phase-delay zone (in the late night/early morning hours), and a non-responsive dead.  zone (when melatonin levels are high) (46). The melatonin PRC is useful for the clinical administration of melatonin as a chronobiotic agent and treatment of sleep circadian and mood  disorders (47). In addition to the circadian chronobiotic effects, melatonin importantly has chronobiotic seasonal effects and acts as a circannual synchronizer, one season determined by increasing duration of the nocturnal melatonin production (in the direction of the winter solstice) and the other season defined by the reduction of nocturnal melatonin production (in the direction of the summer solstice) (8, 48). Interestingly, most of the clock genes are expressed in the pars tuberalis with a 24h rhythmicity different from their expression in the SCN (49), and these clock genes are influenced by melatonin with numerous potential seasonal effects. However, whether melatonin modulates the activity of the SCN via regulation of clock genes is unclear. Also, a central clock, independent of the SCN, can be entrained by food availability, temperature variations, forced activity and rest, drugs (melatonin itself), and timing (50).

 

MELATONIN PHYSIOLOGY AND PATHOPHYSIOLOGY

 

As previously stated, melatonin can act through several mechanisms and at almost all levels of the organism. Therefore, it has multiple and diversified actions with immediate (endogenous melatonin, during the night) or prospective effects (exogenous melatonin, during the previous day).

 

Hypomelatoninemia is more common, and it can be due to factors that affect directly the pineal gland, innervation, melatonin synthesis as a result of congenital disease; or secondary as a consequence of environmental factors and/or medications (shift work, spinal cord cervical transection, sympathectomy, aging, neurodegenerative diseases, genetic diseases, β-blockers, calcium channel blockers, ACE inhibitors). Hypermelatoninemia is less common, and except for pharmacological effects, few conditions have been associated with high melatonin production: spontaneous hypothermia, hyperhidrosis syndrome, polycystic ovary syndrome, hypogonadotropic hypogonadism, anorexia nervosa, and Rabson-Mendenhall syndrome that induces pineal hyperplasia.

 

Melatonin During Puberty, Menstrual Cycle and Reproductive Function

 

Neuroendocrine control of sexual maturation is influenced by the pattern of melatonin secretion as a consequence of the light-dark cycle, and in some species the photoperiod via melatonin secretion determines the timing of puberty (51). In vitro studies, in cultured prepuberal rat pituitary gland, showed that melatonin inhibits gonadotrophin-releasing hormone and therefore luteinizing hormone release, providing evidence for a potential causal role of melatonin in the timing of developmental stages (52) (Figure 2). The mechanism by which melatonin inhibits GnRH secretion is not clear; recently, kisspeptin was suggested to mediate this effect. Lower melatonin levels have been associated with precocious puberty and higher levels in delayed puberty and hypothalamic amenorrhea compared to age-matched controls; however, a causal role of melatonin in pubertal development has not been described (53, 54). Data on circulating melatonin, and its variation during the menstrual cycle, are inconsistent (55). In males, high melatonin doses (100 mg daily) potentiate testosterone-induced LH suppression, and a negative correlation between nocturnal serum LH and melatonin have been reported (56, 57). Nevertheless, attempts to develop melatonin as a contraceptive pill in combination with progestin have not been successful (58). Interestingly, people living near the Arctic circle present lower conception rates during winter darkness, when melatonin levels are high, than in summer. In summary, the overall perception in human studies is that melatonin is inhibitory to human reproductive function.

 

Melatonin and Core Body Temperature

 

Melatonin plays a role in circadian thermoregulation. In particular, the melatonin peak is associated with the nadir in body temperature, together with maximum tiredness, lowest alertness and performance (59) (Figure 6). Exogenous administration of melatonin during the daytime reproduces this association, increasing fatigue and sleepiness and decreasing body temperature, especially if the subject is seated (posture dependent effect) (59). Along these lines, the rise in temperature during the ovulatory phase of the menstrual cycle is associated with a decline in the amplitude of melatonin.

 

Melatonin and Energy Metabolism and Glucose Homeostasis

 

Melatonin is an important player in the regulation of energy metabolism and glucose homeostasis. It is responsible for the daily distribution of energy metabolism functions (daily phase of high insulin sensitivity, glycogen synthesis, and lipogenesis and a sleep phase associated with the usage of stored energy) (60). Interestingly, administration of melatonin in post-menopausal women induced a reduction in fat mass and increase in lean mass compared to placebo (61). Nocturnal melatonin secretion facilitates diurnal insulin sensitivity and preservation of beta cell mass and function (62) and low melatonin secretion has independently been associated to a higher risk for type 2 diabetes (T2DM) (63). In short sleepers or when there is a misalignment between waking time and melatonin secretion (melatonin secretion is not interrupted), insulin resistance and hyperglycemia can be observed. Melatonin administration can result in iatrogenic insulin resistance and hyperglycemia in the morning, depending when it is administered and on the metabolizing characteristics of the subject. Moreover, recently, some variants of the gene encoding for the MT-1B have been associated with reduced beta-cell function and increased risk for T2DM (64). Several cardiovascular effects have also been attributed to melatonin, such as, antihypertensive properties, regulation of heart rate, and vascular resistance (65, 66).

 

Melatonin, Antioxidant Properties and Cancer

 

Antioxidant and anti-aging properties have also been attributed to melatonin. Melatonin acts as a potent free radical scavenger and antioxidant in vitro, independently of the presence of the receptor (38, 67, 68) protecting lipids, protein and DNA from oxidative damage. It is more effective than glutathione in reducing oxidative stress under many circumstances, being highly concentrated in the mitochondria. Although most of these effects in humans have been observed in supraphysiological doses of melatonin, the quantity of exogenous melatonin required to generate relevant antioxidant activity is not well established. Moreover, the clinical benefits of antioxidant supplements are not clear.  Quote “research has not shown antioxidant supplements to be beneficial in preventing diseases”, https://www.nccih.nih.gov/health/antioxidants-in-depth#:~:text=Antioxidants%20are%20man%2Dmade%20or,be%20beneficial%20in%20preventing%20diseases.

 

Also, there is growing recent evidence for anti-tumoral activity of melatonin (69, 70). Melatonin seems to be useful as an oncostatic agent at the cellular level and slows the progression of cancer (most of the studies are focused on breast and prostate cancer). Interestingly, in rats when a carcinogen is given at night during the highest levels of melatonin, DNA damage is significantly lower (20%) than in rats that receive a carcinogen exposure during the day (71). Pinealectomy stimulates cancer initiation or growth in animals, and pineal calcification, that leads to a decline in melatonin production, have been associated with an increase in pediatric primary brain tumors (72).

 

An ’Umbrella review’, whereby the results of numerous meta-analyses are combined, has provided important data on which of the numerous claims for melatonin therapeutic benefit stand up to scrutiny. A simplified version is provided here (see below).

 

As mentioned previously, exposure to light during the “biological night” can suppress melatonin production, and it is also associated to a deleterious effect on health (i.e., increased risk of cancer in most epidemiology studies in night shift workers) (70, 73). Night-shift workers present a higher incidence of hormone-dependent cancer which has been related to light-induced melatonin suppression which consequently increases estrogen production. A 50% increased risk to develop breast cancer in nurses exposed to rotating shift work has been documented (74). In contrast, a reduced risk for breast cancer was observed in blind women, with potentially higher levels of melatonin throughout all day (although there is no evidence that blind people produce more melatonin than sighted people) However, the mechanisms by which melatonin exerts any of oncostatic effects remains to be established.

 

Miscellaneous

 

The circadian annual rhythm of prolactin secretion depends on the circadian melatonin signal. Melatonin regulates pars tuberalis timer cells, and coordinates prolactin-secreting cells which together function as an intrapituitary pacemaker timer system. Interestingly, several clock genes are expressed within the pituitary with a circadian rhythm independent of the SCN (75).

 

Melatonin might act on the adrenal glands as an endogenous pacemaker. Glucocorticoids levels are low after the onset of darkness and rise after the middle of the night concomitantly with a decrease in melatonin levels. This is consistent with an inhibitory effect of melatonin through MT1 on glucocorticoid production. Interestingly, many antidepressant drugs increase the availability of the precursors (tryptophan and serotonin) and the major pineal neurotransmitter NE and therefore stimulate melatonin secretion. A melatonin agonist has been developed as an antidepressant through its actions on serotonin 2C receptors (76). If there is a link between this increase in melatonin production and the efficacy of antidepressant medications this needs further evaluation.

 

Several conditions (congenital or acquired) might induce a dysregulation of the melatonin synthesis/signaling and affect rhythmic melatonin production:

 

Pathological or traumatic sympathetic denervation (i.e., injury to the spinal cord) of the pineal gland or administration of β-adrenergic antagonists abolishes the rhythmic synthesis of melatonin and the light-dark control of its production (77, 78). NE is the major transmitter via β1 adrenoceptors with potentiation by α1 stimulation. NE levels are higher at night, approximately 180 degrees out of phase with the serotonin rhythm. Both availability of NE and serotonin are stimulatory for melatonin synthesis. However, several other factors have been related to the secretion and production of melatonin (13) (Table 1). In humans, administration of atenolol suppresses melatonin and enhances the magnitude of light-induced phase shift (79). In fact, several related observations suggest that endogenous melatonin acts to counter undesirable abrupt changes in phase. Pinealectomy i.e., abolishment of the melatonin rhythm, leads to a more rapid circadian adaptation to phase shift in rats (80).

 

Craniopharyngioma patients often display low melatonin secretion as a result of SCN impairment associated with alterations in sleep pattern (81).

 

Smith-Magenis syndrome (a congenital disorder due to a haplo-insufficiency of the retinoic acid-induced 1 gene, involved in the regulation of the expression of circadian genes) patients present with an inverted rhythmic melatonin secretion and sleep difficulties that can be managed with melatonin administration in the evening and β-adrenergic blockers during the day to reduce melatonin secretion (82). Low levels of melatonin have been consistently associated with autism spectrum disorders (ASD) (83). The last enzyme in the synthesis of melatonin, acetylserotonin-O-methyltransferase, has been associated with susceptibility for ASD which could be an explanation for low melatonin levels in ASD (84). Interestingly, administration of melatonin has shown to be efficacious for insomnia in children with ASD (85).

 

MELATONIN, CLINICAL APPLICATION AND THERAPEUTIC USE

 

Literature on the clinical use of melatonin is extensive and has increased exponentially over the last decade. Melatonin effects on sleep are the most well-known; however, the finding of increased cancer risk in shift workers and that patients with neurodegenerative diseases, autism or depression present abnormal melatonin rhythms, have recently increased attention on the role of melatonin.

 

Melatonin and Sleep

 

Numerous factors and comorbidities are associated with chronic insomnia in the adult population, different from the circadian sleep-wake rhythm disorders. Some of these risk factors and comorbidities involve psychiatric conditions (i.e. depression, anxiety, posttraumatic stress disorder), medical conditions (i.e. pulmonary diseases, chronic pain, heart failure, hyperthyroidism, nocturia, gastroesophageal reflux, cancer, pregnancy, pruritus, HIV infection, obstructive sleep apnea syndrome), neurological conditions (i.e. Alzheimer’s, Parkinson’s disease, peripheral neuropathies, strokes, brain tumors, headache syndromes), and medications (i.e. central nervous system stimulants or depressants, bronchodilators, antidepressants, diuretics, glucocorticoids, caffeine, alcohol). All these conditions should be ruled out before establishing the suspicion of a circadian sleep-wake rhythm disorder. The importance of melatonin levels for human sleep was apparently demonstrated by studies on pinealectomized subjects. These patients presented a disrupted 24h circadian rhythm, and a reduction of sleep time and quality, that were reversed after the administration of melatonin (86, 87). However, another careful, prospective study using polysomnography before and following pinealectomy found no effect on sleep and similar data are reported in rats (88-90). Thus, the question is not resolved. Clearly it is not a sleep hormone since in nocturnal animals it is secreted during the active periods. Overall, exogenous melatonin has been shown consistently to reduce sleep latency, and less consistently increase total sleep time, reduce night awakenings, and ultimately improve sleep quality (91). The most obvious action is to optimize sleep timing with respect to the circadian clock: we sleep better when melatonin production (and thus the circadian clock) and sleep are correctly aligned. Diagnostic criteria of every circadian sleep-wake rhythm disorder were fully described by the American Academy of Sleep Medicine (2014) (92).

 

CIRCADIAN SLEEP WAKE-RHYTHM DISORDERS EVALUATION

 

The origin of a circadian rhythm disorder can be related either to an intrinsic abnormality in the circadian system itself (misalignment of the intrinsic circadian timing with the desired sleep schedule) or to external factors, as when individuals must be awake at times that are not synchronized with their intrinsic rhythms. Overall, sleep disorders result in clinically significant symptoms of insomnia, daytime sleepiness and impaired physical, neurocognitive (concentration, processing speed, memory), emotional and social functioning, as well as impaired functioning at work or school (92). Age and associated comorbidities can help in the differential diagnoses. Symptoms across sleeping disorders are non-specific; the key to properly identify them is the recognition of abnormal sleep-wake patterns using sleep diaries beyond the complaint of insomnia or daytime sleepiness (93). Time domain is very important for melatonin actions (immediate or prospective effects, chronobiotic, or seasonal effects); therefore, adequate melatonin measurement and time of administration are crucial. Actigraphy can supplement self-reported sleep diary information or be useful in situations of neurodevelopmental disorders where a sleep diary cannot be completed.

 

Melatonin concentrations typically increase 90 to 120 minutes prior to the habitual bedtime if bright light (>10 lux) is absent. Preferentially frequent blood sampling every 30 to 60 minutes from six hours prior to and one hour following habitual bedtime is collected to assess the individual’s dim light melatonin onset (DLMO) and circadian phase. Samples should be collected in a dark environment (red light <10 lux) without interfering with sleep. In research studies a DLMO protocol is used as a valid and reliable indicator of circadian phase position, according to the time at which melatonin levels rise, and is extremely helpful to guide the timing for melatonin therapy in each individual; however, this is rarely feasible for routine use in clinical settings (94). Alternatively, urinary excretion of 6-sulfatoxymelatonin is a good index of nocturnal melatonin production and it should be collected as sequential urine samples for 48h in order to calculate the timing of the peak or acrophase. Also, it is possible to measure melatonin in saliva, during the evening before sleep and this approach has been frequently used. But, in the case of for example very delayed melatonin rise the time of onset can be missed during sleep. Importantly, melatonin values vary individually and according to age and sex. Although, endogenous melatonin production is closely related with the onset and offset of sleep, few associations have been found between melatonin production and sleep stages (95), and sleep deprivation does not abolish the melatonin rhythm.

 

CIRCADIAN SLEEP WAKE-RHYTHM DISORDERS

 

The primary goal of treatment of circadian sleep-wake rhythm disorders is to realign the circadian timing of sleep and wake with the required sleep-wake period. Appropriately timed and dosed melatonin, melatonin receptor agonists, and light therapy seem to be useful approaches (96, 97). Following the melatonin PRC and taking the individual DLMO as the reference phase to decide the most suitable time of melatonin administration, according to the desirable effect, seem the most appropriate guide to decide when to start chronic melatonin therapy (98-100).

                                             

Delayed and advanced sleep-wake phase disorders, non-24 hour, and irregular sleep-wake rhythm disorders are considered intrinsic circadian disorders; in contrast, jet lag and shift work disorders are due to an environmentally imposed misalignment. The most potent phase shifting factor (zeitgeber) is the environmental light-dark cycle (101).

 

Light exposure during the last hours of the usual sleep period moves the circadian rhythm forward (phase advanced). On the contrary, light exposure in the evening and the first half of the usual sleep period moves the circadian rhythm back (phase delayed) (Figure 7) (96). Advanced sleep-wake phase disorder is partially due to the physiologic advance that occurs with aging together with the weakening of circadian rhythms observed with aging. It can be genetically determined in some families as well. Delayed sleep-wake phase disorder is more often seen in children with some neurodevelopmental disorders; these patients cannot sleep during the dark time and delay sleep onset until the early hours of the morning, sleeping much of the day. In these situations both bright light in the early morning and evening melatonin 5 hours before endogenous melatonin onset secretion (0.5-5 mg) has been shown to advance sleep time significantly (102), the magnitude of the advanced shift being dose-dependent. In contrast early “biological morning” administration of melatonin induces delayed shifts. The non-24-hour sleep-wake rhythm disorder is most commonly seen in blindness, since the light-dark cycle is the most powerful cue for synchronizing the hypothalamic pacemaker to the 24-hour day (103). Therefore, this disorder is characterized by a failure to maintain stable alignment to the 24-h day, and a “free-running” circadian rhythm system which usually shifts to a later and later phase position. Timed melatonin treatment has been successful in entraining free-running blind patients.

 

The irregular sleep-wake rhythm disorder is seen in patients with dementia, which a neurodegenerative process might induce a disruption of the circadian system with a consequent loss of modulatory influence on sleep and wakefulness (104).

 

 

Jet lag occurs when crossing time zones, and one needs to sleep and be awake at times that are not aligned with one’s own circadian system. It is more severe when more time zones are crossed and if the direction of the travel is eastbound, as it is more difficult to advance than delay the natural circadian cycle. Melatonin accelerates the phase shift if given at the appropriate time prior to bedtime at destination, and the benefits of melatonin administration in the alleviation of jet lag seems to be greater with larger numbers of time zones (105). If melatonin is appropriately time-administered, self-rated jet lag can be reduced by 50 percent (106). The maximum advance shift obtainable with a single treatment of oral melatonin (3-5 mg) is approximately 1-1.5 h.

 

For night shift workers wakefulness is required at the time that melatonin secretion is rising, and alertness is dissipating, and the opposite for the next day when melatonin secretion decreases, and circadian rhythms promote alertness. While some studies suggested that the use of melatonin improved sleep and increase daytime alertness in night shift workers compared to placebo (107), other studies have not shown beneficial effects (108). More data are needed before recommendations about melatonin as a therapy for shift workers can be made. However anecdotal evidence suggests that it is widely used for daytime sleep.

 

 

Melatonin and the CNS

 

Individuals with neurodegenerative disorders (i.e., Alzheimer’s and Parkinson’s disease, Huntington’s disease, autism) had significant flattened and attenuated melatonin rhythms compared to age-matched controls (37, 109). Melatonin showed efficacy in managing the insomnia in elderly people or improving cognitive function associated with neurodegenerative disorders (110). It has reported effects on anxiety, cognitive function and memory. Knowing the wide distribution of melatonin receptors in the CNS, melatonin seems one of the promising neuroprotective agents to be tested in humans. However other studies have found deleterious effects of melatonin in elderly demented patients (111).

 

Melatonin: Therapeutic Use

 

Almost 200 randomized clinical trials on the use of melatonin and evaluation of clinical effects have been published. Moreover, several patents have been registered in relation to the therapeutic applications of melatonin and melatonin analogues (sleep disorders, neuroprotection, cancer). The American Academy of Sleep Science recommends the use of melatonin for jet lag, delayed sleep phase syndrome, and non-24h sleep wake disorder syndrome seen in blind people; however, there were few consensuses acknowledging its therapeutic benefits (112) until an ‘Umbrella’ review of meta-analyses identified consistent therapeutic targets (91). Interestingly, at least in animal models, appropriate melatonin dose administration can reverse most of the effects after a pinealectomy.

 

Table 2. Melatonin’s Reported Significant Clinical Effects

• Breast cancer, risk of death down at 1 year • Nocturnal hypertension, systolic and diastolic reduced • Sleep latency shortened • Sleep duration increased • Melatonin onset advanced • Core body temperature decreased • In rodents, protective effects of melatonin in ischemic stroke

Combined meta-analyses of studies meeting strict criteria (simplified from Posadzki PP et al. BMC Med (2018) 16: 18.)

 

Several melatonin receptor agonists have been synthesized, some of them have higher affinity for the receptor than endogenous melatonin. Agomelatine (activity at the serotonin-2C receptor) functions as an anti-depressant, ramelteon (selective MT1/MT2 agonist) is marketed for use in sleep onset insomnia, tasimelteon (MT1/MT2 agonist), is commercialized for the treatment of circadian rhythm disorders particularly non-24h sleep wake disorder (113, 114). Also, slow-release formulations of the natural biologic melatonin have been developed.

 

The time of melatonin administration is critical, especially in chronic treatments, and the melatonin profile shows large interindividual variation; however, the profile of an individual is highly reproducible from day to day. Also, absorption, metabolism and excretion of melatonin vary between individuals and should be considered to get the desired clinical efficacy of the therapy. Ideally, although not feasible in a daily clinical practice, DLMO should be determined for each individual, and used as a timing reference for the prescription of melatonin. Alternatively, the time each individual goes to sleep could determine the time of administration of melatonin; it is advisable to take oral melatonin around 45 minutes to 1 hour before the usual bedtime (time to reach maximal plasma concentrations following oral immediate-release formulations). Moreover, the duration of the pharmacological profile should last until the usual wake time of the patient; thus, the type of pharmaceutical formulation (slow or fast release) is also an important consideration. However, there is little evidence for greater benefit of slow-release preparations. Route of administration, as well as age, liver function and potential drug interactions (since melatonin is metabolized in the liver), may influence plasma melatonin levels and should be taken into account (115). Sensitivities and pharmacokinetics of melatonin vary between individuals, and a lower dose of 0.3-0.5 mg might be more effective than higher doses in many subjects.

 

There is no general consensus regarding dosage. A wide range of dose formulations are available, and the usage varies depending on the clinical application. The usual advice is to start with the lowest dose available. Low doses 0.1 to 0.3 mg/d that produce near physiological melatonin concentrations can be used for central clock synchronization; doses ranging from 0.6 to 5 mg/d for sleep disorders, or doses as high as 300 mg/d for neurodegenerative disorders (amyotrophic lateral sclerosis) (116, 117). There is a current tendency to recommend high pharmacological doses for ‘protective’ or antioxidant effects. What consequences these might have for the circadian aspects of melatonin function is not known. It should not be forgotten that melatonin has profound effects on the reproductive function of photoperiodic seasonal breeders and there is good evidence for residual photoperiodicity in humans. However in most clinical trials or studies using melatonin, it is well accepted that in general melatonin lacks toxic adverse events, and it is a safe drug at most of the usual tested doses from 0.5 to 5 mg/d (118). Whether dosage should be changed in chronic melatonin treatment according to the annual season requires further investigation; further studies are needed on the undesirable consequences of melatonin suppression in the long term (for example by beta blocking drugs or shiftwork).

 

To summarize, benefits of melatonin and its analogues on circadian sleep disorders are consistently reported but for more generalized “insomnia” are often of low strength. The dose must be timed and individually adjusted as optimization will vary among individuals. More studies are required to develop treatment guidelines for different conditions. The long-term consequences of taking high dose melatonin especially in pediatrics need careful evaluation. Melatonin mechanisms of action and effects need much further work, to better understand the therapeutic value of melatonin and its potential applications.

 

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ABSTRACT

 

Pituitary adenomas comprise approximately 10-20% of intracranial tumors. Non-functioning pituitary adenomas (NFPAs) are benign adenohypophyseal tumors not associated with clinical evidence of hormonal hypersecretion. NFPAs comprise different histological subtypes, classified according to their immunostaining to different adenohypophyseal hormones and transcription factors. The silent gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (POU1F1) gene lineage, and null cell tumors. Patients with NFPAs usually come to medical attention as a result of “mass effects” symptoms such as headaches, visual disorders, and/or cranial nerve dysfunction caused by lesions large enough to damage surrounding structures. Hypopituitarism, caused by the compression of the normal anterior pituitary, and hyperprolactinemia due to pituitary stalk deviation can also be present. Some cases may be diagnosed incidentally through imaging studies performed for other purposes. Patients with NFPAs should undergo hormonal, clinical, and laboratory evaluation to rule out hyper and hypopituitarism. Assessment of prolactin and IGF-1 levels have been recommended in all patients whereas screening for cortisol excess is suggested in the presence of clinical symptoms. Impairment of pituitary function should be assessed by baseline hormonal measurements and/or stimulatory tests, if needed. Patients in whom the tumor abuts the optic chiasm should be submitted to visual field perimetry. Surgical resection is the primary treatment for symptomatic patients with NFPAs, i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Visual deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach. For patients with residual NFPAs following transsphenoidal surgery, a therapeutic attempt using cabergoline can be made according to clinical judgment in individual cases. Radiotherapy in the postoperative period is not consensual and is generally reserved for cases of tumors not completely resected by surgery, those cases that present progressive tumor growth during follow-up, or for patients who, at diagnosis, already have tumors with aggressive features. Highly aggressive tumors need special care during follow-up, including temozolomide with or without radiotherapy complementation or new potential emerging treatments. For patients with asymptomatic NFPAs a “watch and wait” option is reasonable. Follow up is individualized and should consider tumor size, prior treatments, and clinical symptoms.

 

INTRODUCTION

 

Pituitary adenomas are common, predominantly indolent neoplasms comprising approximately 10-20% of intracranial tumors (1,2). Non-functioning pituitary adenomas (NFPAs) are benign neoplasms that originate from the adenohypophyseal cells and are not associated with clinical evidence of hormonal hypersecretion (3). They comprise a large and heterogeneous group, representing a sizeable proportion (22% to 54% in different series) of all pituitary adenomas (4-7). Malignant transformation in a non-functioning pituitary adenoma (pituitary carcinoma) is extremely rare and is characterized by craniospinal or systemic dissemination (8).

 

NFPAs can be further classified according to their pituitary hormone and transcription factor profile, as defined by the 2017 World Health Organization (WHO) classification for endocrine tumors (9). Tumors that express one or more anterior pituitary hormones or their transcription factors with immunohistochemistry (IHC) but do not secrete hormones at a clinically relevant level can be referred to as silent pituitary adenomas (SPAs) (10,11). As a consequence, the definition of “null cell adenoma” is now limited to an exceptionally rare primary adenohypophyseal tumor that shows immunonegativity for all adenohypophyseal hormones as well as for cell-type specific transcription factors (12) - Figure 1.

The term “totally silent” has been proposed to be used when a patient with an NFPA presents basal and stimulated serum concentrations of the correspondent hormones within the normal range and there are no clinical signs or symptoms that can be attributed to hormone excess (13). The term “clinically silent” may be used when NFPAs secrete hormonal products that cause an elevation of the serum concentration but do not result in clinical signs or symptoms of hormonal hypersecretion (13). Some cases are referred to as “whispering” adenomas with borderline, mild, often overlooked, clinical symptoms and signs (14,15).

 

EPIDEMIOLOGY

 

The prevalence of NFPAs is variable and is often based upon autopsy or magnetic resonance imaging (MRI) series. Data from Europe, North and South America have estimated that the prevalence of clinically relevant NFPAs is 7–41.3 cases per 100,000 of population (16). This is likely an underestimate of the true prevalence, as many NFPAs go undiagnosed until they are large enough to cause mass effect or are accidentally discovered. Data are discordant about gender predominance and the peak occurrence is from the fourth to the eighth decade. A recent population-based study from South Korea showed an annual incidence of 3.5 cases/ 100,000 population for NFPAs, which is noticeably higher than the annual incidences previously reported in other countries, such as Sweden, Finland, and Argentina (17). These differences may be related to genetic and environmental factors in Asian populations or inter-study heterogeneity and may also reflect the good local access to diagnostic evaluations using magnetic resonance imaging and computed tomography.

 

CLINICAL PRESENTATION

 

The absence of clinical manifestations of hormonal hypersecretion usually results in significant diagnostic delay and therefore NFPAs may not be diagnosed until they cause mass effects to surrounding structures (3), causing symptoms such as headaches, visual disorders, and/or cranial nerve dysfunction. Other manifestations are hormone deficiencies or hyperprolactinemia due to pituitary stalk deviation and, less frequently, pituitary apoplexy (18,19). Additionally, some cases may be diagnosed incidentally through imaging studies performed for other purposes, the so-called pituitary incidentaloma.

 

Neurologic Manifestations

 

VISUAL IMPAIRMENT 

 

Impaired vision, caused by suprasellar extension of the adenoma that compresses the optic chiasm, is the most common neuro-ophthalmological symptom (19). Different types of visual defects depend on the degree and site of optic nerve compression. Both eyes are usually affected, although a significant proportion of patients may have unilateral or altitudinal problems in 33 and 16% of the cases, respectively (20). Diplopia, induced by oculomotor nerve compression resulting from parasellar expansion of the adenoma may occur, and the fourth, fifth and sixth cranial nerves may also be occasionally involved when there is parasellar expansion (16). Nevertheless, the typical visual field defect associated with pituitary tumors is bitemporal hemianopia, reported in approximately 40% of the patients.

 

HEADACHE

 

Headaches, the second most common neurologic symptom, occur in 19-75% of patients with pituitary tumors, regardless of size (21). In a retrospective case series of incidentally-discovered NFPAs, headache was present in approximately 20% of the cases (22). In a recent prospective series, 138 out of 269 patients with NFPAs complained of tumor-related symptoms and, among them, 23% presented with headaches (23). Although it is not always clear whether the presenting headache is related to the tumor, proposed mechanisms for headache include increased intrasellar pressure, stretching of dural membrane pain receptors, and activation of trigeminal pain pathways (16,24). Cerebrospinal fluid (CSF) rhinorrhea, associated or not with headache, can occur in cases where the tumor causes erosion of the sellar floor and extends inferiorly to the sphenoid sinus.

 

PITUITARY APOPLEXY

 

Pituitary apoplexy (sudden hemorrhage into a pituitary adenoma) is rare. According to a retrospective case series of 485 Mexican patients with NFPAs, pituitary apoplexy was the initial presentation in 8% of the cases (25). It causes acute onset of a severe headache associated with visual disturbances and can occur in all types of pituitary tumors, although some series suggest pituitary apoplexy might be more common in NFPAs than other adenomas subtypes (26). It has been suggested that the combination of the high metabolism of pituitary adenomas combined with their special blood supply would make them more prone to vascular events (27). Pituitary apoplexy may occur without an identified risk factor, but it has also been reported as being related to pregnancy, use of anticoagulants, surgical procedures, as well as in association with dynamic tests, such as thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), and insulin-tolerance stimulation tests (28).

 

The actual long-term risk of apoplexy in NFPAs has not been clearly defined (29). In a Japanese prospective cohort study of 42 asymptomatic patients with NFPAs (mean initial tumor size of 18.3 mm) followed-up for approximately four years, pituitary apoplexy was reported in 9.5% of the cases (30). A systematic review and meta-analysis evaluating the outcomes of patients with NFPAs and pituitary incidentalomas who were treated conservatively and followed-up for mean period of 3.9 years showed that the development of pituitary apoplexy was rare (0.2/100 patients-year), although a trend for a greater incidence of pituitary apoplexy was seen in macroadenomas compared with microadenomas, with a reported incidence of apoplexy of 1.1% per year (31). These findings are in line with two recent retrospective studies including a Korean series of 197 patients with NFPAs without mass effect symptoms or pituitary apoplexy at baseline who were followed-up for a median of 37 months (32) and an Italian series of 296 patients with incidentally discovered NFPAs (macroadenomas in 49% of the cases) who we followed-up for approximately three years (33). Five out of 169 patients with macroadenomas developed pituitary apoplexy with an overall incidence of 0.83 per 100 patients-years in the Korean series while no cases of pituitary apoplexy were reported in the Italian series.

 

Endocrine Manifestations

 

HORMONAL DEFICIENCIES

 

Most patients with nonfunctioning pituitary macroadenomas present with deficiency of at least one pituitary hormone resulting from the compression of the normal anterior pituitary and/or pituitary stalk, preventing the stimulation of pituitary cells by hypothalamic factors. Hypogonadism can result from either a direct compressive effect on gonadotropic cells or by stalk compression-induced hyperprolactinemia that inhibits the pulsatile secretion of gonadotropin releasing hormone via interfering with hypothalamic kisspeptin-secreting cells (34). This “disconnection hyperprolactinemia” usually <2000mIU/L (95 ng/mL) (35) is characterized by compression of the pituitary stalk, which prevents the arrival of dopamine to the anterior pituitary, the main inhibitor of prolactin (stalk effect). GH and gonadotroph axes are most commonly affected, followed by adrenal insufficiency and central hypothyroidism (16,23,32).

 

HORMONAL EXCESS

 

Gonadotroph adenomas are usually considered to be "nonfunctioning" although they can secrete intact gonadotropins, as they do not generally result in a clinical syndrome. Occasionally, gonadotroph adenomas secrete primarily FSH but also LH in quantities high enough to raise serum gonadotropin levels, which in turn, may lead to the development of some specific symptoms, such as ovarian hyperstimulation in young women (36-38) or, more rarely, precocious puberty or testicular enlargement in men. In addition, low serum LH:FSH ratios (usually < 1.0) have been described in clinically-secreting gonadotroph adenomas (39). Measurement of α-subunit may also contribute to a pre-operative diagnosis in clinically silent but biochemically-secreting NFPAs, as it may be the sole biochemical marker of the gonadotroph subtype in a number of cases. In addition, circulating FSH, LH and α-subunit levels can help the post-operative surveillance of these patients (39).

 

HISTOPATHOLOGICAL CLASSIFICATION

 

The 2017 WHO classification for endocrine tumors (9) defines pituitary adenomas, including NFPAs, according to their pituitary hormone and transcription factor profile. It is noteworthy that in this classification system, IHC forms the basis of the new categorization, in which an adenohypophyseal cell lineage designation of the pituitary adenoma has replaced the concept of a “hormone-producing pituitary adenoma” (40) -Table 1.

 

Table 1 – Classification of Silent Pituitary Adenomas According to Adenohypophyseal Hormones and Transcription Factors.
Cell Lineage	Hormone Staining	Transcription Factors and Other Co-Factors
Somatotroph adenoma      Sparsely granulated      Densely granulated	GH, α-subunit Weak and patchy Diffuse and strong	PIT-1
Lactotroph adenomas      Sparsely granulated      Densely granulated      Acidophil stem-cell adenoma	PRL Perinuclear Diffuse PRL Focal and variable PRL, GH	PIT-1, ERα
Thyrotroph adenomas	TSHβ, α-subunit	PIT-1, GATA2
Corticotroph adenomas     Densely granulated (type I)     Sparsely granulated (type II)     Crooke-cell	ACTH Diffuse and strong ACTH Weak and patchy ACTH Periphery (ring-like)	TPIT
Gonadotroph adenomas	FSHβ, LHβ, α-subunit	SF1, GATA2, ERα
Null cell adenomas	None	None
Plurihormonal      PIT-1-positive adenomas      Adenomas with unusual IHC combinations	GH, PRL, TSHβ±, α-subunit Various combinations	PIT-1

Adapted from Mete et al, 2017.

 

Indeed, the prevalence of the different histological subtypes of NFPAs is dependent on the extent of the IHC profile. According to a large retrospective case series (11), the gonadotroph adenoma is the most common subtype, followed by corticotroph, PIT1 (GH/ Prolactin/TSH) lineage and null cell - Figure 2.

 

Pituitary hormones gene expression analysis using real-time quantitative PCR (RT-qPCR) has been shown to complement the IHC classification of pituitary tumors, according to a large observational, cross-sectional study evaluating 268 patients (41). Addition of RT-qPCR analysis reduced the proportion of null-cell subtype in this series from 36% to 19%. Lower specific adenohypophyseal hormone gene expression was observed in some SPA variants when compared with their secreting counterparts, including somatotroph, lactotroph, mixed somatotroph-lactotroph and plurihormonal adenomas, which could contribute to the absence of endocrine manifestations.

 

The European Pituitary Pathology Group has recently proposed a standardized report for the diagnosis of pituitary adenomas which includes a multi-step approach, comprising the summary of clinical and neuroimaging features, IHC for hormones and transcription factors, assessment of proliferation and, when indicated, the use of markers predictive of treatment response (42). This proposition highlights the role of the pituitary pathologist as part of a multidisciplinary pituitary team helping the clinician define the most appropriate post-operative strategy for each patient, including appropriate follow-up and early recognition and treatment of potentially aggressive tumors (43).

 

Subtypes of NFPAs

 

NULL CELL ADENOMAS AND SILENT GONADOTROPH ADENOMAS

 

Null cell adenomas and silent gonadotroph adenomas (SGAs) were previously misunderstood to be the same type of tumor. The addition of immunostaining for steroidogenic factor 1 (SF1) as a tool in the diagnosis of pituitary lesions has shown that many LH/FSH immunonegative adenomas are in fact SGAs (44), which comprise almost 80% of resected NFPAs. The distinction between SGAs and null cell adenomas is of clinical relevance because true null cell adenomas are rare and likely to be more invasive and aggressive than SGAs (45). In addition, these rare cases of ‘null cell adenomas’ may require a broad panel of immunohistochemistry in order to exclude a sellar metastasis of a neuroendocrine tumor (NET) from another organ. Several markers, including TTF1, serotonin, ATRX, DAXX and CDX2 may be useful in the differential diagnosis between an NFPA and a metastatic NET in the sellar regionData from a retrospective case series of 516 patients with NFPAs have shown that from the 23% of the tumors initially classified as null cell adenomas by using only classical pituitary hormone IHC, only 5% of them remained as true null cell type. Indeed, immunostaining using lineage-specific markers, namely, PIT-1 (coded by the POU class 1 homeobox 1 (POU1F1) gene), SF1, TPIT (coded by the T-Box transcription factor 19 (TBX19) gene) and estrogen receptor-α (ERα) provided tumor reclassification in 95% of cases (11).

 

SILENT CORTICOTROPH ADENOMAS

 

Silent corticotroph adenomas (SCAs) are characterized by the absence of clinical features of Cushing syndrome, along with normal circadian cortisol secretion (totally silent) or elevated ACTH (clinically silent) (5,47-49). They currently account for approximately 15% of NFPAs, an underestimated proportion, since IHC for the transcription factor TPIT, a marker of corticotroph differentiation which regulates the proopiomelanocortin (POMC) lineage giving origin to the corticotrophs, is still not widely available (50). For instance, in a recent retrospective series, inclusion of IHC for TPIT allowed identification of eight SCAs out of 18 (44%) pituitary tumors previously classified as null cell adenomas (51).

 

SCAs often present themselves as macroadenomas associated with mass-related symptoms. In comparison with SGAs, SCAs show female preponderance, are more frequently giant adenomas, and are more often associated with marked cavernous sinus invasion (49). Importantly, the presence of multiple microcysts in T2-weighted pituitary MRI sequences in a NFPA has a high specificity (> 90%) for the corticotroph subtype. The first study showed that multiple microcysts were present in 76% (13/17) of SCAs as opposed to 5% (3/60) of SGAs (52) while a more recent study showed comparable results, as multiple microcysts were present in 58% (7/12) SCAs but in only 9.5% of SGAs (53).Histologically, SCAs can be further divided into type 1 (densely granulated), type 2 (sparsely granulated) and Crooke-cell adenoma. Type 1 SCAs show strong ACTH immunoreactivity whereas type 2 SCAs resemble the rare chromophobe corticotroph adenoma and show weak and focal ACTH immunoreactivity. Type 2 SCAs seem to be more common and are likely to display higher expression of migration and proliferation factors compared with type 1 SCAs (5,11,40). Clinically silent Crooke-cell adenoma is a rare yet highly aggressive subtype as it carries significant risk of morbidity (54). Since SCAs are nonfunctional, an important point to note is the absence of Crooke’s hyalinization in the normal surrounding pituitary gland, as there is no exposure to high circulating glucocorticoid levels to promote hyaline deposits of cytokeratin filaments in the cytoplasm of normal corticotrophs (49).

 

The transformation of a silent corticotroph tumor into Cushing disease has been described, although the mechanism involved in this phenomenon is not yet well understood (55,56). It has been proposed that the clinical manifestations of Cushing disease are dependent on the processing of the pro-hormone POMC in corticotrophs. The pro-hormone convertase 1/3 (PC1/3) is involved in the post-translational processing of POMC into mature and biologically active ACTH. SCAs show a decrease in PC1/3 expression associated with a down-regulation of PC1/3 genes compared with corticotroph adenomas associated with Cushing disease (47). An attractive and more likely hypothesis involves epigenetic mechanisms: DNA hypermethylation of regulatory regions could lead to reduced expression of POMCtranscripts impairing the production of secreted ACTH. Differences in methylation status were observed when comparing ACTH-secreting pituitary tumors and SCAs: the second promoter was highly methylated in SCAs, partially demethylated in normal pituitary tissue and highly demethylated in pituitary and ectopic ACTH-secreting tumors (57).

 

SILENT SOMATOTROPH ADENOMAS

 

Silent somatotroph adenomas are PIT1 and GH-immunoreactive tumors without clinical and biological signs of acromegaly. They represent approximately 2-4% of all pituitary adenomas in surgical case series (58). Patients with silent somatotroph adenomas usually present with normal pre-operative GH and IGF-1 levels but there have been few reports of "clinically silent" cases, with non-suppressible serum GH and elevated IGF-1 levels (59).

 

Similar to secreting somatotroph adenomas, silent somatotroph adenomas are classified into densely granulated and sparsely granulated types, based on the presence and pattern of the low molecular weight cytokeratin staining. More than 50% of silent somatotroph adenoma cases constitute mixed GH–PRL adenomas, a proportion twofold higher than somatotroph adenomas causing acromegaly (60). Unlike clinically functioning somatotroph tumors, the silent ones are more frequently sparsely granulated with more aggressive behavior and a lower response to somatostatin analog therapy (61). Furthermore, silent somatotroph adenomas are more frequent in females, present at a younger age, are larger, more invasive, and recur earlier and more frequently than their secreting counterparts (62).

 

SILENT THYROTROPH ADENOMAS

 

Silent thyrotroph adenomas usually present TSHβ, α-subunit, and PIT-1 expression on IHC in a variable manner (40). They are more frequent when compared with their functioning counterparts and seem to behave similarly regarding treatment outcomes and recurrence rates (63). In a recent retrospective series of 20 patients with silent thyrotroph adenomas who underwent transsphenoidal surgery, 95% were macroadenomas and 85% showed extrasellar growth. Gross total tumor resection was achieved in 45% while major tumor progression or recurrence occurred in 10% of the patients over a median follow-up period of 18.5 months (64). These results show that, although silent thyrotroph adenomas tend to be large and invasive tumors, good overall outcomes with low complication rates can be achieved.

 

SILENT LACTOTROPH ADENOMAS

 

Clinically silent lactotroph adenomas are rare. The positive prolactin staining by IHC with no clinical signs of hyperprolactinemia is usually encountered concomitantly with

GH positive staining (silent mixed somatotroph-lactotroph adenoma) (61). They can also express ERα on immunostaining (9).

 

PLURIHORMONAL ADENOMAS

 

According to the WHO 2017 classification, plurihormonal adenomas can be classified on the basis of their transcription factor expression into two groups: 1) “PIT1-positive adenomas” (previously known as silent subtype 3 pituitary adenoma); and 2) plurihormonal with more than one transcription factor, termed “plurihormonal adenomas with unusual immunohistochemical combinations” (PAWUC) (9,40). In a recent large retrospective series comprising 665 patients who underwent endoscopic endonasal transsphenoidal surgery, plurihormonal adenomas were identified in 4% of the cases (18 patients had PAWUC and 9 patients were diagnosed with PIT-1 positive adenomas); 24 (89%) were macroadenomas (including 6 giant adenomas) and cavernous sinus invasion was found in 12 patients (44%) (65).

 

PIT1-positive plurihormonal adenomas are a distinct entity, with reportedly aggressive behavior. A single-center retrospective case series reported a prevalence of 0.9% for ‘silent adenomas type 3’ among resected pituitary tumors over a period of 13 years. All tumors were macroadenomas, aggressive, invasive, with a high rate of persistent/recurrent disease (66). Interestingly, these tumors may present clinical symptoms of hormone excess, such as acromegaly, hyperthyroidism or marked hyperprolactinemia (67) and the diagnosis of a PIT-1 positive plurihormonal adenoma should be kept in mind in case of large and invasive NFPA tumors in young patients, particularly those showing TSH and often minor reactivities for PRL and GH as well as cytologic atypia in conjunction with an elevated Ki-67 labelling index (66).

 

PAWUC are also characterized by aggressive behavior and higher rates of cavernous sinus invasion. A recent single-center retrospective series comparing clinical characteristics, outcome parameters and rate of invasiveness of 22 PAWUC to 51 SGAs showed that patients with PAWUC were younger, were more likely to present with intraoperatively signs of tumor invasion and less likely to achieve gross-total resection than patients with SGAs, suggesting a more aggressive behavior of NFPAs if additional transcription factors other than SF1, GATA2/3 and ER α are expressed within the tumor cells (68).

 

EVALUATION

 

According to current practice guidelines on incidentally discovered sellar masses, all patients, including those without symptoms, should undergo hormonal, clinical, and laboratory evaluation for hyper and hypopituitarism (69). The extent of the evaluation is still debatable and has commonly relied on clinical experience - assessment of prolactin and IGF-1 levels have been recommended, whereas screening for cortisol excess (i.e., overnight 1mg dexamethasone and/or late-evening salivary cortisol and/or urinary-free cortisol) is suggested in the presence of clinical symptoms, while measurement of ACTH levels is not routinely recommended. (69).

 

Patients with macroadenoma, especially those >3 cm and with normal or slightly elevated prolactin, must have their serum prolactin diluted in order to exclude the "hook effect" (70,71). This effect occurs when excessively high levels of this hormone interfere with the formation of the complex antibody-antigen-antibody sandwich, and a prolactinoma might be mistaken for a NFPA (72).

 

It is also suggested to check for pituitary hormone deficiencies in patients with non-functioning tumors, irrespective of symptoms. Macroadenomas can cause impairment of pituitary function by the involvement of the normal gland or pituitary stalk compression, and the risk of hypopituitarism is directly related to tumor volume. Microadenomas eventually lead to pituitary dysfunction, particularly if larger than 5mm (32,69). A large retrospective cohort of 218 patients with NFPAs found at least one pituitary deficiency at diagnosis in 33.3% of microadenomas (73) although other studies have failed to identify hormonal deficiencies in microadenomas. An interesting and cost-effective approach for microincidentalomas could be no hormonal evaluation for cystic and solid lesions smaller than 5 mm. For solid microadenomas (between 6-9 mm), it is suggested a simple investigation with measurements of PRL and IGF1 (74).

 

Concerning macroadenomas, current guidelines suggest that serum cortisol levels at 8-9 AM should be done as the first-line test for diagnosing central adrenal insufficiency (75). Despite well recognized limitations of most commercial cortisol immunoassays (76), baseline cortisol levels <3 µg/dL (83 nmol/L) are suggestive of central adrenal insufficiency, while baseline cortisol levels >15 µg/dL (414 nmol/L) likely exclude it, and levels between 3 and 15 µg/dL require a corticotropin/insulin stimulation test. Central hypothyroidism is assessed by measuring serum TSH and free T4 (fT4), and fT4 level below the laboratory reference range in conjunction with a low, normal, or mildly elevated TSH in the setting of pituitary disease usually confirms the diagnosis (75). Diagnosing GH deficiency (GHD) is relatively straightforward in patients with IGF1 concentration lower than the gender- and age-specific lower limit of normal and structural pituitary disease. Low IGF1 in patients with non-functioning tumors who have at least three pituitary hormone deficiencies is also suggestive of GHD (29). Nevertheless, some adults with suspected GHD may have normal IGF1, in such cases GH stimulation testing may be useful. Central hypogonadism in males, manifests with low serum testosterone, low or inappropriately normal FSH/LH and features of testosterone deficiency. For females, pre-menopausal women with oligomenorrhea or amenorrhea should be screened with serum E2, FSH, and LH. In postmenopausal women, if the patient is not in hormonal therapy, the absence of high serum FSH and LH is sufficient for a diagnosis of central hypogonadism (75).

 

Sellar MRI with gadolinium is the best imaging study for suspected adenomas because it provides images of high resolution of the mass as well as its relationship with surrounding structures. Pituitary adenomas usually appear hypo or isointense compared to normal pituitary tissue in T1 images on MRI. In addition, while the normal pituitary tissue enhances earlier with contrast, pituitary adenomas commonly present with delayed contrast uptake (77). Patients whose tumor abuts the optic chiasm should have visual field perimetry, preferably by the Goldmann method, to assess for visual deficits (69).

 

Functional (FDG- and/or SSTR- positron emission tomography (PET) imaging studies should only be considered in aggressive pituitary adenomas/carcinomas in the setting of site-specific symptoms (neck/back pain or neurological complaints), and/or where laboratory measures are discordant with known visible extent of disease (78). Recently, 37 patients diagnosed with NFPAs enrolled in a clinical trial underwent ⁶⁸Ga-DOTATATE PET/computed tomography (CT) of the head. ⁶⁸Ga-DOTATATE uptake was positive in 34/37 patients (92%), demonstrating in vivo SSTR expression in NFPAs and ultimately showing superior sensitivity of PET imaging when compared with ¹¹¹In-DTPA-octreotide scintigraphy (79).

 

The pre-operative differential diagnosis of NFPAs includes several additional primary non-hormonal-secreting lesions (80). This distinction is challenging in many cases because the clinical presentations and radiological aspects may be similar between adenomas and other pituitary lesions. The presence of diabetes insipidus (DI) is common in tumors of non-pituitary origin and indicates that the sellar mass is most likely not a pituitary adenoma (81). An increase in α subunit of the glycoprotein hormones (elevated in 30% of NFPA patients) can help in the differential diagnosis although normal values do not rule them out (82).

 

TREATMENT

                                   

In spite of current knowledge of definite NFPAs pathologic subtypes, initial treatment strategies are similar regardless of the subset. A small number of studies have reported treatment outcome results taking into consideration adenohypophyseal hormone immuno-profile (82-84) and, for the future, precise pathologic characterization utilizing adenohypophyseal hormones and transcription factors may potentially aid in predicting the response to specific adjunctive therapies.

 

Surgery

 

Surgical resection is the primary treatment for symptomatic patients with NFPAs (85), i.e., those with neuro-ophthalmologic complaints and/or tumors affecting the optic pathway. Surgery is also urgently indicated for patients with apoplexy who develop neuro-ophthalmologic complaints. In some experts’ opinion, tumors larger than 2cm should also be considered for surgery due to their propensity for growth (86). Treatment for hypopituitarism, primarily central adrenal insufficiency, and central hypothyroidism, should be commenced prior to surgical resection. Mortality rate is low (<1%) and reported surgical complication rates are acceptable. Postoperative complications such as CSF leakage, fistula, meningitis, vascular injury, persistent diabetes insipidus (DI), or new visual field defect occurred in ≤ 5% of patients, as reported by a systematic review and metanalysis (87). Accomplishment of total or near-total resection can be challenging and varies in different series, ranging from 20% to 80% (88,89). According to a recent large retrospective series evaluating 254 patients who underwent endoscopic endonasal surgery for a macroadenoma (including 72 patients with NFPAs), cavernous sinus invasion as assessed by the modified Knosp classification (90)effectively predicted surgical outcomes. Gross total resection was negatively correlated with tumor grade and success rates of gross-total resection were between 30% and 56% for tumors extending beyond the internal carotid artery and into the cavernous sinus compartments (grades 3A and 3B) (91).

 

A new “shape grading system” (Figure 3) has been recently proposed for predicting outcomes in patients with NFPAs operated by transsphenoidal surgery. In a retrospective single center study, 191 NFPAs were assessed according to different radiological growth patterns: spherical (Shape I), oval (Shape II), dumbbell (Shape III), mushroom (Shape IV), and polylobulated (Shape V) (92). Gross total resection was achieved in 53% of the patients, with decreasing likelihood of accomplishment in higher shape grades - shape I (82%), shape II (74%), shape III (24%), shape IV (17%) and shape 5 (0%). Furthermore, shape grades predicted resection rate better than Knosp grades. During a mean follow-up of 59 months, tumor recurrence or regrowth was observed in 6% and 12% of the patients, respectively. Higher shape grades also correlated with higher risk of tumor recurrence/regrowth as well as the need for reoperation and/or radiotherapy. Of note, SCAs more often grew as shape IV or V, while silent somatotroph adenomas were more often detected in the shape V group. Thus, the shape grading system seems to be a complimentary tool to better predict NFPA surgical outcomes, however, these findings need validation in other cohorts.

 

Visual field deficits and, less commonly, hormone deficiencies may improve following surgical treatment although new hormone deficiencies may also occasionally develop after a surgical approach (87,93). The incidence of DI after endoscopic transsphenoidal surgery to remove NFPAs was recently investigated in 168 patients. Seventy-seven (45.8%) patients experienced postoperative DI and 10 (6.0%) patients suffered from permanent DI. A large cephalocaudal tumor diameter (cut off value-of 2.7 cm) was predictive of postoperative DI in such patients (94). Moreover, younger age and the absence or intrasellar location of the bright spot (as opposed to suprasellar location) on preoperative T1-weighted MRI were further factors predicting postoperative DI in a Japanese series of 333 patients with NFPAs undergoing transsphenoidal surgery (95).

 

Pituitary function should be reassessed one to three months after surgery and treatment of hypopituitarism introduced according to hormone deficiencies. Whether or not GH deficiency should be replaced requires a thoughtful and individualized evaluation of risks and benefits (96,97). Current data supports the absence of any stimulation of remnant or induction of recurrence by growth hormone replacement in patients with NFPAs solely treated by surgical removal (98). In a retrospective series, tumor regrowth occurred in 38/107 (36%) of non-GH treated subjects and in 8/23 (35%) of GH-treated subjects, followed up for a mean period of 6.8 years. The Cox regression analysis showed that after adjusting for sex and age at tumor diagnosis, cavernous sinus invasion at diagnosis, and type of tumor removal, GH treatment was not a significant independent predictor of recurrence.

 

A sellar MRI should be obtained three to six months after surgery to assess the extent of tumor resection. Also, as a significant number of patients with NFPAs may develop tumor re-growth, long-term imaging surveillance is recommended. In a retrospective analysis of 155 patients with NFPAs treated solely by surgery and followed-up for a mean period of six years (twenty-nine were followed up for more than 10 years), re-growth was reported in 34.8% of the cases, with 20% of relapse/re-growth occurring after 10 years (88). Likewise, in patients with NFPAs who present with classical apoplexy, tumor re-growth rate is not negligible. In a retrospective series of thirty-two patients with NFPAs who underwent surgery for pituitary apoplexy, tumor re-growth was reported in 11% of the cases at just over five years (99). Therefore, it is advisable that patients with NFPAs, particularly those with post-operative tumor remnants, be closely monitored following surgery, and follow-up surveillance needs to certainly be continued for more than 10 years.

 

Radiation Therapy

 

Radiation therapy (RT) has been shown to be effective as an adjunct to surgical resection in cases of post-operative residual tumor or recurrence. However, it carries a major long-term risk of hypopituitarism (85,100,101). Stereotactic techniques such as stereotactic radiosurgery or fractionated stereotactic radiotherapy have been developed with the purpose of delivering more localized irradiation and reducing long-term side-effects. Both techniques provide excellent tumor control in patients with NFPAs, ranging from 85% to 95% at five to ten years (102). However, at the present time, there is no consensus concerning the systematic use of RT in the postoperative period for patients with incompletely resected NFPAs (29) and whether an earlier approach would be preferable over conservative management. In general, RT is reserved for cases with large tumor remnants and for those cases that present progressive tumor growth during follow-up (85). Adjuvant RT may also be considered for patients who, at diagnosis, already present aggressive tumors, such as those invading parasellar structures or with extensive positive immunostaining for Ki-67, a proliferative index significantly associated with recurrence in NFPAs (9,103). Furthermore, NFPA subtype may be a relevant factor in the expected response to radiotherapy. A retrospective multicenter study demonstrated that overall tumor control rate after radiosurgery was lower in SCAs compared to other NFPA subtypes (104) suggesting that, in SCAs, an elevated margin dose may be considered in order to achieve a better chance of tumor control. In line with these findings, a recent and large retrospective surgical series showed a significantly lower progression-free survival in patients with SCAs compared to other NFPAs (24.5 vs 51.1 months) (105). Among the SCA cohort, progression was noted despite the use of adjuvant radiosurgery in one third of the patients. Notwithstanding, a recent systematic review and meta-analysis showed no evidence supporting higher recurrence rate after primary treatment of SCAs compared to other NFPAs (106), however the evaluated study samples included only a small number of patients who had been offered adjuvant radiotherapy.

Primary treatment of NFPAs with medical therapy is not currently recommended (15,85). Small series have demonstrated significant tumor volume stabilization under medical treatment in non-operated patients with NFPAs due to contraindications or refusal (110), whereas other studies have revealed tumor volume progression in the long term (111). The main medical agents that have been evaluated in NFPAs are dopamine agonists (DA) and somatostatin analogues (SAs), mainly in patients with residual tumor after transsphenoidal surgery. Figure 4 shows a suggested algorithm for the management of patients with NFPAs.

 

DOPAMINE AGONISTS

 

Dopamine receptor type 2 (D2R) expression has been demonstrated in patients with NFPAs. In a small series of 18 patients with hormone-negative NFPAs, two thirds of them (12/18) expressed D2R by real-time polymerase chain reaction. Patients who presented residual tumor (9/18) were treated with cabergoline up to 3mg per week. After 12 months of treatment, tumor shrinkage was observed in 56% of the patients and tumor reduction was significantly greater in those expressing D2R (112). A historical cohort analysis on the adjunctive role of DA in adult patients with GH and ACTH negative NFPAs showed favorable results (83). The treatment group consisted of patients who were either initiated on DA upon post-surgical residual tumor detection or when tumor growth was subsequently detected on follow-up, while the control group received no medication after surgical treatment. Tumor control was significantly superior in patients who were treated upon detection of post-operative residual tumor, compared to those who were treated after presenting tumor progression or the control group, 87% vs. 58% vs. 47%, respectively. The requirement for additional treatment (surgery and/or radiotherapy) during follow-up was significantly decreased from 47% to 16% with adjunctive DA therapy. In this series, there were no correlations between clinical response to DA and D2R expression, the isoform type or their expression levels.

 

A randomized open-label clinical trial was recently conducted aiming to compare cabergoline with non-intervention in 116 Brazilian patients with residual NFPA after transsphenoidal surgery followed-up for over two years. NFPAs were classified solely based on IHC for anterior pituitary hormones and included 59% hormone-negative adenomas and 34% silent gonadotroph adenomas. Silent corticotroph adenomas were excluded from this study. By the end of the study, residual tumor shrinkage was significantly more frequently observed in the medical-therapy group compared to patients in the control group (28.8% vs. 10.5%). Tumor response was not associated with D2R expression (113). Thus, although cabergoline has not been a consensual treatment for patients with NFPAs, a therapeutic attempt can be made according to clinical judgment in individual cases; particularly in patients with large extrasellar remnants after surgery and a high probability of tumor progression (114). On the other hand, most studies published so far included only patients with hormone-negative or silent gonadotroph adenomas, such that this proposition should not be extended to silent corticotroph adenomas or the other rarer silent tumors. It is also important to bear in mind the natural history of untreated NFPAs, which may show a spontaneous decrease in tumor volume in up to 30% of them(115).

 

Cabergoline doses are variable, but usually started at 0.5 mg weekly, increasing 0.5 mg each week until a maximum dose of 3.0 mg/week is reached. For these patients, tumor shrinkage is not the major target although it can occur in 38% of them; otherwise, prevention of tumor growth is the main treatment goal. Tumor progression can be assessed by a sellar MRI every six months for the first two years, and annually thereafter. Once stability is achieved without significant side effects, the drug can be maintained indefinitely, taking into account individual cost-benefits (82,116).

 

SOMATOSTATIN RECEPTOR LIGANDS 

 

The finding of somatostatin receptors (SSTRs) expression by NFPAs has raised the possibility that the use of somatostatin receptor ligands (SRLs) could be an effective treatment strategy (117,118). The SRL octreotide which binds with high affinity to SSTR2 was not effective in controlling tumor size or improving visual field in a small group of patients with NFPAs (119). A case-control study evaluated the results of long-acting octreotide in patients harboring post-surgical NFPA residues and it demonstrated tumor remnant stabilization in 81% (21/26) of patients in the treated group compared to 47% (6/13) of patients in the control group after a mean follow-up of 37 months (120). However, neither visual field nor pituitary function changed in any of the groups. This cohort of 39 NFPAs consisted of a heterogeneous group as IHC revealed positivity for pituitary hormones in 28 cases (20 of those showing positivity for FSH and/or LH) whereas the remaining 11 cases were negative for adenohypophyseal hormones. SSTR5 was the predominant SSTR expressed (84%), followed by SSTR3 (61%), while SSTR2 was expressed in 46% of the cases.

 

The expression of SSTRs and zinc finger protein 1 (ZAC1), a protein regulating apoptosis and cell cycle arrest, were assessed in a group of NFPAs (SGAs and hormone-negative adenomas), active somatotroph adenomas, and normal pituitary. SSTR2 and ZAC1 expression was reduced whereas SSTR3 expression was increased in SGAs compared to active somatotroph adenomas and normal pituitary (121). Likewise, other studies have suggested that SSTR3 is the predominant SSTR expressed in most NFPAs, both by IHC studies (118,122) and mRNA levels (11,122). However, a few other studies have demonstrated higher SSTR2 expression than SSTR3 or SSTR5 expression in SGAs and hormone-negative adenomas (103,123). Regarding corticotroph pituitary tumors, SSTR5 expression was significantly less frequently found in SCAs as compared to their secreting counterparts (2/23 vs. 24/39) (124). Furthermore, amongst the 23 SCAs, somatic ubiquitin-specific protease 8 (USP8) mutation was detected in only two tumors, in line with previous reports showing a significantly lower prevalence of USP8 mutations in SCAs compared to functioning corticotroph tumors (125,126).

 

There is an ongoing multicenter, randomized, double-blind, placebo-controlled trial (GALANT study) evaluating the effectiveness of first generation SRLs in patients with NFPAs and suprasellar extension, either surgery-naive or with a postoperative remnant. Forty-four patients with positive results on 68Ga-DOTATATE PET are being randomized to treatment with either the SRL lanreotide or placebo (127).

 

Pasireotide is a universal SRL with action on SSTR1, SSTR2, SSTR3 and STR5 subtypes and, therefore, seems more attractive than first-generation SRLs as an alternative medical treatment for NFPAs. A head-to-head comparison of octreotide and pasireotide in MENX-affected rat harboring a mutation I the gene encoding p27, an in vivo model of NFPAs, was recently performed. Pasireotide showed superior anti-tumor effect vs. octreotide, especially in females, which also showed higher SSTR3 expression (128). There are two phase 2 clinical trials evaluating the safety and efficacy of pasireotide for the treatment of NFPAs. Both trials have been recently completed but the results are not yet available. Passion 1 (NCT01283542) is an open-label single arm study evaluating tumor volume response to pasireotide in naive patients with NFPAs >1cm. Another phase 2 clinical trial (NCT01620138) is currently comparing the response of patients with NFPAs and surgical remnants to cabergoline vs. pasireotide. Notwithstanding, until further data are available, the use of SSRLs is not currently recommended for the treatment of patients with NFPAs.

 

Treatment of NFPAs with dopastatin, i.e., chimeric compound with dopamine and somatostatin agonist activity, has also been under investigation. BIM-23A760, a compound with potent agonist activity both at D2R and SSTR2 effectively inhibited cell proliferation in primary cultures of NFPAs (129). Interestingly, TBR-760 (formerly BIM-23A760)was recently tested in a mouse model of aggressive NFPA and resulted in nearly complete inhibition of tumor growth (130). A one year, randomized, double-blind, placebo-controlled phase 2 study of TBR-760 in adult patients with residual NFPAs > 1cm after transsphenoidal surgery (NCT04335357) is being conducted and is expected to be completed by 2023.

 

TEMOZOLOMIDE

 

Temozolomide (TMZ) was the first alkylating chemotherapeutic drug to show significant response rates in aggressive pituitary tumors (131). Notwithstanding, TMZ seems to be less effective in NFPAs compared to their secreting counterparts as 45% of 110 clinically functioning pituitary tumors showed regression on first-line TMZ while only 17% of 47 NFPAs did (132).

 

Responsiveness to TMZ is probably dependent on the immuno-expression of O (6)-methylguanine DNA methyltransferase (MGMT), a DNA repair protein that acts by removing the alkyl group and therefore is associated with TMZ resistance (133). Low immuno-expression of MGMT by pituitary tumors has been associated with higher response rates to TMZ (131). MGMT immuno-expression was assessed in a group of 45 NFPAs and the degree of expression was correlated with tumor aggressiveness (133). Low MGMT expression was observed in 50% of the aggressive NFPAs compared to 24% of the non-aggressive NFPAs. These findings suggest that aggressive NFPAs with low MGMT expression could be potential candidates for treatment with TMZ.

 

Regarding dosing regimens and indications, current guidelines suggest using TMZ (150-200 mg/m²daily for 5/28 days) as an alternative treatment for patients with aggressive NFPAs presenting tumor progression despite radiotherapy and other therapeutic measures, and for exceptional cases of pituitary carcinomas (29,132). Furthermore, in the case of rapid tumor growth in patients who have not previously reached maximal doses of radiotherapy, the use of the STUPP protocol (six weeks of concomitant fractionated radiotherapy and TMZ 75 mg/m² daily, followed by TMZ alone 150-200 mg/m² daily for 5/28 days) has been suggested (132). Studies on long-term administration of TMZ are scarce but clinical observations indicate that TMZ should be continued for as long as it is effective and well tolerated. Some authors suggest continuing TMZ at standard dosage for two years and later reducing to half-dose (134). Patients receiving TMZ chemotherapy are at risk for hematologic toxicity, occurring in approximately 15-20% of the cases. The most common non-hematologic side effects of TMZ are nausea, anorexia, and fatigue (135).

 

PEPTIDE RECEPTOR RADIONUCLIDE THERAPY

 

In vivo SSTR expression by NFPAs has been previously demonstrated both by positive uptake on somatostatin receptor scintigraphy (120) and on 68Gallium DOTATATE PET/ CT (79) providing a rationale for the administration of PRRT in these patients. So far, PRRT has been studied in few patients with aggressive pituitary tumors (clinically functioning and NFPAs) with different response patterns (136,137). Among the six patients with nonfunctioning pituitary adenoma or carcinoma, three patients (not previously treated with TMZ) showed stable disease (138-140), two patients had progressive disease (138,141) and one patient died in the following months while information on tumor volume was lacking (142).

 

QUALITY OF LIFE AND LONG-TERM MORBIDITY AND MORTALITY

 

A substantial number of patients with NFPAs suffer from morbidities related to the tumor itself, as well as to the treatments offered. Standardized mortality ratios in these patients seem to be higher than that of the general population with deaths associated mainly with circulatory, respiratory, and infectious causes (143). Until now, there was no consensus on predictive factors of mortality but those most consistently described are older age at diagnosis (144,145) and high doses of glucocorticoid replacement therapy (146). A retrospective series of 546 patients operated on for a macro NFPA between 1963 and 2011 and followed up for a median period of 8 years reported a standardized mortality ratio of 3.6 (95% CI, 2.9–4.5) (144). After adjustment for factors proven to be significant in univariate analysis - radiotherapy, tumor regrowth, and untreated growth hormone deficiency - age at diagnosis was an independent predictor of mortality, with shorter survival observed in older patients.

 

Following NFPA treatment, patient-reported health-related quality of life (HR-QoL) substantially improves.evertheless, there are conflicting findings about HR-QoL normalization, which may also be related to the lack of a disease-specific HR-QoL questionnaire for NFPAs (147). Some studies have described a persistent decreased HR-QoL compared to healthy controls and reference data (148,149), while others have not (150). In the latest study, HR-QoL was evaluated in 193 consecutive patients with NFPAs followed up in a tertiary endocrine referral center (150). The overall health-related quality of life and perception of subjective health in patients with NFPAs was not compromised although specific groups, such as females, patients with tumor recurrences, and with visual defects, were shown to be affected in various dimensions. Altered sleep-wake rhythmicity has also been described in a cohort of 69 patients with NFPA in long term remission after transsphenoidal surgery on stable replacement treatment for hypopituitarism (151). NFPA patients reported severely impaired QoL, sleep quality, and increased daytime sleepiness. Preoperative visual field defects were associated with sleep-wake rhythm fragmentation and vasopressin deficiency was associated with decreased sleep efficiency, independent of age, hypopituitarism, or radiotherapy.

 

More recently, the use of the Wilson–Cleary model, a conceptual biopsychosocial model of HR-QoL, suggested that elements at each stage of this model could be contributing to the impairment in HR-QoL observed in patients with a NFPA (147). The authors concluded that currently available biomedical treatments, i.e., surgery, radiotherapy, and hormone replacement therapy, are clearly not sufficient for achievement of good HR-QoL in patients with a NFPA, and further improvement should be supported by a pituitary specific care trajectory, targeting not only biological and physiological variables, but also psychosocial care.

 

PROGNOSTIC FACTORS AND FUTURE PERSPECTIVES

 

There has been a search to identify reliable factors related to aggressiveness and the risk of recurrence in NFPAs. A single-center retrospective study which evaluated 108 surgically-resected NFPAs followed for up to 15 years (152)showed that 22% of the patients required further treatment, either second surgery or radiotherapy. Factors determining recurrence were the presence of residual tumor, tumor growth rate (>80 mm³/year), and suprasellar extension (152). According to another retrospective case series evaluating patients with NFPAs who presented tumor regrowth after primary treatment, the NFPA subtype, categorized by anterior pituitary hormone immunostaining, was not a predictive factor for the requirement of secondary treatment or tumor regrowth. Significant risk factors were female gender and treatment approach (monitoring vs. interventions); secondary progression was significantly higher in those patients who were followed conservatively (63%) as compared to those who received surgery (36%), radiotherapy (13%), and surgery/adjuvant radiotherapy (13%) (84). In patients with SGAs, ERα seems to be a prognostic factor for re-intervention (reoperation or radiation) in males - the combination of the absence of ERα expression and young age served as good predictive markers of aggressiveness (122).

 

The role of cellular markers, associated with cell proliferation and apoptosis, in predicting the recurrence of NFPAs has also been investigated (153). Proliferative indexes such as a high Ki-67 index, assessed by IHC, were significantly associated with a tumor size greater than 3 cm, as well as with tumor recurrence (103). Evaluation of tumor proliferation by using Ki-67 IHC is widely available and is recommended as part of the assessment of NFPAs (40). According to a large retrospective series evaluating 601 patients with surgically resected pituitary tumors, including approximately 30% NFPAs, the optimal cut-off points of the Ki-67 proliferation index that predicted recurrence was 2.5% with 84.6% sensitivity and 47.4% specificity (154). Moreover, a recent retrospective analysis of 120 patients operated on for an NFPA showed that invasive and proliferative tumors, i.e., grade 2b tumors. according to the clinicopathological classification of Trouillas et al. (155) showed an overall likelihood of recurrence that was approximately 9 times greater than those of grade 1a, i.e., non-invasive and non-proliferative tumors (156). Further risk factors associated with an increased risk of recurrence in this cohort were a younger age and the presence of residual tumor.

 

Minichromosome maintenance 7 (MCM7), a cell-cycle regulator protein, has been recently proposed as a marker of tumor progression in NFPAs. In a cohort study of 97 surgically treated NFPAs, the probability for reintervention within 6 years for patients harboring residual tumors with high MCM7 expression was 93% (157). Ki-67 expression >3%, age ≤55 years and mitotic index≥1, but not tumor subtype, were also associated with reintervention. Attempts to develop clinical nomograms to predict post-operative recurrence of NFPAs have recently been demonstrated based on age, tumor size, cavernous sinus invasion, sphenoid sinus invasion, and surgery extension. The nomogram model proposed by Lyu et al. (158) showed an area under the ROC curve of 0.953 and correlation analyses indicated that sphenoid sinus invasion, cavernous sinus invasion and tumor size could promote the recurrence of NFPA while advanced age and gross total resection could effectively inhibit it.

 

Genetic and epigenetic mechanisms underlying the development and aggressiveness of NFPAs have not been fully elucidated (159). The mutational landscape of a cohort of pituitary tumors including 37 NFPAs was recently published (160). Aside from demonstrating gonadotroph signatures in seven out of eight SCAs, retrieving the question whether a subset of SCAs arise from a specific pituitary lineage distinct from other corticotroph (161), this study showed that most NFPAs displayed no functional somatic variant and no chromosome alterations. Further characterization of these tumors with techniques such as whole genome sequencing coupled to chromatin structure analysis may disclose mutations in non-coding regions affecting chromatin opening and/or the binding of specific transcription factors. allowing for the development of novel therapeutic strategies.

 

An adverse pituitary adenoma phenotype is defined not only by the intrinsic activity of tumor cells but also by the infiltrated immune cells in the tumor microenvironment (162). The study of the immune profile of pituitary tumors for predicting immunotherapy responsiveness is an evolving field. Wang et al (163) have recently proposed classification of these tumors on three clusters based on tumor-infiltrating immune cells and the expression of immune checkpoint molecules This study cohort included 57 “unspecified“ NFPAs since they were not pathologically classified with pituitary transcription factors. The majority of them exhibited a “hot” immune microenvironment and were predicted to exhibit higher immunotherapy responsiveness.

 

CONCLUSION

 

NFPAs are frequent in endocrine practice. Clinically they range from being completely asymptomatic (incidental findings on head MRI or computed tomography scans) to causing significant hypothalamic/pituitary dysfunction and visual symptoms due to mass effect., For microadenomas and asymptomatic/relatively small macroadenomas (1–2 cm), a “watch and wait” option is reasonable. If tumor growth, development of visual field defects, or progressive pituitary dysfunction is detected during follow up, then surgery is indicated. It is now recommended that NFPAs are classified according to their pituitary hormone and transcription factor profiles along with proliferation markers such as Ki-67. This refined stratification may potentially aid in predicting disease course and in selecting adjunctive therapies. Radiotherapy is an effective treatment, although usually reserved for those patients with aggressive tumors and significant tumor remnant after pituitary surgery as considerable side effects may occur. Medical treatment with dopamine agonists stands as an alternative in selected cases, despite the lack of placebo-controlled trials. Highly aggressive tumors need special care during follow-up, including treatment with TMZ with or without RT complementation or new potential emerging treatments. Close collaboration of a multidisciplinary pituitary team is crucial to better serve this challenging group of patients.

 

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